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Till Johanna och Isak
List of Papers
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.
I
Neuronal nitric oxide synthase-deficient mice have impaired
renin release but normal blood pressure.
Sällström J, Carlström M, Jensen BL, Skøtt O, Brown RD,
Persson AEG.
Am J Hypertens, 21: 111-6, 2008.
II
Neuronal nitric oxide synthase supports renin release
during sodium restriction through inhibition of
phosphodiesterase 3.
Sällström J, Jensen BL, Skøtt O, Xiang G, Persson AEG.
Manuscript.
III
Diabetes-induced hyperfiltration in adenosine A1-receptor
deficient mice lacking the tubuloglomerular feedback
mechanism.
Sällström J, Carlsson PO, Fredholm BB, Larsson E,
Persson AEG, Palm F.
Acta Physiol (Oxf), 190: 253-9, 2007.
IV
Inhibition of sodium-linked glucose reabsorption in the
kidney normalizes diabetes-induced glomerular hyperfiltration in conscious adenosine A1-receptor-deficient mice.
Sällström J, Engström T, Fredholm BB, Persson AEG, Palm F.
Manuscript.
Reprints were made with permission from The Nature Publishing Group and
Wiley-Blackwell, respectively.
Contents
Introduction...................................................................................................11
Regulation of glomerular filtration...........................................................12
The myogenic response .......................................................................13
The TGF mechanism ...........................................................................14
Proximal reabsorption..........................................................................14
Nitric oxide...............................................................................................15
Nitric oxide synthases..........................................................................15
Modulation of TGF response by nitric oxide.......................................15
Nitric oxide and hypertension..............................................................16
The renin angiotensin aldosterone system................................................16
Renin release........................................................................................17
Intracellular regulation of renin release ...............................................19
Nitric oxide and renin release ..............................................................20
Glomerular hyperfiltration and diabetic nephropathy ..............................20
Renal glucose handling........................................................................21
Mechanisms causing diabetes-induced hyperfiltration........................21
Aims of the investigation ..............................................................................23
Methods ........................................................................................................24
Animals ....................................................................................................24
nNOS knockout mice...........................................................................24
A1AR knockout mice ...........................................................................24
Experimental protocols ............................................................................25
Protocol for study I ..............................................................................25
Protocol for study II.............................................................................26
Protocol for study III ...........................................................................27
Protocol for study IV ...........................................................................27
Telemetric blood pressure measurements ................................................27
Implantation of transmitters (Study I&II)............................................27
Data processing of chronic data (Study I) ...........................................28
Acute blood pressure response (Study II)............................................29
Measurements of renin and aldosterone...................................................29
Plasma renin concentration (Study I&II).............................................29
Plasma aldosterone concentration (Study I) ........................................30
Renin mRNA levels in the renal cortex (Study I)................................30
Renin genes (Study I) ..........................................................................30
Renal excretion and plasma potassium concentration..............................31
Urine collection in metabolic cages (Study I&III) ..............................31
Measurement of plasma potassium concentration (Study I)................31
Measurements of cAMP (Study II) ..........................................................31
cAMP concentration in the renal cortex ..............................................32
cAMP concentration in cultured As4.1 cells .......................................32
Acid dissection of renal tissue (Study II) .................................................32
Measurements of GFR and renal plasma flow .........................................33
GFR during anaesthesia (Study III) .....................................................33
GFR in conscious mice (Study IV)......................................................34
GFR and renal plasma flow in conscious mice (Study II) ...................34
Histology (Study III) ................................................................................35
Statistics ...................................................................................................36
Results...........................................................................................................37
nNOS in renin and blood pressure regulation (Study I&II) ....................37
Blood pressure and heart rate regulation .............................................37
Renin and aldosterone regulation ........................................................38
Renal excretion ....................................................................................38
PDE3 and renin regulation...................................................................39
cAMP measurements ...........................................................................41
PDE5 and renin regulation...................................................................42
Effect of nNOS deletion on JG cell index ...........................................42
Diabetes-induced hyperfiltration (Study III&IV).....................................43
TGF and diabetes-induced hyperfiltration...........................................43
Glucose reabsorption and diabetes-induced hyperfiltration ................44
Discussion .....................................................................................................45
The role of nNOS in renin regulation.......................................................45
Aldosterone regulation in nNOS-/- with impaired renin regulation ..........47
Cardiovascular consequences of nNOS deficiency..................................47
TGF and diabetes-induced hyperfiltration ...............................................49
Glucose reabsorption and diabetes-induced hyperfiltration .....................50
Summary and conclusions ............................................................................52
Future perspectives .......................................................................................53
Sammanfattning på svenska..........................................................................54
Kväveoxid, hypertension och renin (Arbete I&II) ...................................55
Diabetes, hyperfiltration och nefropati (Arbete III&IV) ..........................56
Acknowledgements.......................................................................................54
References.....................................................................................................61
Abbreviations
7-NI
A1AR-/AC
cAMP
bw
cGMP
eNOS
GFR
GU
JG
JGA
Kf
nNOS-/PAH
PBS
PGC
Pprox
PDE
PRC
SEM
SGLT
SNGFRprox
SNGFRdist
TGF
BS
GC
7-nitroindazole
adenosine A1-receptor knockout mice
adenylate cyclase
cyclic adenosine monophosphate
body weight
cyclic guanosine monophosphate
endothelial nitric oxide synthase
glomerular filtration rate
Goldblatt units
juxtaglomerular
juxtaglomerular apparatus
filtration coefficient
neuronal nitric oxide synthase knockout mice
para-amino hippuric acid
hydrostatic pressure in Bowman's space
hydrostatic pressure in the glomerular capillaries
hydrostatic pressure in the proximal tubule
phosphodiesterase
plasma renin concentration
standard error of the mean
sodium-glucose-linked transporter
single nephron GFR measured in the proximal tubule
single nephron GFR measured in the distal tubule
tubuloglomerular feedback
oncotic pressure in Bowman's space
oncotic pressure in the glomerular capillaries
Introduction
The kidneys regulate the body’s fluid balance and are therefore important for
blood pressure regulation. This is achieved by controlling the amount of
water and solutes excreted, and by the release of hormones. The tubuloglomerular feedback (TGF) mechanism is a negative feedback loop located
in the juxtaglomerular apparatus (JGA) of each nephron, that regulates the
glomerular filtration rate (GFR) to match tubular transport capacity. Nitric
oxide locally generated in the macula densa of the JGA by neuronal nitric
oxide synthase (nNOS), has been shown to have an important effect on the
sensitivity of the TGF mechanism. Earlier data from several models of experimental hypertension shows that an increased TGF sensitivity is observed
during the development of hypertension1,2, suggesting that TGF can play a
causal role in the development of the disease.
Renin is a proteolytic hormone that is secreted from the granulated juxtaglomerular (JG) cells in the wall of the afferent arterioles of the kidney. In
the circulation, renin is the rate-limiting step in the formation of the sodiumretaining and vasoconstrictive peptide angiotensin II, thus constituting an
important regulator of blood pressure and sodium balance. The close proximity of the JG cells and the nNOS-rich macula densa, suggests a possible
role of NO in renin regulation. However, studies on this issue have provided
conflicting results.
Nephropathy as a consequence of long-term diabetes is a significant
health problem3. The mechanisms causing nephropathy in diabetes have
however not yet been elucidated. Early in the disease, diabetic patients are
commonly found to exhibit glomerular hyperfiltration4,5. However, in one
third of the diabetic patients, GFR starts to decline after some time, as a result of a progressive diabetic nephropathy. It has been demonstrated that
diabetic patients that hyperfiltrate early in the disease, have an increased risk
of developing microalbuminuria and nephropathy6-8. According to one hypothesis, hyperfiltration is caused by a reduced activity of the TGF mechanism, caused by increased proximal sodium-glucose reabsorption that will
reduce the NaCl concentration at the macula densa9.
In the studies presented in this thesis, knockout mice were used to investigate the influence of different aspects of the JGA on the regulation of GFR,
blood pressure, and renin release. Effort has been made to improve the experimental techniques and, whenever it was possible, to perform measurements in conscious animals to avoid the confounding effects of anaesthesia.
11
Regulation of glomerular filtration
GFR is tightly regulated to match tubular reabsorption in order to maintain
fluid and electrolyte homeostasis. Without such careful control, fluctuations
in arterial blood pressure would either lead to fluid loss or retention. The
autoregulation of GFR is performed by two different mechanisms; the myogenic response and TGF. All nephrons work as single autoregulating units
and possess both mechanisms.
Like the filtration in other vessels, the filtration process in the glomeruli is
dependent on two factors; the net filtration pressure and the filtration coefficient (Kf). The relationship between these factors and GFR can be expressed
by the Starling equation10 (Figure 1). The filtration pressure, which is the
sum of the factors favouring filtration and those opposing filtration, can be
affected by several parameters. The glomerular capillary hydrostatic pressure
(PGC) is higher than the hydrostatic pressure in Bowman’s space (PBS), and
this pressure difference (PGC - PBS) favours filtration. Since the glomerular
membrane does not filter molecules larger than 50 Å, the ultrafiltrate is virtually protein-free and the oncotic pressure in Bowman’s space (BS) is
therefore close to zero. Accordingly, the oncotic pressure difference between
the glomerular capillaries and Bowman’s space (GC-BS) opposes filtration.
Figure 1. Illustration of the factors affecting net filtration pressure and GFR.
The oncotic pressure difference will increase from the afferent to the efferent
side of the glomerular capillaries when GC increases due to continuous filtration. Since the hydrostatic pressure difference will be essentially un12
changed, the filtration will reduce along the capillary network when approaching the efferent side.10 Munich-Wistar rats have been extensively used
for micropuncture, due to the frequent occurrence of superficial glomeruli.
In this strain, filtration equilibrium eventually occurs when the factors favouring and opposing filtration balance each other11. However, the more
commonly used Sprague-Dawley rats do not reach filtration equilibrium due
to the higher PGC12, while mice, however, have not yet been investigated.
Filtration pressure is directly influenced by renal plasma flow, the tonus
of the afferent and efferent arterioles, and the rate of reabsorption in the
proximal tubule. An increase in renal plasma flow will slow down the increase of plasma proteins in the glomerular capillaries, thus reducing GC,
which consequently will increase filtration pressure and GFR. An increased
contraction of the afferent arteriole will reduce GFR by decreasing plasma
flow and PGC. The effects of the tonus of the efferent arteriole are more difficult to predict. An increased contraction will reduce glomerular plasma flow,
which will act to reduce GFR, but will also increase PGC, which in turn will
counteract the decreased GFR.10 The rate of reabsorption in the proximal
tubule affects filtration pressure by changing PBS, i.e. an increased reabsorption will decrease PBS thus favouring filtration13.
The afferent and efferent arterioles are innervated by sympathetic adrenergic nerves. Nerve stimulation will cause a contraction of both arterioles,
which in turn will decrease GFR and renal plasma flow14. The contractile
effect of norepinephrine is mediated by adrenergic 1 receptors15. Adrenergic
1 receptors are also present, and apart from their role as a trigger of renin
release, they also appear to dampen arteriolar contraction induced by 1 receptors16. Glomerular ultrafiltration is also affected by many hormones and
other substances. Angiotensin II acts on angiotensin-II AT1 receptors in both
arterioles, which will reduce renal blood flow. However, since the contractile
effects are larger on the efferent side, GFR will not change to a large extent
during high plasma levels of angiotensin II.17 Adenosine is formed in the
kidney by ATP metabolism, and can constrict the afferent arterioles by activation of adenosine A1 receptors (A1AR)18. Adenosine plays an important
role in the TGF mechanism which will be discussed in detail below.
The myogenic response
The myogenic response is the ability of the afferent arteriole to contract in
response to increased arterial pressure, which will reduce blood flow to the
glomerulus. This is probably mediated by stretch-sensitive cation channels in
the vascular smooth muscle of the arteriole, that increase Ca2+ influx19. Interestingly, the myogenic response of the afferent arteriole has a special ability
to respond to fast systolic blood pressure oscillations, most likely in order to
protect the glomeruli from damage20.
13
The TGF mechanism
The TGF mechanism is a negative feedback loop located in the JGA of each
nephron that controls GFR. If the macula densa detects increased tubular
NaCl concentration, TGF is activated, constricting the afferent arteriole21,
thus matching the tubular sodium load to the reabsorption capacity. The relation between the luminal perfusion rate and the feedback response is nonlinear22 and the normal tubular flow rate is located on the steep portion of the
curve, which makes the system sensitive to small changes in flow rate23. In
1980, it was suggested that adenosine was the mediator of the TGF response24, which provided a link between the metabolic demand from the
NKCC2 transporters in the macula densa cells and vasoconstriction. The
afferent arterioles have a predominant expression of A1ARs25,26 which mediates vasoconstriction27 by Gi protein-mediated activation of phospholipase
C28, that will increase [Ca2+]i29. Further studies in A1AR-/- confirmed that the
TGF mechanism is dependent on A1ARs, since these mice completely lack
the TGF response30,31.
Proximal reabsorption
The rate of proximal reabsorption influences GFR, as demonstrated mainly
in studies where proximal reabsorption has been inhibited by carbonic anhydrase inhibitors. These inhibitors reduce GFR by direct effects on proximal
intratubular pressure as well as via TGF activation as a consequence of the
increased distal NaCl load. Based on the different effects of carbonic anhydrase inhibition on SNGFR measured proximally and distally, TGF effects
accounted for approximately 50% of the GFR decrease32. Further studies
demonstrated that removal of the influence of TGF by co-administration of
dopamine that dilates the afferent arteriole, was only able to partially restore
GFR after carbonic anhydrase inhibition, despite a higher glomerular capillary pressure than animals only receiving the inhibitor33. Moreover, carbonic
anhydrase inhibition was recently found to decrease GFR in A1AR-/- lacking
a functional TGF mechanism34. In hyperfiltering diabetic rats, several studies
have demonstrated a reduced proximal intratubular pressure35-38, which
might be secondary to the increased glucose reabsorption, but also due to
lower hydraulic flow resistance in the distal nephron segments during high
tubular flow rates13. Consequently, it is possible that the low PBS contributes
to the elevated GFR found during diabetes.
14
Nitric oxide
Nitric oxide is a gaseous, short-lived, potent vasodilator that affects vascular
smooth musculature via a cyclic guanosine monophosphate (cGMP) dependant pathway39. The substance was first shown to be produced from vascular
endothelium and was known as the endothelium-derived relaxing factor40
until it was identified as NO41,42.
Nitric oxide synthases
Nitric oxide is produced enzymatically when L-arginine is cleaved into
citrulline at a wide range of places in the body by three types of nitric oxide
synthases; endothelial (eNOS), inducible (iNOS) and neuronal (nNOS) nitric
oxide synthase43. Nitric oxide deficiency has been shown to have a role in
hypertension, both in animal experiments and in studies of human genomics.
eNOS expressed in blood vessels is important for maintaining normal endothelial function, and mice with targeted deletion of the gene for eNOS, develop hypertension44,45, whereas polymorphism of the gene in humans is
associated with an increased incidence of hypertension46. nNOS was first
detected in the brain47, but has later been found in several other tissues such
as the heart and kidneys. In the kidneys, nNOS is abundant in the macula
densa cells of the JGA48,49, where it regulates the tonus of the afferent arteriole48 and modulates the TGF mechanism50.
Modulation of TGF response by nitric oxide
The focused expression of nNOS at the macula densa indicates a functional
role of the enzyme in the JGA. Tubular infusion of an unspecific NOS antagonist decreased the glomerular capillary pressure, whereas an NO donor
increased the pressure, an effect that was abolished by the presence of an NO
scavenger48. Similar observations were made in isolated JGAs51. Consequent
studies demonstrated that NOS inhibition increased the sensitivity of the
TGF response, shifting the TGF curve to the left50,52 and also demonstrated
that NOS inhibition reduced the SNGFR at a given perfusion rate53. More
evidence of a specific role of nNOS was provided by experiments using the
relatively selective nNOS inhibitor 7-nitroindazole (7-NI), which caused a
similar sensitization as unspecific NOS blockade54. In nNOS-/-, SNGFRdist
was reduced and the proximal-distal SNGFR difference was increased, supporting the notion that nNOS-derived NO sensitizes TGF. However, the
maximum stop-flow response was not found different between the strains.55
The effects of NO appears dependent on cGMP56, and can be mediated either
through a direct vasodilatory action on the smooth muscle cells in the afferent arteriole, or alternatively, via inhibition of the NKCC2 transporters in the
macula densa cells57, thus reducing NaCl influx. Physiologically, nNOS15
derived NO might be important when there is a need to excrete excess volume, e.g. during volume expansion58.
Nitric oxide and hypertension
Intravenous infusion of an unspecific NOS inhibitor leads to an elevation of
the systemic blood pressure, due to an increase in peripheral vascular resistance caused by inhibition of eNOS in the vessels. There are some new findings indicating that peripheral resistance also might be affected by nNOS59,
but this isoform is not generally considered involved in the short-term blood
pressure regulation, and acute nNOS inhibition does not increase blood pressure54. However, long-term nNOS inhibition by 7-NI caused hypertension in
rats, which was characterized by increased TGF sensitivity and reduced GFR
in the initial phase. After the hypertension had developed, these parameters
were normalized.60 Interestingly, this development has similarities to that
seen in two hypertensive strains of rats; the spontaneously hypertensive
rat1,61 and the Milan hypertensive strain2,62, which exhibited volume retention
and an increased TGF sensitivity during the development phase of hypertension. The cause of hypertension in those strains has further been localized to
the kidney, due to the fact that transplantation of a kidney from a hypertensive rat to a normotensive control rat also transferred the hypertension63,64. In
light of these findings, it appears interesting to study mice with targeted deletion of nNOS. However, blood pressure in mice with nNOS deficiency has
not been found to be different from that in wild-type mice65,66, but the blood
pressure has only been measured during anaesthesia, which is known to influence cardiovascular parameters and might mask differences.
The renin angiotensin aldosterone system
The renin angiotensin aldosterone system is important for the normal regulation of blood pressure and electrolyte homeostasis, and is stimulated in situations of low blood pressure or low sodium intake. Renin is a proteolytic enzyme that is produced from the granulated JG cells in the wall of the afferent
arteriole. Already back in 1898, Robert Tigerstedt at the Karolinska Institute
discovered that a crude renal extract was able to increase blood pressure
when it was injected67. They realized that the extract contained an active
compound and called it renin, but the general interest of the finding was low,
and it took almost 40 years before the full renin angiotensin system was described.68 Renin is the central player in the system, and converts the physiological inactive protein angiotensinogen to angiotensin I. Angiotensin I itself
displays no vasoactive properties, but is rapidly transformed into the highly
active peptide angiotensin II by the action of the angiotensin converting enzyme (ACE). Angiotensinogen is continuously released from the liver, and
16
ACE is mainly localized to the vascular endothelium, particularly in the
lungs. The classical physiological effects of angiotensin II include general
vasoconstriction, increased renal electrolyte reabsorption, and thirst. Angiotensin II will increase aldosterone release from the adrenal gland, which, in
turn, will promote sodium retention. All these effects are mediated through
angiotensin AT1 receptors and will act to prevent a fall in blood pressure.69
Angiotensin II also acts on angiotensin AT2 receptors, which appear to have
opposite effects as the AT1 receptors. This is illustrated by studies in knockout mice, where deletion of the gene encoding AT1A receptors*, decreased
blood pressure and abolished the pressor response to angiotensin II infusion70. Conversely, deletion of the gene encoding AT2 receptors has been
shown to increase blood pressure slightly71,72, and augmented the pressor
response to angiotensin II infusion72. Angiotensin II has a short plasma halflife and is further degraded by different peptidases to shorter peptides (angiotensin III-IV) that are less vasoactive but have other properties that are
still not fully described. Humans and most mice strains only have one renin
gene (Ren-1), however some mice strains contain a duplicated copy of the
gene (Ren-2) that mainly expresses renin in the submandibular glands73. The
129 strain, which many genetically altered mice are derived from, expresses
this extra copy. If the targeted gene is close to the Ren-2 gene in the genome,
the extra renin gene might follow the mutated gene even when the strain is
inbred to another strain for several generations, which could complicate the
interpretation of renin data. However, a recent study could not find a clear
influence of the Ren-2 gene on the circulating renin levels74.
Renin release
The pathways regulating renin release can be assigned to four categories;
renal baroreceptors, macula densa, renal nerves, and hormones. Intracellularly, an increased cAMP concentration promotes renin exocytosis from the
JG cells, whereas an increased Ca2+ concentration inhibits exocytosis.
The renal perfusion pressure affects renin release via renal baroreceptors75,76, although no direct evidence of those receptors has been presented so
far. Experiments in whole animals suggest that the mechanism is independent from the macula densa pathway, since it is preserved in the non-filtering
kidney and during NKCC transport inhibition77,78. However, experiments
using isolated afferent arterioles have yielded conflicting results. One study
demonstrated a clear pressure dependency79, while a study from another
group failed to show this dependency80. The latter experimental protocol was
however slightly different, utilizing a model without perfusion. Mechanistically, increased perfusion pressure could reduce renin release via increased
*
Mice express two types of AT1-receptors; AT1A and AT1B, respectively, of which the AT1A
subtype appears most important and best mimics the human AT1-receptor.
17
intracellular Ca2+ concentration in the JG cells. The pressure could either be
sensed directly by the JG cells, or by the endothelial cells. The presence of
gap junctions between the endothelial cells and the JG cells81 suggests that
Ca2+ might be transferred between those cells. Connexin 40 knockout mice
do not reduce their plasma renin concentration (PRC) in response to an increased perfusion pressure, which suggests a specific role of gap junctions
composed by this connexin in endothelial-JG cell communication.82
The macula densa cells in the distal tubule regulate renin release (as well
as GFR) by sensing the NaCl concentration of the filtrate83. Increased NaCl
concentration at the macula densa will increase salt transport across the
NKCC2 carriers, which will increase adenosine exposure of the smooth
muscle and JG cells in the afferent arteriole. The activated A1ARs increases
[Ca2+] in the smooth muscle cells29, but also in the JG cells*, probably due to
the presence of gap junctions84. Furthermore, direct activation of A1ARs in
the JG cells reduces renin release85,86, possibly via [Ca2+] increase87, or as a
consequence of the receptor’s coupling to the Gi protein which will reduce
the intracellular cyclic adenosine monophosphate (cAMP) concentration88.
The macula densa pathway’s role is supported by recent data from knockout
mice, showing that deletion of the NKCC2A gene causes an abolished renin
reduction after an acute sodium load89. Furthermore, the basal PRC has been
reported to be increased in A1AR-/- mice30,90,91, and the acute response to a
saline load is abolished92, however the basal PRC was unaltered in the latter
study. Interestingly, the macula densa pathway also seems to be important
for the renin-suppressing effects of an increased renal perfusion pressure,
since this response is impaired in A1AR-/- mice91.
The macula densa cells have a high expression of COX-2 and nNOS,
which may act stimulatory on renin release, as indicated by the increased
expression of both enzymes during conditions with a high PRC93-95. During a
low luminal sodium chloride concentration at the macula densa, the [Ca2+]
has been shown to increase in the macula densa cells96. PGE2 is a powerful
stimulant of renin release97, which acts on EP2 and EP4 receptors on the JG
cells98. A high Ca2+ concentration will increase the activity of cytosolic
phospholipase A2, which would increase the availability of arachidonic
acid99 that is the rate-limiting step in prostaglandin production. This mechanism is supported by a recent study where release of PGE2 from macula
densa cells correlated negatively with the luminal sodium chloride concentration100. For long-term adaptation, evidence indicates that p38 and p44/42
mitogen-activated protein kinases could be activated by a low extracellular
[Cl-], which would promote COX-2 expression101. NO derived from nNOS
could also be involved in prostaglandin signalling, since it may increase the
activity of COX-2102. NO also directly affects renin release via its second
messenger cGMP, and the main effect seems to be stimulatory, even though
*
Lai E, Patzak A, Liu R, Persson AEG, unpublished observation.
18
NO during some circumstances could have an inhibitory role, which will be
discussed in detail below.
The JGA is innervated by sympathetic nerve fibres103, and renal nerve
stimulation increases renin release104, while bilateral denervation reduces
PRC94,105. Furthermore, administration of isoproterenol increases renin release, while propranolol decreases the release106. Adrenergic 1 receptors on
the JG cells will activate adenylate cyclase (AC) via the Gs protein, which
will increase renin release107. Sympathetic nerve stimulation of the JG cells
also appears to stimulate renin synthesis, which is demonstrated by the low
renin mRNA levels in denervated kidneys105 or kidneys from animals deficient of 1/2 receptors108. The degree of renal sympathetic nerve activity is
affected by baroreceptors in the circulatory system. High-pressure baroreceptors in the carotid sinus and low-pressure baroreceptors in the atria, will
increase renal sympathetic nerve activity and renin release during a fall in
systemic blood pressure107. The physiological mechanism that appears to
rely most on renal nerves for a normal PRC response, is loss of blood volume, since this response is reduced by renal denervation109, and abolished if
the pressure fall in the atria is prevented110. The response to alteration in
dietary sodium intake is relatively well-preserved after renal denervation94,105, or in animals deficient of 1/2 receptors108, indicating that other
pathways are more important for the adaptation to salt intake.
Renin release is affected by several hormones, of which angiotensin II
and atrial natriuretic peptide (ANP) probably are the most important. Angiotensin II serves as a negative feedback loop, limiting the renin secretion by
binding to AT1 receptors on the JG cells that increase [Ca2+]i87,111. Conversely, AT1 receptor antagonists increase renin secretion112. An increased
distension of the atrium, secretes ANP113,114, which reduces renin release115
by acting on the guanyl cyclase-coupled natriuretic peptide receptor-A116.
Accordingly, inhibition of renin release by ANP in isolated JG cells, is associated with an increased intracellular cGMP concentration117.
Intracellular regulation of renin release
Renin is predominantly formed and released from the JG cells, which are
mainly located in the distal end of the afferent arteriole. JG cells are modified smooth muscle cells, but they have a sparse content of myofibrils and a
low degree of myosin expression118. Renin is stored in vesicles in the cytoplasm, that are secreted by exocytosis. The main intracellular factors regulating renin release are the concentrations of cAMP and Ca2+. Cyclic AMP
stimulates exocytosis, and the production is regulated by G protein-coupled
receptors that control AC activity. JG cells express various receptors that
stimulate AC activity through the Gs protein, e.g. 1, EP2/EP4 and IPreceptors. The [cAMP]i is also regulated by the degree of degradation by
phosphodiesterases, and data indicates that the most important are
19
PDE3&4119. Cyclic GMP acts as a competitive inhibitor of PDE3, since this
isoform also degrades cGMP, but at a much lower catalytic rate120. Cyclic
AMP mediates exocytosis through a protein kinase A-dependent pathway,
but the exact mechanisms are not yet understood. Ca2+ acts inhibitorily on
renin release, in opposition with the role in most other secretory cells. This
might be explained by the presence of the Ca2+ inhibitable AC5 and 6 isoforms in the JG cells121. In the long-term, renin release can also be affected
by the number of JG cells. This is achieved by metaplasia, i.e. smooth muscle cells are replaced by JG cells122. For example, this occurs in mice with a
deficient angiotensinogen production in order to increase the production of
Angiotensin II123.
Nitric oxide and renin release
The mRNA levels of nNOS and renin in the renal cortex correlate directly,
which suggests a causal association94,95. However, the role of NO in renin
regulation has been a matter of controversy, and over the last years, in vivo
evidence has been presented for both a stimulatory124-126 and an inhibitory127130
role of the substance. The disparate results may have been caused by the
use of different NOS inhibitors as well as different doses. In vitro data has
shed some light on the issue. In cortical kidney slices, NO was shown to
inhibit renin release131, while in isolated JG cells, NO initially had an inhibitory role, but after some hours had a stimulatory role132. A stimulatory role
of NO was also observed in isolated perfused rat kidneys133. In isolated perfused JGAs, NO at the macula densa appears to support renin release,
whereas NO in the arterioles appears to have an inhibitory role134, which
illustrates that the effects of NO vary to a large extent with the place of action. The effects can be different in various anatomical structures, and also
when different NOS isoforms are blocked. In 1994, it was suggested that NO
from the macula densa could increase renin secretion by inducing production
of cGMP, that competitively inhibits PDE3 in the JG cells, thus increasing
the cAMP concentration and the renin release in response to diverse stimuli135. Conversely, the inhibitory effect might be caused by cGMP-mediated
activation of G kinase, that directly inhibits renin exocytosis133.
Glomerular hyperfiltration and diabetic nephropathy
Diabetes is the leading cause of end-stage renal disease worldwide136. During
end-stage renal disease, GFR has reached such low levels that death will
occur without dialysis. A strong predictor of future nephropathy and increased mortality is microalbuminuria, which is defined as the excretion of
30-300 mg albumin/24 hours137-140. The source of albumin is leakage from
the glomerulus which might be caused by a decrease in negatively charged
20
heparan sulphate proteoglycans, that are important for charge-based permeability of the glomerular basement membrane, in particular since albumin is
negatively charged141,142. Early in the disease, before the development of
microalbuminuria and nephropathy, glomerular hyperfiltration is commonly
found4,5. The increase is largest in newly diagnosed, untreated diabetes, and
GFR is returned towards normal values by intensive insulin treatment143.
Hyperfiltration has clinical importance since the presence of early hyperfiltration correlates with an increased risk of developing microalbuminuria
and nephropathy later on in the disease6-8.
Renal glucose handling
Glucose is reabsorbed in the proximal tubule by secondary active transport.
At the apical membrane, glucose and sodium are transported into the cell by
sodium-glucose-linked transporters (SGLT), driven by the low [Na+]i caused
by the basolateral Na-K pump. The accumulated glucose leaves the cell
through the basolateral membrane by facilitated diffusion through GLUT
proteins. In the early proximal tubule, glucose is reabsorbed by the highcapacity/low-affinity SGLT2 protein (1Na+/glucose), while the lowcapacity/high-affinity SGLT1 protein (2Na+/glucose) reabsorbs glucose in
the late proximal tubule. When the plasma glucose reaches approximately 11
mM, the SGLTs starts to saturate and glucosuria will occur if the concentration exceeds this level.144 During diabetes, the chronically high glucose levels will increase the proximal glucose reabsorption capacity145,146, probably
due to proximal hypertrophy and upregulation of SGLT1/2147,148. Since glucose is reabsorbed by cotransport, the reabsorption of sodium will increase
as well. This has been demonstrated in perfused proximal tubules, where
increased tubular glucose concentration increased sodium uptake significantly149. Furthermore, data obtained by lithium clearance in diabetic patients, indicates an increased proximal reabsorption of sodium150.
Mechanisms causing diabetes-induced hyperfiltration
The diabetic kidney is characterized by growth, and increased GFR and
blood flow143. The renal enlargement can be caused by several factors, but
the most central is probably the increased filtration of glucose which will
upregulate glucose transporters and cause growth of the proximal tubule.
The relation to the glucose load is demonstrated by the finding that there is a
positive correlation between kidney size and insufficient glycaemic control151.
Increased renal blood flow is generally found during diabetes and could
be the factor responsible for hyperfiltration, but the mechanisms are not fully
understood. Renal blood flow can increase either in response to a primary
vascular, or to a primary tubular event. A primary vascular event is a direct
21
relaxation of the afferent arteriole (e.g. due to increased production of a
vasodilator), which consequently will increase blood flow. A primary tubular
event will instead dilate the afferent arteriole indirectly, via the tubuloglomerular feedback mechanism.
A proposed mechanism for a tubular cause for diabetes-induced hyperfiltration is the tubular hypothesis of glomerular filtration9. According to this
theory, glomerular hyperfiltration is secondary to increased proximal reabsorption. The resulting reduction in the NaCl load to the macula densa will
reduce the TGF response, consequently increasing the GFR. Several investigators have found an increased proximal reabsorption in diabetes, both in
patients and in models of experimental diabetes38,150,152. Mechanisms that can
cause this include increased expression of SGLTs147,148, and hypertrophy of
the proximal tubule, increasing the reabsorption area153. The dependence on
renal growth was confirmed by blocking ornithine decarboxylase, which
prevented the development of hyperfiltration in diabetic rats9. Furthermore,
by micropuncture experiments, it has been confirmed that the NaCl concentration close to the macula densa is reduced38,154.
As discussed above, the degree of proximal reabsorption affects GFR directly by affecting the Pprox and the net filtration pressure. Consequently, it
has been proposed that hyperfiltration could directly result from the increased proximal reabsorption found during diabetes155, which is supported
by the findings that Pprox actually is reduced during the disease35-38.
22
Aims of the investigation
This thesis deals with functional aspects of the JGA. The first two studies
focus on the role of nNOS in blood pressure and renin regulation, whereas
the two following studies investigate mechanisms for the development of
diabetes-induced glomerular hyperfiltration.
Specifically, the aims were to:
¾ Investigate if nNOS deficiency can cause hypertension.
(Study I)
¾ Investigate the role of nNOS in the regulation of renin
release. (Study I & II)
¾ Determine the roles of TGF and renal glucose reabsorption in
diabetes-induced hyperfiltration. (Study III & IV)
23
Methods
Animals
All experiments were approved by the animal ethics committee of Uppsala.
nNOS knockout mice
The nNOS-deficient mice used in Study I & II were bred at the BMC animal
department by using heterozygous breeding pairs (B6.129S4-Nos1tm1Plh, The
Jackson Laboratory, Bar Harbor, ME). Their genotype was determined by
taking a small biopsy specimen from their tail or ear at the age of three
weeks. DNA was extracted from the tissue using a preparation kit (DNeasy
Tissue Kit, Qiagen, Hilden, Germany) whereby their genotype was determined using PCR. The primers used were as follows: 5’-tca gat ctg atc cga
gga gg-3’; 5’-ttc cag agc gct gtc ata gc-3’; 5’-ctt ggg tgg aga ggc tat tc-3’;
5’-agg tga gat gac agg aga tc-3’. Amplification was carried out for 37 cycles;
denaturation at 94°C for 35 s, annealing initially at 64°C for 45 s with a decrease in temperature of 0.5°C per cycle until 58°C, extension at 72°C for
45 s per cycle with a final extension time of 2 min. The animals in study I
were given two diets (TD90228 & TD92034, Harlan Scandinavia, Denmark)
containing different sodium concentrations; low sodium (0.01% NaCl) and
high sodium (4% NaCl), whereas the animals in study II were only given
low sodium diet (0.01% NaCl; Lantmännen, Kimstad, Sweden).
A1AR knockout mice
The A1AR knockout mice developed by Johansson and co-workers156 used in
Study III & IV were bred and genotyped at The Karolinska Institute. The
strain has been inbred on a C57BL/6J background (Jackson Laboratories,
Bar Harbor, MA). The animals in study III & IV were given standard rodent
chow (0.7% NaCl; Lantmännen, Kimstad, Sweden). Diabetes was induced
by a single bolus injection of alloxan (75 mg/kg bw) in the tail vein. Animals
with a plasma glucose concentration 20 mM were considered diabetic.
24
Experimental protocols
Protocol for study I
Series 1 – Telemetric blood pressure measurements
Low sodium
diet
(10 days)
Telemetry
recording
(2-4 days)
High sodium
diet
(10 days)
Telemetry
recording
(2-4 days)
Series 2 – Plasma renin concentration
Low- or high sodium diet
(10 days)
Blood sample
Series 3 – Plasma aldosterone, potassium and renin mRNA
Low- or high
sodium diet
(10 days)
Blood sample
Renal cortex
harvested
Series 4 – Renal excretion
Low- or high sodium diet
(10 days)
Metabolic cage
(24 h)
Normal cage
(1-2 days)
Repeated 4 times
25
Protocol for study II
Series 1 – Plasma renin concentration
Milrinone/Vehicle
or
Zaprinast/Vehicle
injection
Low sodium diet
(10 days)
Normal sodium
diet
(14 days)
Blood sample
Repeated once (cross-over design)
Series 2 – Glomerular filtration rate and renal plasma flow
Milrinone/Vehicle
or
Zaprinast/Vehicle
injection
Low sodium diet
(10 days)
Normal sodium
diet
(14 days)
PAH/Inulin
clearance
Repeated once (cross-over design)
Series 3 – Telemetric blood pressure measurements
Telemetry
recording
(30 min)
Low sodium diet
(10 days)
Milrinone/
Zaprinast/
Vehicle injection
Telemetry
recording
(30 min)
Repeated
Series 4 – Acid dissection and tissue cAMP concentration
Low sodium diet
(10 days)
Renal cortex harvested
from one kidney
Other kidney
placed in acid
Series 5 – Cell culture
Cells seeded and
cultured
(24 h)
26
New medium with
either vehicle or
milrinone
Incubation
(2 h)
Cells
harvested
Washout
(2 days)
Protocol for study III
Series 1 – Glomerular filtration rate and histology
Alloxan
injection
Diabetes
2-3 weeks
Surgery
Inulin bolus
and infusion
(40 min)
Urine collection
and blood
sampling
(60 min)
Left kidney
formalin fixed
Series 2 – Renal excretion
Alloxan
injection
Diabetes
2-3 weeks
Metabolic
cage
(24 h)
Normal cage
(1-2 days)
Repeated once
The normoglycemic controls were treated in the same manner, except for
the alloxan injection.
Protocol for study IV
Series 1 – TGF and diabetes-induced hyperfiltration
GFR
measurement
Alloxan
injection
Diabetes
4-5 weeks
GFR
measurement
Series 2 – Glucose reabsorption and diabetes-induced hyperfiltration
Alloxan
injection
Diabetes
4-5 weeks
Phlorizin/Vehicle
injection
+ GFR measurement
Recovery
(4 days)
Phlorizin/Vehicle
injection
+ GFR measurement
The normoglycemic controls were treated in the same manner, except for
the alloxan injection.
Telemetric blood pressure measurements
Implantation of transmitters (Study I&II)
All surgical equipment was sterilized. Animals were anaesthetized with ~2%
Isoflurane (Abbott, Solna, Sweden) in 40% O2 and 60% N2, and a midline
incision was made from the lower mandible to the sternum. A telemetric
blood pressure transmitter (TA11PA-C10, Data Sciences International, St.
27
Paul, MN) was introduced into the left carotid artery and advanced to the
aortic arch. The catheter was secured with a 5-0 silk ligature and a drop of
tissue glue (Vetbond; 3M Animal Care Products, St Paul, MN). The body of
the transmitter was placed subcutaneously on the right flank and the incision
was closed using 5-0 sutures. The animals were allowed to recover for ten
days before experiments commenced.
Data processing of chronic data (Study I)
During every experimental period, data for blood pressure and heart rate was
sampled for 5 s every second minute during a period of at least 48 hours,
using a computer program (PC-Lab 5.0, AstraZeneca, Mölndal, Sweden).
Physical activity was measured by the telemetric system during 30 s every
second minute, by analyzing variations in the received radio signal during
animal movement. To minimize the impact of animal movement on blood
pressure and heart rate measurements, new mean values were calculated only
from measurements sampled when the animals had been physically inactive
for at least 10 minutes. Sampling was continued until the animal moved once
again. Figure 2 shows a plot of a heart rate recording demonstrating how the
selection was performed. To study the heart rate and blood pressure during
physical activity, mean values were calculated in the same manner but instead using values sampled when concomitant activity was being recorded.
Figure 2. The green-dashed peaks show activity, the red/blue line heart rate. The
line turns blue (—, round dots) and data is saved when the animal has been inactive
for at least 10 minutes. When the animal moves again, data is discarded and the line
turns red (—, square dots).
28
Acute blood pressure response (Study II)
The telemetric system was connected to an analog-to-digital converter
(MacLab; ADInstruments Pty Ltd, Bella Vista, NSW, Australia), whereby
blood pressure was measured for 30 minutes in undisturbed nNOS+/+ and
nNOS-/- mice maintained on a low sodium diet, whereupon they were administered an i.p. injection of milrinone (0.5 or 3 mg/kg bw), zaprinast (10
mg/kg bw) or their corresponding vehicles. The pressure was followed for
another 30 minutes, whereupon 5-minute mean values were calculated.
Measurements of renin and aldosterone
Plasma renin concentration (Study I&II)
In study I, nNOS+/+ and nNOS-/- were placed on either a low or high sodium
diet for ten days after which they were briefly anaesthetized with isoflurane.
A blood sample (~100 μl) was taken from the retro-orbital plexus and immediately spun down using a cooled centrifuge, whereby plasma was isolated and frozen in liquid nitrogen.
In study II, animals were given a low sodium diet for ten days, whereupon
either a PDE inhibitor (Sigma-Aldrich Sweden AB, Stockholm, Sweden) or
vehicle was given as an i.p. injection. After 15-20 minutes, a blood sample
was taken as described above. Animals were then returned to a normal sodium diet for two weeks, whereafter they once again were given low sodium
diet for ten days. The experiment was repeated, but animals that had been
given milrinone, were instead given vehicle, and vice versa, allowing us to
calculate the effects of milrinone on PRC in every subject. Three different
series were performed where the animals were given the PDE3 inhibitor
milrinone (0.5 & 3 mg/kg), the PDE5 inhibitor zaprinast (10 mg/kg) or their
corresponding vehicles.
The PRC was measured by radioimmunoassay, using the antibody trapping technique157. In brief, 10 μl plasma from each sample was serially diluted (25, 50, 100 and 200X). Five microlitres of each dilution of plasma
was incubated in duplicate for 24 hours together with a mixture of rabbit
angiotensin I antibody and renin substrate (angiotensinogen equivalent to
1200 ng angiotensin I/ml) from rats. After the incubation step, the reaction
was stopped by addition of 1 ml cold barbital buffer, an angiotensin I tracer
was added, and radioimmunoassay was performed. Renin values were standardized by reference to renin standards obtained from The Institute for
Medical Research (Holly Hill, London, UK), and the values are expressed in
standard Goldblatt units (GU). One Goldblatt unit is defined as the amount
of renin needed to increase blood pressure in a dog by 30 mmHg in 3 minutes and was formerly called “dog unit”158.
29
Plasma aldosterone concentration (Study I)
The large plasma volume needed (~500μl) for the aldosterone analysis required a different blood sampling technique. nNOS+/+ and nNOS-/- maintained on a low sodium diet for ten days were anaesthetized by Isoflurane,
whereafter the abdomen was opened by a midline incision. Using a needle, a
blood sample was quickly taken from the vena cava to a syringe containing
EGTA as anticoagulant. Plasma aldosterone concentration was measured
with a commercial radioimmunoassay (COAT-A-COUNT, Diagnostic Products, Los Angeles, CA).
Renin mRNA levels in the renal cortex (Study I)
The renal cortex from nNOS+/+ and nNOS-/- maintained on a low sodium diet
for ten days was dissected, snap frozen in liquid nitrogen and stored
at -80ºC. Total RNA was isolated with mini RNeasy columns (Qiagen, Albertslund, Denmark), quantified by spectrophotometry, and stored at -80oC.
cDNA for amplification was obtained by reverse transcription of 1 μg total
RNA with a reverse transcriptase kit (iScript cDNA synthesis kit, BioRad).
Primer sequences were: Renin sense 5´gct-atg-tga-aga-agg-ctg-3´ antisense
5´ttc-tct-tct-cct-tgg-ctc-3´, 113 bp, covering bases 858-970 (GENEBank
BC061053); Ribosomal 18S subunit sense 5´ctg-tgg-taa-ttc-tag-agc-3´ antisense 5´-agg-tta-tct-aga-gtc-acc-3´,158 bp covering bases 151-308 of rat
sequence (GenBank V01270). For qPCR, 50 ng cDNA in duplicate was used
as a template and mixed with primers and iQ-SYBR Green Supermix (BioRad) in a final volume of 25 μl. The mixture was denatured for 3 min at
95°C and 44 cycles were run on an iQ-Thermocycler (BioRad): denaturation
30 s at 95°C, annealing and extension 45 s at 60°C. Standard was linearized
plasmid containing a fragment of mouse renin cDNA. The standard curve
was constructed by plotting threshold cycle (Ct-values) against serial dilutions of plasmid standard. Specificity was established post-run for each plate
setup by melting curve analysis. Negative controls were water instead of
cDNA and reverse transcriptase-negative samples. Random samples from
each plate were loaded on agarose gels to confirm the amplification product.
Renin genes (Study I)
The nNOS-/- are originally derived from the 129 mouse strain and later inbred on the C57Bl/6J strain for at least ten generations. To exclude the possibility that the mice used in the present study carry a second renin gene that
could confound the results, PCR was performed on genomic DNA using
primers for the Ren-1 and Ren-2 genes, published by Hansen and coworkers74. DNA from A1AR-/-, known to carry both genes, was used as a
positive control.
30
Renal excretion and plasma potassium concentration
Urine collection in metabolic cages (Study I&III)
For the renal excretion measurements, mice were placed in metabolic cages
for 24 hours with access to food and water ad libitum (Figure 3). This procedure was repeated on the same animal; four times in study I, and two times
in study III. Between the measurements, the animals were put back into their
normal cage for 1-2 days. The urine volumes were measured gravimetrically
and urine osmolality was determined with an osmometer (Fiske 210 MicroSample Osmometer, Fiske Associates, Norwood, MA), whereas sodium and
potassium concentrations were determined by flame photometry (IL943,
Instrumentation laboratory, Milan, Italy). The water consumption was calculated from the weights of the water bottle and water spillage collection
chamber. In study III, urinary protein excretion was measured by a standard
laboratory assay based on the Lowry method (Bio-Rad DC Protein Assay,
Bio-Rad Laboratories, Hercules, CA).
Figure 3. Schematic illustration of a metabolic cage
used for urine collections; a) water bottle b) water
spillage collection chamber c) feed container d)
feed spillage collection chamber e) urine collection
chamber f) faeces collection chamber.
Measurement of plasma potassium concentration (Study I)
The potassium concentration in plasma was measured using flame photometry (IL943, Instrumentation Laboratory, Lexington, MA) from the samples
used for aldosterone measurement.
Measurements of cAMP (Study II)
The concentration of cAMP was determined in cortical tissue from nNOS+/+
and nNOS-/- maintained on a low sodium diet for ten days. Also, the milrinone
response was investigated in cultured As4.1 cells, a renin producing cell line.
31
cAMP concentration in the renal cortex
The renal cortex from kidneys from nNOS+/+ and nNOS-/- was separated and
mortared in liquid nitrogen and transferred to an Eppendorf tube with 200 μl
5% trichloroacetic acid in H2O. The tube was vortexed and sonicated for 5
minutes, whereupon it was centrifuged at 10000 rpm for 10 minutes. The
supernatant was moved to a new tube where the trichloroacetic acid was
removed by ether extraction. The cAMP concentration was measured in
acetylated samples using an ELISA (Cayman Chemicals, Ann Arbor, MI).
The tube with the pellet was dried and the weight of the tissue was calculated by subtracting the weight of the empty tube. The cAMP concentration
was related to the dry weight of the tissue.
cAMP concentration in cultured As4.1 cells
The renin-producing cell line As4.1 was acquired from ATCC (Manassas,
VA) and cultured in Dulbecco’s Modified Eagle’s Medium with 10% fetal
bovine serum, penicillin (178 units/ml) and streptomycin (0.178 mg/ml)
according to the manufacturers’ instructions. For the experiments, 240 000
cells/well were seeded in a 24-well plate and cultured for 24 hours, whereupon the medium was exchanged with new medium with added forskolin (5
μM) and either milrinone (100 μM) or vehicle (DMSO). The total DMSO
concentration in all wells was 0.15%. Cells were incubated for two hours,
whereafter the medium was discarded and 100 μl 0.1 M HCl added. After 20
minutes, the cells were harvested using a cell scraper and transferred to Eppendorf tubes. After 5 minutes of sonication, the tubes were spun at 10000
rpm for ten minutes, whereupon the supernatant was collected. The cAMP
concentration was measured in acetylated samples using an ELISA as described above and related to the protein concentration which was determined
using the Bio-Rad DC Protein Assay (Bio-Rad Laboratories, Hercules, CA).
Acid dissection of renal tissue (Study II)
The acid dissection technique was used to visualize the degree of renin activation159. By this technique, the JG cells can easily be identified without
using any staining, due to the different optical properties of JG cells and
smooth muscle cells. The kidneys from nNOS+/+ and nNOS-/-, maintained on
a low sodium diet for ten days, were removed and placed into 5M HCl at
37ºC for one hour. The tissue was thereafter placed in distilled water at 4ºC
for at least 24 hours before the dissections were performed. Using a dark
field stereo microscope, the afferent arteriolar branches were classified into
three categories by a blinded observer; JG cells in juxtaglomerular position,
midafferent JG cell recruitment, or no visible JG cells. The ratio between
32
each category and the total number of visible arterioles was calculated for
every dissected branch. The arterioles from 3-10 branches from each kidney
were counted twice, whereby a mean value from each kidney was calculated
for the statistics.
Measurements of GFR and renal plasma flow
GFR was measured by two different techniques. In the first experiments
(Study III), we measured inulin clearance in anaesthetized animals as the
relation between the plasma concentration and the excreted amount. When
we performed the study, we realized that it would be an advantage to be able
to perform repeated measurements in the same animal, before and after an
intervention, and also avoid anaesthesia. We evaluated different techniques,
and found that the single bolus injection method was most appropriate. By
this technique, inulin clearance in study IV was measured in conscious mice
using only the plasma disappearance curve after the injection. In study II, we
injected both inulin and para-amino hippuric acid (PAH), which also allows
estimation of the renal plasma flow.
GFR during anaesthesia (Study III)
The mice were anaesthetized with Isoflurane (~2 % in 40 % oxygen;
Univentor 400; AgnThos, Stockholm, Sweden) and placed on a servocontrolled heating pad maintaining body temperature at 37.5 °C. Polyethylene catheters were placed in the carotid artery for blood pressure measurements, and in the jugular vein for the infusion of Ringer’s solution. An additional catheter was placed in the bladder for urine collection.
A bolus dose of [3H]methoxy-inulin (26 kBq in 0.1 ml saline; American
Radiolabeled Chemicals, St. Louis, MO) was given followed by the continuous infusion of fluid containing 260 kBq/ml. The infusion rate was set to 0.3
ml/h in control animals and 0.6 ml/h in diabetic animals, in order to compensate for the increased urinary flow rate in the diabetic animals. Clearance
measurements were started after an initial 40-min recovery period. Urine was
collected during two consecutive 30-min periods. Blood samples (20 μl) were
taken from the carotid artery in heparinized glass capillary tubes in the middle
of each period. The 3H-activities in urine and plasma samples were determined by a liquid scintillation counter (Wallac 1409, Wallac Oy, Turku,
Finland) and the GFR calculated as the clearance of inulin (CLinulin) from the
urine flow rate (UF) and the activities of inulin in urine and plasma, respectively:
CLinulin = (UF × [inulin]urine ) /[inulin] plasma
33
GFR in conscious mice (Study IV)
GFR in conscious mice was estimated by measurements of inulin clearance
with a technique modified from the method described by Qi and coworkers160. [3H]methoxy-inulin was dissolved in saline and thereafter dialyzed in 2000 ml of saline at +4C overnight using a 1000 Da cut-off dialysis
membrane (Spectra/Por® 6 Membrane; Spectrum Laboratories Inc, Rancho
Dominguez, CA). The dialyzed inulin was filtered through a 0.22 μm syringe filter before use, and injected (~85 kBq in 200 μl) into the tail vein of
the conscious mice. The syringe was weighed before and after the injection
to calculate the exact dose. Approximately 5 l blood was collected from a
small cut at the tip of the tail at 1, 3, 7, 10, 15 and approximately 35, 55 and
75 minutes after the bolus injection, and the plasma concentration of inulin
was determined by liquid scintigraphy. GFR was calculated as the plasma
clearance of inulin using standard formulas161 by dividing the given i.v. dose
by the total area under the plasma inulin time curve (AUC0-):
CLinulin = Doseiv / AUC0 − ∞
The AUC0-∞ was estimated using non-compartmental pharmacokinetic data
analysis161 from summation of the trapezoid areas that are formed by connecting the data points (trapezoids), a procedure referred to as the linear
trapezoidal rule. The area of the terminal phase (AUC
), i.e. from 15 minutes
to infinity (including the residual area after the last measurement), was calculated based on the equation for the slope. The area before the first measurement (AUCback-extr) was calculated using linear back-extrapolation.
AUC 0−∞ = AUC back −extr + Σtrapezoids + AUC λ
A plot demonstrating the disappearance of inulin and PAH in plasma and the
areas used for the calculation is shown in figure 4.
GFR and renal plasma flow in conscious mice (Study II)
The effect of milrinone (0.5 mg/kg) on GFR and renal plasma flow was estimated by simultaneous measurements of inulin- and PAH clearances, respectively. Similar to the technique described above, animals were given a
bolus injection of [3H]methoxy-inulin (~110 kBq) and of [14C]PAH (~40
kBq; PerkinElmer Sverige AB, Upplands Väsby, Sweden) dissolved in 200
μl saline into the tail vein, whereupon blood samples were taken from the
tail tip, as described above. The 3H and 14C activities in each sample were
determined by liquid scintigraphy. Inulin- and PAH clearances were calculated using non-compartmental pharmacokinetic data analysis as described
34
above. Each animal served as its own control using a similar experiment
design as in the renin series.
Figure 4. Representative semi-logarithmic plot of plasma activity of [3H]MethoxyInulin and [14C]Para-Amino Hippuric Acid after a bolus injection of the substances.
The solid lines represent values taken during the actual experiments, whereas the
dashed lines represent the interpolated residual data for the end of the terminal
phase.
Histology (Study III)
The left kidney was fixed by the infusion of 4% phosphate-buffered formalin
via the carotid artery while the renal vein was cut open. The infusion was
continued for 10 minutes, whereafter the kidneys were dissected and
weighted. The middle part of the left kidney was placed in formalin and embedded in paraffin. Sections (5 μm) were stained with haematoxylin-eosin,
periodic acid-Schiff and Picro-Sirius. Different compartments of the sections
(glomeruli, tubular system, medulla and papilla) were investigated for morphological changes by a blinded observer. The parameters scored were; fibrosis/sclerosis, inflammation, and tubular dilatation. A score of 0–3 was
given, with 0 being no alterations and 3 given for severe alterations. Special
attention was given to the papillary–medullary transition which is especially
susceptible to develop fibrosis.
35
The entire renal cortical area of one section from the first five animals in
each group was photographed. Computer software (Scion Image; Scion Corporation, MD) was then used to manually determine the area of all visible
glomeruli (40-98 glomeruli/section) and the surrounding capsule in the
cross-section, respectively. Mean values for the area of the glomeruli and
capsules were calculated for every section. Assuming that the glomeruli are
circular, the mean diameters (d) were calculated from the areas. From the
calculated mean diameters, an approximation of the diameter around the
waist (D) was calculated using the following equation162:
D=
4
π
×d
The glomerular and capsular volumes were then calculated using standard
formulas. The volume of the urinary space was calculated as the difference
between the volume of the capsule and the glomerulus.
Statistics
All values are expressed as mean±SEM, and for all comparisons P<0.05 was
considered statistically significant. The calculations were performed in
GraphPad Prism 4 (GraphPad Software, La Jolla, CA), MINITAB Release
14 (Minitab, State College, PA) and Microsoft Office Excel 2003 (Microsoft, Redmond, WA).
In study I, the cardiovascular response to an increased sodium intake was
examined using a paired t-test, whereas comparisons between the two genotypes were performed using an unpaired t-test. Excretion, PRC and aldostone
data were examined using the unpaired t-test, whereas the renin mRNA data
was evaluated by a one-way ANOVA followed by the Tukey–Kramer multiple comparison post hoc test.
In study II, the renin, blood pressure, GFR, and blood flow responses to
PDE inhibition were examined using a paired t-test, whereas comparisons
between the two genotypes were performed using an unpaired t-test. Differences in cAMP concentration and the degree of JG cell activation between
the different groups were tested using an unpaired t-test.
In study III, GFR, excretion, body weight, and stereological data were examined using one-way ANOVA and, when appropriate, followed by Fisher’s
post hoc test. The results from the histological evaluation were examined
with the Kruskal-Wallis test.
In study IV, GFR and blood glucose data was analyzed using one-way
ANOVA and, when appropriate, followed by Fisher’s post hoc test.
36
Results
nNOS in renin and blood pressure regulation
(Study I&II)
Blood pressure and heart rate regulation
No difference in blood pressure between nNOS-/- and nNOS+/+ mice was
found on any of the sodium diets or in any experimental period (Figure 5).
When animals were transferred from a low to a high sodium diet, the blood
pressure increased slightly in both nNOS-/- and nNOS+/+ mice, but no difference in the magnitude of change was found between the genotypes. During
the day, but not the night, the heart rate in nNOS-/- mice was reduced on both
diets. However, when the animals were inactive (i.e. when data associated
with physical activity was removed), the heart rate in nNOS-/- was found to
be reduced both during the day and night (Figure 6A).
Figure 5. 24-hour systolic, mean and diastolic arterial blood pressure during different sodium diets. For the inactive parts, measurements associated with activity have
been removed. * denotes P<0.05 compared to low sodium diet, same genotype.
37
Figure 6. 24-hour heart rate (A) during different sodium diets. For the inactive parts,
measurements associated with activity have been removed. Panel B shows the mean
activity level during the day and night. * denotes P<0.05 compared to inactive data,
# denotes P<0.05 compared to inactive nNOS+/+, same diet, † denotes P<0.05 compared to the daytime, same genotype and diet.
Renin and aldosterone regulation
Analysis of genomic DNA demonstrated that nNOS+/+ and nNOS-/- mice
used in the present study only carry the Ren-1 gene. Figure 7C shows a representative gel with all investigated genotypes. During high sodium conditions, PRC was similar between the genotypes but under low sodium conditions, nNOS-/- mice had a reduced PRC compared to nNOS+/+ mice (Figure
7A). During the low sodium diet, PRC in nNOS+/+ mice was elevated compared to that under high sodium conditions, while PRC in nNOS-/- mice was
unaffected. Renin mRNA level in the renal cortex was significantly stimulated by a low salt intake in nNOS+/+ mice, whereas the mRNA levels were
unaffected in the nNOS-/- mice (Figure 7B). Plasma aldosterone concentration was increased by low salt intake in nNOS-/- and nNOS+/+ mice (Figure
7D) but there were no significant differences between the strains of mice.
Also, the plasma potassium concentration was similar between the genotypes
on both low- (nNOS+/+ 4.3±0.2 vs. nNOS-/- 4.1±0.1 mM) and high sodium
diets (nNOS+/+ 4.0±0.1 vs. nNOS-/- 4.1±0.1 mM).
Renal excretion
During low sodium diet, the urine flow rate, osmolar and potassium excretion were found to be increased in nNOS-/- compared to nNOS+/+ (Table 1).
However, when animals were given a high sodium diet, no significant
changes were found.
38
Table 1. 24-hour urinary excretion data from metabolic cages.
Parameter
Urine flow rate (μl/24 h/g bw)
Osmolar excretion (μOsm/24 h/g bw)
Sodium excretion (μmol/24 h/g bw)
Potassium excretion (μmol/24 h/g bw)
Sodium diet
nNOS+/+
Low
High
Low
High
Low
High
Low
High
44 ±4
90 ±12
55 ±5
97 ±14
0.46 ±0.13
25 ±4.6
7.8 ±1
8.3 ±1.3
nNOS-/78 ±8*
118 ±9
75 ±5*
128 ±10
0.45 ±0.18
33 ±2.2
11±0.69*
10±0.62
* denotes P<0.05 compared to nNOS+/+, same diet.
Figure 7. Plasma renin (A), kidney renin mRNA (B) and plasma aldosterone (D) in
relation to different sodium loads. Panel C shows an agarose gel from the genomic
DNA analysis of the renin genes in these mice. DNA derived from A1AR-/- mice was
used as a positive control for the Ren-2 gene. * denotes P<0.05.
PDE3 and renin regulation
During vehicle treatment, PRC in the nNOS-/- was reduced compared to
wild-type littermates (Figure 8A). Administration of milrinone (0.5 mg/kg)
increased PRC significantly in both genotypes, but the increase was significantly larger in the nNOS-/- mice (Figure 8B), and resulted in similar renin
levels.
39
Figure 8. Paired measurements of plasma renin concentration after milrinone or
vehicle administration, respectively (A). Mean values of the renin increase caused
by milrinone (B). * denotes P<0.05 compared to vehicle, whereas
# denotes p<0.05 compared to nNOS+/+.
The blood pressure responses to milrinone and vehicle injections are shown
in figure 9A. No differences were found in the basal blood pressure between
the genotypes. The low milrinone dose (0.5 mg/kg) and vehicle injection,
increased blood pressure similarly in both strains of mice. In contrast, a
higher dose of milrinone (3 mg/kg) caused a marked and significant blood
pressure reduction. Administration of the higher dose of milrinone increased
PRC well above the levels found after 0.5 mg/kg, demonstrating that a
maximum PRC was not reached during the lower dose (Figure 9B).
Figure 9. Blood pressure responses to milrinone and vehicle injections (A). Demonstration of the renin responses to the depressor and the non-depressor doses of milrinone (B). * denotes P<0.05 compared to vehicle, same genotype and dose, whereas
# denotes P<0.05 compared to milrinone 0.5 mg, same genotype.
40
Low-dose milrinone (0.5 mg/kg) reduced GFR and PAH clearance similarly
in both genotypes (Figure 10). During milrinone treatment, the nNOS-/- had a
reduced GFR compared to the nNOS+/+, but this difference was not significant during the vehicle period (P=0.17).
Figure 10. Paired measurements of
glomerular filtration rate and PAH
clearance after administration of
milrinone or vehicle, respectively.
* denotes P<0.05 compared to vehicle, same genotype, whereas # denotes P<0.05 compared to nNOS+/+.
cAMP measurements
Administration of milrinone (100 μM) to the As4.1 cells caused approximately a 4-fold increase in intracellular cAMP concentration (Figure 11A).
When cAMP concentration in cortical tissue from nNOS-/- and wild-type
mice was analyzed, there was no difference between the genotypes (Figure
11B).
Figure 11. cAMP concentration in As4.1 cells treated with milrinone compared to
cells only given forskolin (A). cAMP concentration in renal cortex (B). * denotes
P<0.05 compared to cells only given forskolin.
41
PDE5 and renin regulation
In a separate experimental series, blood pressure and PRC were determined
in response to a PDE5 inhibitor. Zaprinast had no effect on blood pressure
compared to vehicle treatment. Also in this series, PRC in sodium-restricted
wild-type mice injected with vehicle was significantly higher compared to
nNOS-/-. However, zaprinast injection did not change PRC in wild-type or in
nNOS-/- mice.
Effect of nNOS deletion on JG cell index
Approximately 3500 arterioles from microdissected renal microvasculature
were evaluated from each genotype (Figure 12). The microvessels from
nNOS-/- exhibited an increased percentage of renin-positive arterioles (Total
activated). Furthermore, an elevated percentage of JG cells in juxtaglomerular position was observed, while no difference was found in the degree of
recruitment.
Figure 12. Visualization of renin cells by acid dissection. * denotes P<0.05 compared with nNOS+/+.
42
Diabetes-induced hyperfiltration (Study III&IV)
TGF and diabetes-induced hyperfiltration
The baseline GFR was similar in both genotypes, and diabetic animals of
both genotypes developed a pronounced and similar glomerular hyperfiltration (Figure 13A).
The glomerular volume was similar in all investigated groups (Figure
13B), while a tendency to increased fibrosis was observed in the diabetic
groups compared to normoglycaemic animals, however no statistically significant differences were found between the groups.
Figure 13. Glomerular filtration rate (A) and glomerular volume (B) in mice with
different blood glucose status. * denotes P<0.05.
In both genotypes, the induction of diabetes resulted in a similar increase in
water intake, urinary flow rate, and protein and osmolar excretion, compared
to normoglycemic animals (Table 2). During diabetes, the excretion of sodium and potassium was lower in the A1AR-/- compared to the A1AR+/+.
Table 2. 24-hour urinary excretion data from metabolic cages.
A1AR+/+
Water intake
(μl/24 h/g bw)
Urinary flow rate
(μl/24 h/g bw)
Sodium excretion
(μmol/24 h/g bw)
Potassium excretion
(μmol/24 h/g bw)
Osmolal excretion
(μOsm/24 h/g bw)
Protein excretion
(mg/24 h/g bw)
A1AR-/-
Control
Diabetes
Control
Diabetes
108 ±14
500 ±114*
83 ±7
462 ±47*
63 ±7
479 ±84*
55 ±8
457 ±56*
7.1 ±0.4
15.8 ±1.3*
6.4 ±0.9
11.3 ±1.5†*
11.3 ±0.8
25.4 ±2.3*
10.5 ±1.6
15.3 ±2.0†
99 ±6
531 ±74*
86 ±13
448 ±60*
0.80 ±0.05
2.01 ±0.27*
0.76 ±0.10
1.35 ±0.16*
* denotes P<0.05 vs. corresponding normoglycemic control group, whereas † denotes P<0.05 vs.
corresponding A1AR+/+ group.
43
Glucose reabsorption and diabetes-induced hyperfiltration
In study 4, we investigated the role of TGF and sodium-linked glucose transport on diabetes-induced hyperfiltration. GFR was measured by the singlebolus technique in conscious mice. The diabetic A1AR-/- demonstrated a
similar degree of hyperfiltration as their wild-type controls, thus confirming
the results obtained in anaesthetized animals.
Administration of phlorizin reduced GFR in the diabetic groups, but not
in the normoglycemic groups (Figure 14). Notably, the decrease in GFR was
more pronounced in A1AR-/- compared to the A1AR+/+. Phlorizin lowered the
plasma glucose levels in all groups, although the reduction did not reach
statistical significance in the normoglycemic A1AR-/- group (Figure 14).
Figure 14. Effect of phlorizin on glomerular filtration rate and plasma glucose concentration. * denotes P<0.05 compared to corresponding normoglycemic group,
# denotes P<0.05 compared to vehicle within the same genotype and plasma glucose
status, and † denotes P<0.05 compared to phlorizin-treated A1AR+/+ group.
44
Discussion
The role of nNOS in renin regulation
The studies in this thesis demonstrate that nNOS-deficient animals have an
impaired renin regulation that prevents the normal renin increase during
sodium restriction. Since blood pressure and GFR were similar in
nNOS-/- and nNOS+/+ mice, we can rule out the possibility that the absent
renin response is caused by changes in these parameters. The presence of the
possibly confounding Ren-2 gene was also excluded by genotyping the animals. Consequently, it appears that the regulation of renin by sodium restriction depends critically on local generation of NO by nNOS in the JGA.
The stimulatory effects of NO on renin release have been proposed to be
caused by cGMP-mediated competitive inhibition of PDE3, which hydrolyze
cAMP. As a result, the cAMP levels will increase, and renin secretion will
thus be stimulated135. A schematic illustration of the proposed hypothesis is
shown in figure 15. In order to test whether this model explains the inability
of appropriate renin regulation in the nNOS-/-, we examined the renin response to PDE3 inhibition in these genotypes. Supporting the hypothesis,
the nNOS-/-, compared to the nNOS+/+, were found to exhibit a more pronounced renin increase after administration of the PDE3 inhibitor, suggesting that the PDE3 isoform in JG cells is more active due to the absence of
nNOS-derived NO. Consequently, during PDE3 inhibition, the PRC levels
were similar in both genotypes. This is supported by earlier findings, where
milrinone blocked the renin-suppressing effects of NOS inhibition119. In
patch clamp experiments on single JG cells, administration of cGMP increased renin exocytosis, and this was not further increased during PDE3
inhibition, supporting the notion of a common target163. Furthermore, data
from the perfused isolated kidney suggests that the stimulatory effect of NO
on renin secretion is mediated through cAMP, since inhibition of protein
kinase A attenuates the stimulatory effect of NO133.
Earlier studies have suggested that PDE5 inhibitors could increase PRC
by increasing the cGMP levels in JG cells164,165. Since the NO-cGMP levels
in JG cells from nNOS-/- are expected to be reduced, inhibition of PDE5 in
these mice is predicted to have no or minor effects on renin. However, even
administering the highest sub-depressor dose possible in mice, PRC did not
increase in any of the groups, suggesting that PDE5 does not play a major
role in renin release. One possible explanation might be that previous ex45
periments164,165 were performed during anesthesia, whereas the present series
was done without. This view is supported by data from another study, using
conscious rats, where PDE5 inhibition did not affect the PRC levels166.
The renin mRNA levels were also reduced in the nNOS-/- during sodium
restriction, which indicates that nNOS-derived NO might have a stimulatory
role in renin transcription. Earlier findings from cultured JG cells indicate
that cAMP stimulates renin transcription167. Following the discussion above,
the cAMP concentration in JG cells of nNOS-/- is assumed to be reduced.
Accordingly, it appears plausible that the low cAMP concentration might
contribute to the observed low renin mRNA levels.
Macula densa
cell
nNOS
L-citrulline
+
NO
L-arginine
GPCRs
AC5/6
Gs
PDE3
NO
ATP
AMP
cAMP
Renin
2+
transcription Ca
cGMP
Renin
GC
PKA
Renin
Exocytosis
GTP
PKG
Juxtaglomerular cell
Figure 15. Schematic demonstration of the mechanisms that couples nitric oxide
production to increased renin exocytosis135. The alternative pathway by which nitric
oxide could inhibit renin release is also denoted. The solid lines denote stimulatory
effects, whereas the dashed lines denote inhibitory effects. Denotations; GPCRs, G
protein-coupled receptors, PKA, protein kinase A, PKG, protein kinase G, GC,
guanyl cyclase.
The number of JG cells could also contribute to the regulation of PRC. Since
a reduced number of JG cells hypothetically could have contributed to the
deficient renin regulation in the nNOS-/-, we measured the JG cell index.
Interestingly, the nNOS-/- mice, despite their inability to regulate renin, had a
higher degree of renin-positive JG cells. This suggests that nNOS-/- have
increased the number of JG cells in order to compensate for their reduced
ability to secrete renin. Importantly, this shows that the inability of
46
nNOS-/- to increase PRC during sodium restriction is not caused by a reduced number of JG cells.
Taken together, the experiments support the notion that nNOS-derived
NO increases the cAMP levels in JG cells via cGMP-mediated inhibition of
PDE3, which would increase renin synthesis and also directly stimulate renin
exocytosis.
Aldosterone regulation in nNOS-/- with impaired renin
regulation
Despite the impaired regulation of renin release in the nNOS-/- during sodium restriction, the plasma aldosterone concentration was interestingly
elevated to a similar degree as the nNOS+/+. This was surprising, since normally, the renin concentration is considered as the rate-limiting step in the
formation of angiotensin II, which regulates aldosterone secretion. Furthermore, the plasma potassium concentration was similar in both genotypes,
indicating that this parameter had not affected the aldosterone concentration.
ACTH was not measured, but this factor is only considered important in
short-term regulation of aldosterone. These findings demonstrate that the
aldosterone concentration, under certain circumstances, could be regulated
independently of renin. It is possible that the adrenal expression of angiotensin II-AT1 receptors has an active role in the aldosterone response, since
the receptor concentration is normally upregulated during sodium restriction168.
Cardiovascular consequences of nNOS deficiency
The telemetric system used in this study, enabled us to monitor blood pressure, heart rate, and physical activity simultaneously in conscious animals
kept in their normal environment. It is essential that physical activity is
monitored, as it is an important determinant of the measured blood pressure
and heart rate in conscious mice44. As expected, the activity was higher during the night than in the daytime, but did not differ between the genotypes.
As all parameters were sampled simultaneously, the data could be analyzed
for investigations of cardiovascular parameters during the day and night as
well as in periods of rest and physical activity. By doing this, we were also
able to rule out the possibility that minor differences in activity between
genotypes could confound the results. However, we found no differences in
blood pressure between the genotypes either during activity or inactivity,
during night or day, or during different sodium diets. These results are in
accordance with measurements performed on anaesthetized nNOS-/47
mice65,66, but in contrast with two studies using pharmacological nNOS inhibition in rats, where blood pressure was found to increase60,128. The blood
pressure increase was associated with a sensitized TGF mechanism that was
normalized when hypertension had developed60. However, in another study,
using a similar experimental protocol in rats, blood pressure was unaffected130.
When comparing the present results obtained in genetically altered mice
to the former data from 7-NI-treated rats, it is important to point out that
there are obvious differences between the models. In rats, the hypertension
was accompanied by an increase in renin release, while the nNOS-/- mice
instead displayed a reduced renin concentration. The main difference between the nNOS-/- mice and the pharmacological approach used in the rats, is
the degree of blockade. The dose of 7-NI administered to the rats gives a
very low degree of blockade169, while the nNOS-/- have an almost* complete
inhibition. Interestingly, in another study, when a substantially higher concentration of 7-NI was used, the renin response to sodium restriction was
abolished124, resembling the present results obtained in nNOS-/- mice. Furthermore, the exposure of an antagonist to different cells might differ, depending on the properties of the substance. Also, 7-NI is only a relatively
selective nNOS inhibitor, and might inhibit other NOS isoforms to some
degree43.
The heart rate was found to be decreased in nNOS-/- mice during the day
and, when data associated with activity was removed from the analyses, the
decrease was also significant during the night. Earlier studies have shown
that isolated cardiac myocytes from nNOS-/- mice have increased contractility, but hearts from such mice also display left ventricular hypertrophy,
which increases with age66. Under resting conditions, it is possible that in the
nNOS-/- mice, as a result of increased cardiac contractility with a slower
heart rate, the same cardiac output is reached as in nNOS+/+. Consequently,
the reduced heart rate does not appear to affect blood pressure levels.
In view of the deficient renin regulation during the low sodium diet, it is
interesting that the nNOS-/- are able to maintain a normal blood pressure. In
general, inhibition of the renin-angiotensin system lowers the blood pressure
during sodium restriction. However, the normal aldosterone levels in the
nNOS-/- must clearly contribute to the conserved blood pressure regulation.
Furthermore, the more sensitive TGF mechanism in the nNOS-/- could help
maintain blood pressure when renin levels are low55. Taken together, the
mechanisms that maintain blood pressure during sodium restriction in these
mice are not clear and need further investigation.
*
The nNOS-/- have some residual nNOS activity in the brain, due to expression of two alternative splice variants of the gene, nNOS & nNOS. Importantly, the macula densa cells in
the nNOS-/- have been shown to lack nNOS expression.
48
In conclusion, nNOS-derived NO appears to have a stimulatory role in renin
release via cGMP-mediated inhibition of PDE3. Consequently, the normal
renin response to sodium restriction is abolished in nNOS-/- mice. Furthermore, the presented data indicates that genetic deletion of nNOS does not
cause hypertension.
TGF and diabetes-induced hyperfiltration
Several publications have presented arguments explaining diabetes-induced
hyperfiltration as a TGF-mediated event9,38,152. The theory presented, “The
tubular hypothesis of glomerular hyperfiltration”, stipulates that hyperfiltration is caused by a series of events starting with increased sodium-glucose
reabsorption in the proximal tubule. This will consequently reduce the NaCl
load to the macula densa, resulting in a reduced TGF response and an elevated GFR.
In Study III, we investigated the effects of 2-3 weeks of alloxan-induced
diabetes in A1AR-/- mice, known to lack a functional TGF mechanism. As a
general characterization of the model, we measured urinary excretion and
performed a histological evaluation of the kidneys. As expected, the diabetic
animals were characterized by an increased diuresis and electrolyte excretion. Kidneys from the diabetic animals displayed only a tendency to increased fibrosis, which is in line with earlier studies demonstrating that the
C57BL/6J background is relatively resistant to development of diabetic
nephropathy170.
Our main finding is that A1AR-/- mice develop a similar degree of hyperfiltration as wild-type animals with a functional TGF mechanism, which
demonstrates that diabetes-induced hyperfiltration is an event independent of
TGF. The glomerular volumes were not altered by diabetes, indicating that
the hyperfiltration was independent from an increased filtration area. Two
other groups have also studied hyperfiltration in A1AR-/- mice. In the study
by Faulhaber-Walter et al, A1AR-/- mice were made diabetic by crossbreeding the animals with a hyperglycemic mouse strain, which caused an
even more pronounced hyperfiltration than their wild-type controls171. The
other study by Vallon et al used A1AR-/- that were made diabetic with multiple injections of streptozotocin. Interestingly, those mice did not develop
hyperfiltration.172 However, the hyperfiltration in the wild types was very
small, which indicates that this experimental model differs from the one used
in the present studies.
Furthermore, when SNGFRprox is measured without perfusion of the macula densa, the TGF mechanism is inactivated. Consequently, when SNGFR
was assessed in this way in diabetic rats, hyperfiltration was still observed154,173, which supports the TGF independency of diabetes-induced
hyperfiltration.
49
Glucose reabsorption and diabetes-induced
hyperfiltration
Since the TGF mechanism does not appear to mediate the development of
diabetes-induced hyperfiltration, the explanation for hyperfiltration must be
sought elsewhere. In Study IV, we investigated whether the increased
proximal reabsorption during diabetes can contribute directly to hyperfiltration. Hypothetically, the increased reabsorption could reduce the pressure in
the proximal tubule and Bowman’s space which in turn can increase the net
filtration pressure. We therefore found it of interest to investigate the effects
of SGLT inhibition. If hyperfiltration in diabetic mice depends on an elevated degree of sodium-glucose transport, they should reduce their GFR
more than normoglycemic animals. In fact, diabetic animals are more sensitive to transport inhibition38,174, but the GFR reduction has been ascribed to
the TGF mechanism, as a consequence of increased distal NaCl delivery38.
In order to study the direct effects of transport inhibition, without the autoregulatory influence of TGF, it was necessary to perform the experiments in
A1AR-/-. To eliminate the possible influence of anesthesia, a new method for
GFR assessments was used, which allowed measurements in conscious mice
kept in their usual environment.
Wild-type and A1AR-/- mice developed a similar degree of hyperfiltration,
which confirms the results previously found in study III. Administration of
phlorizin had no effect on normoglycemic mice, but reduced GFR in both
A1AR+/+ and A1AR-/-, demonstrating that the reduction is TGF-independent.
The reduction could instead be a direct consequence of the inhibited proximal
reabsorption, which would increase proximal intratubular pressure, thus reducing net filtration pressure. This is supported by earlier findings demonstrating
that intratubular perfusion by phlorizin is associated with an increased pressure
in Bowman’s space in diabetic rats, while control rats were unaffected38. The
fact that diabetic animals are more sensitive to SGLT1/2 inhibition, supports
the idea that the upregulated proximal reabsorption directly causes hyperfiltration. The proposed mechanism is depicted in figure 16.
Interestingly, phlorizin reduced GFR to a larger extent in diabetic
A1AR-/- mice than diabetic wild types. The reduced GFR could decrease
tubular NaCl concentration sensed by the macula densa. A functional TGF
mechanism will partially preserve GFR via a dilation of the afferent arteriole. Thus, the reduction will be greater in the A1AR-/- animals, since they
lack this compensatory component. Consequently, TGF does not appear to
have a causative role in the development of hyperfiltration, but rather functions to stabilize the high GFR.
50
Figure 16. Schematic illustration demonstrating the proposed hypothesis of hyperfiltration occurring without a functional TGF mechanism.
In conclusion, despite that the mechanisms are not fully understood, the experiments show that tubular glucose reabsorption plays a pivotal role in
maintaining elevated GFR during diabetes, at least in the short-term perspective. These findings suggest that the observed increased reabsorption during
diabetes, directly increases GFR by lowering proximal intratubular pressure.
51
Summary and conclusions
¾ Deficiency of nNOS causes an inability to increase plasma renin
concentration during low sodium conditions, probably due to a
reduced degree of PDE3 inhibition.
¾ Genetic deletion of the nNOS gene in mice does not cause
hypertension.
¾ The TGF mechanism is not involved in the development of
diabetes-induced hyperfiltration.
¾ Sodium-linked glucose reabsorption appears to directly contribute
to the development of diabetes-induced hyperfiltration.
52
Future perspectives
The methods used in this thesis were shown to be useful in studying vascular
and renal function in genetically modified mice. Especially, the methods for
the assessment of GFR and renin in conscious or briefly anaesthetized mice,
could be used to study other problems in renal physiology.
The present studies indicate an important role of renal glucose reabsorption in the development of diabetes-induced hyperfiltration. Several pharmaceutical companies are currently developing selective SGLT2 inhibitors as a
new solution to the reduction of plasma glucose concentration in type 2 diabetes. Given the GFR-lowering effects of phlorizin described in the present
paper, long-term administration of SGLT2 inhibitors might also be effective
as a renoprotective agent in type 1 diabetes. Consequently, it would be interesting to investigate the effects of long-term SGLT inhibition on GFR and
also renal damage. The newly developed SGLT2 knockout mice could also
prove useful for this experiment.
53
Sammanfattning på svenska
Syftet med undersökningarna som presenteras i denna avhandling har varit
att öka förståelsen kring njurens roll i blodtrycksregleringen samt att utforska mekanismer som orsakar njursvikt vid diabetes.
Genom en vuxen människas njurar passerar varje dygn ungefär 1700 liter
blod. Av detta kommer omkring 180 liter att filtreras i njurens glomeruli.
Dessa fungerar som en slags silar, som stoppar blodkroppar och större proteiner, men släpper igenom vätska, elektrolyter och mindre molekyler. Den
filtrerade s.k. primärurinen passerar därefter rörsystem, tubuli, där ämnen
som behövs för kroppens funktion - som vatten, glukos, elektrolyter och
näringsämnen återtas (reabsorberas) till cirkulationen. När reabsorptionen är
fullständig återstår endast en procent av den ursprungliga filtrerade mängden. Den består av överskott av elektrolyter och oönskade ämnen, och det är
denna volym som är urinen.
På grund av de stora mängderna som filtreras varje dag, räcker det med
små fel för att vår vätskebalans ska bli felaktig. Om njuren reabsorberar för
mycket, kan blodtrycket stiga okontrollerat och på motsvarande sätt leder
minskad reabsorption till blodtrycksfall. Eftersom båda dessa tillstånd är
potentiellt livshotande har däggdjuren utvecklat sofistikerade reglermekanismer som ser till att njurens blodflöde, filtration och reabsorption precis
motsvarar kroppens vätskebehov. En viktig reglermekanism är den tubuloglomerulära återkopplingsmekanismen (TGF) och en förutsättning för dess
funktion finns i njurens anatomi. Varje tubulus återvänder till sin egen glomerulus och bildar där den s.k. juxtaglomerulära apparaten. I tubulusväggen
vid den juxtaglomerulära apparaten finns macula densa, som består av speciella celler som känner av natriumkloridkoncentrationen i tubulusvätskan.
Om koncentrationen stiger är det ett tecken på att filtrationen i glomerulus
ökat, vilket aktiverar en process som begränsar blodflödet till glomerulus.
Filtrationen återgår på detta sätt till den normala.
Njuren kan även påverka blodtrycket genom frisättning av det blodtryckshöjande hormonet renin. Renin är ett enzym och verkar i cirkulationen
indirekt blodtryckshöjande genom att klyva peptiden angiotensin I från proteinet angiotensinogen. Angiotensin I omvandlas snabbt till den aktiva formen angiotensin II som höjer blodtrycket genom kontraktion av kärl samt
ökning av njurens återupptag av natrium.
54
Kväveoxid, hypertension och renin (Arbete I&II)
Kväveoxid är en gas som i kroppen fungerar som signalsubstans i många
olika system. Kroppen producerar kväveoxid med speciella enzymer, så
kallade kväveoxidsyntaser, och den mest uppenbara biologiska funktionen är
att vidga blodkärlen. Kväveoxid verkar på detta sätt blodtryckssänkande.
Upptäckten av kväveoxids roll i biologiska system är relativt ny och forskarna bakom fyndet belönades 1998 med nobelpriset i fysiologi eller medicin.
I njurens juxtaglomerulära apparat produceras kväveoxid av neuronalt
kväveoxidsyntas (nNOS) vilket har visats kunna påverka TGF-mekanismens
känslighet. Vid situationer när kroppen vill göra sig av med mycket elektrolyter, kan kväveoxidproduktionen öka vilket främjar utsöndring av vätskeöverskottet. En felaktig kväveoxidproduktion skulle därmed kunna leda till
bristande blodtryckskontroll. I försök på råttor gavs en nNOS-blockerare
under fyra veckor. Intressant nog fann forskarna att TGF-mekanismen hade
en ökad känslighet, samtidigt som blodtrycket gradvis steg. Denna utveckling påminner om den som har setts i särskilda råttstammar med en ärftlig
benägenhet att utveckla högt blodtryck. På samma sätt skulle vissa former av
högt blodtryck hos människa kunna förklaras.
Kväveoxid har också visats vara inblandat i reninfrisättningen, vilket
skulle kunna bidra till enzymets påverkan på blodtrycket. Detta har studerats
på många sätt, bland annat genom att man gett olika typer av kväveoxidsyntashämmare till råttor. Experimenten har dock inte gett någon entydig bild av
kväveoxids funktion vid reninfrisättning. I vissa arbeten har kväveoxid visat
sig verka stimulerande, medan andra har påvisat en hämmande roll.
I denna avhandling studerades den specifika rollen av nNOS i regleringen
av blodtryck och reninfrisättning med hjälp av en genetiskt modifierad musstam som saknar nNOS.
Blodtrycket mättes med hjälp av s.k. telemetri där trycket sänds ut från en
liten sändare som opererats in under huden på mössen. Fördelen med denna
metod är att djuren befinner sig i sin vanliga miljö när mätningen pågår,
vilket minimerar deras stress. Eftersom mängden salt i kosten kan påverka
blodtrycket, studerades trycket under dieter med både lågt och högt saltinnehåll. När blodtrycksdata analyserades fanns trycket överraskande vara normalt i nNOS-knocken. Resultatet skiljer sig därmed från de tidiga försöken
där råttor fått en nNOS-hämmare i dricksvattnet.
För att utvärdera reninkoncentrationen i mössen togs ett blodprov vid
båda saltdieterna. nNOS-knockarna visade sig då ha en felaktig reninfrisättning, och saknade den normala ökningen man ser vid en lågsaltdiet. Eftersom detta ansågs intressant, gjordes ytterligare en studie för att försöka förstå hur nNOS kan påverka reninfrisättningen. Mössen behandlades med en
hämmare som blockerar fosfodiesteras 3 (PDE3), vilket tidigare visat sig öka
reninfrisättningen. Djuren som saknar nNOS visade sig då vara känsligare
för denna hämmare, och uppvisade en större ökning än mössen med intakt
55
nNOS-gen. Kväveoxids verkan sker genom bildning av signalsubstansen
cGMP. Tidigare försök har visat att cGMP fungerar som en naturlig hämmare av PDE3. Det är välkänt att reninfrisättning till stor del är beroende av
signalsubstansen cAMP. PDE3 fungerar genom att bryta ned cAMP, och
minskar därmed reninfrisättningen. Kväveoxid kan på detta sätt stimulera
reninfrisättning, genom att blockera det nedbrytande enzymet PDE3.
Sammanfattningsvis tyder resultaten på att den normala reninfrisättningen
under saltfattig kost orsakas av en ökning av enzymet nNOS. Den ökade
mängden kväveoxid kommer att bilda cGMP, vilket hämmar PDE3 i de reninproducerande cellerna. Eftersom PDE3 bryter ned cAMP, som är viktigt
för reninfrisättning, kommer njuren att frisätta mer renin och koncentrationen i cirkulationen kommer att stiga vilket förhindrar att blodtrycket faller
vid saltbrist.
Diabetes, hyperfiltration och nefropati (Arbete III&IV)
En vanlig komplikation vid diabetes är njursvikt, d.v.s. en bristande glomerulär filtration. Eftersom njuren är viktig för blodtrycksreglering och utsöndring av slaggprodukter, är njursvikt ett livshotande tillstånd som antingen
kräver livslång dialys eller en ny njure genom transplantation.
I studier har man funnit att många diabetiker på ett tidigt stadium av sjukdomen uppvisar en förhöjd glomerulär filtration, s.k. hyperfiltration. Dessa
patienter löper en ökad risk att senare i livet drabbas av njursvikt. Mekanismerna för detta är oklara, men ökad kunskap skulle kunna möjliggöra utvecklandet av nya läkemedel som i sin tur skulle kunna förhindra hyperfiltrationen och den framtida risken att utveckla nefropati.
En hypotes som tidigare har lagts fram är att hyperfiltration orsakas av
den ökade glukosbelastningen på njuren. Även om en diabetiker kontrollerar
sin blodsockernivå noga är det stor risk att patienten sett över längre tid ligger på högre blodsockernivåer än en frisk person. Detta leder till att en större
mängd glukos kommer att filtreras i njurens glomeruli. När glukosen tas
tillbaka kommer också natriumklorid att följa med, vilket gör att saltupptaget
ökar hos diabetikern. Den ovan beskrivna TGF-mekanismen verkar genom
att känna av saltkoncentrationen i tubulusvätskan vid macula densa. Om
saltkoncentrationen sjunker, tolkar mekanismen det som att filtrationen sjunkit och ökar därför filtrationen. Det ökade saltupptaget hos diabetiker kommer därmed att ge en falsk signal, som får njuren att öka sin filtration över
det normala.
I avhandlingen testades denna hypotes i en djurmodell för diabetes. Djuren gjordes diabetiska genom att de fick en substans som förstör de insulinproducerande -cellerna i bukspottkörteln. För att undersöka betydelsen av
TGF, användes förutom vanliga möss adenosin A1-receptor knockar, vilka
saknar TGF-mekanismen. När den glomerulära filtrationen mättes före och
56
efter diabetes i dessa djur upptäcktes dock att båda typerna möss utvecklat
en liknande grad av hyperfiltration vilket innebär att den framlagda hypotesen troligen inte är korrekt. För att undersöka detta vidare, studerades betydelsen av glukostransportörerna som är ansvariga för återupptaget i proximala tubuli. När dessa hämmades hos friska möss, påverkades filtrationen endast marginellt, men när de istället hämmades hos diabetiska djur föll den
förhöjda filtrationen mot normala värden. Detta tyder på att återupptaget har
en direkt betydelse för upprätthållandet av hyperfiltrationen. Eftersom denna
effekt skulle kunna vara orsakad av TGF-mekanismen undersöktes effekten
av hämning hos adenosinknockarna, som saknar TGF. Även hos dessa djur
föll filtrationen, vilket tyder på att glukosupptag direkt påverkar filtrationen
vid diabetes, utan inblandning av TGF-mekanismen.
Sammanfattningsvis tyder resultaten på att de ökade blodsockernivåerna
och därmed filtrationen av glukos vid diabetes, ökar återupptaget i njuren.
Detta sänker trycket i tubulisystemet, vilket leder till att tryckskillnaden mellan kapillärerna i glomerulus och tubulisystemet stiger, vilket i sin tur ökar
filtrationen. Genom att blockera glukostransportörerna kan filtrationen på
motsatt sätt sänkas. Just nu pågår utveckling av läkemedel som blockerar
njurens återupptag av glukos. Tanken med dessa är att de ska verka
blodsockersänkande, eftersom glukos kommer att förloras med urinen. Dessa
läkemedel skulle också kunna minska graden av hyperfiltration. Eftersom
hyperfiltration är en riskfaktor för utvecklandet av njursvikt, skulle de därmed också kunna minska risken för diabetiker att drabbas av detta allvarliga
tillstånd.
57
Acknowledgements
This work was performed at the Department of Medical Cell Biology,
Uppsala University, and was financially supported by The Swedish
Research Council, The Wallenberg Foundation, Wallenberg Consortium
North, The Swedish Heart-Lung Foundation, The Ingabritt and Arne
Lundberg Foundation, The Gillbergska Foundation, The Swedish Academy
of Pharmaceutical Sciences, and Anna Maria Lundins Stipendiefond.
The thesis work has involved help by many people. Among those, I would
particularly like to thank:
My supervisor Erik Persson, for excellent guidance and support through the
field of renal physiology. Your door has always been open when I had something to discuss. Also, I would like to thank for all the interesting tales about
the extremely advanced experiments that were performed in the past.
My co-supervisors; Peter Hansell, for general support, feedback, and advice, Russell Brown, for teaching me mouse surgery and for a fun time in
Australia.
Fredrik Palm, for introducing me to the field of diabetes-induced hyperfiltration, and for all the encouragement. It has always been fun to work with
you, and apart from science, you have always tried to teach me more about
life.
Mattias Carlström, for being an excellent colleague, friend, room- and
travelmate, and for the continuous encouragement and support during these
years.
Boye Jensen and Ole Skøtt, for managing the renin analyses. Special thanks
to Boye for many good discussions about renin regulation, fast answers to all
my emails, and for teaching me the acid-dissection technique at your lab in
Odense. Furthermore, Gao Xiang is acknowledged for the hard work of
actually performing the acid dissections in this thesis.
Bertil Fredholm, for providing the A1AR-/- mice that rendered the diabetes
studies possible.
58
Erik Larsson, for performing the pathological examinations. Visiting your
lab and listening to your stories is always exciting.
Terese Engström, for skillful assistance during you master thesis project in
our group.
Anna Ollerstam, for taking your time to supervise me during my first summer project at the department. Lena Holm, for inspiring me to apply to the
summer research school and start at the department, and for all social activities at yours’ and Erik’s house.
Angelica Fasching, Britta Isaksson, Astrid Nordin, and Eva Törnelius,
for always being helpful in and outside the lab, showing me experimental
techniques, and guiding me when I am lost.
Johanna Henriksnäs, for introducing me to the secrets of mice breeding.
Markus Fridén, my pharmacokinetic consultant, for help with the GFR
calculations. Daniel Färnstrand, for excellent linguistic revisions of my
work. Martin Skybrand and Jacob Vesterlund, for all advice on computerrelated issues. Malou Friederich, for proofreading the thesis, and Joel
Petersson, for photographing the author portrait.
Nils and Michael Welsh, for letting me use your labs where, at night, Björn
Åkerblom and Johan Olerud often were found, and who provided good
company and also helped me with the PCRs.
The animal department, especially Martina, for taking good care of my
mice during these years.
Nils Wåhlin, for good discussions about small animal surgery during the
first years, and for your adage ”det ska vara så enkelt att en bonde klarar
det”. Andreas Patzak, for NO/adenosine advice and for many fun nights.
Örjan Källskog, for sharing your vast physiological knowledge, and for
help with radioactive matters. Leif Jansson, for just knowing everything.
Per-Ola Carlsson, for good discussions of diabetic mouse models. Mia
Phillipson, for you enthusiastic questions during seminars.
Gunno Nilsson, for all the teaching assistance, especially with the pig lab,
and Göran Ståhl, for various things, like breaking into our office when the
lock jammed. Agneta Sandler Bäfwe, Marianne Ljungkvist and Karin
Öberg, for the administrative help.
My new roommates Ulrika Pettersson and Jenny Edlund for creating a
friendly atmosphere, Cornish Rex knowledge, and the shared Spotify connection.
59
All my colleagues at the department, for all the great times during these
years.
My mother, Monika, for inspiring me to study in Uppsala and for all baby
sitting during the thesis-writing process. My father, Anders, for enthusing
me to explore things and giving me constant support during these years. My
brother, Erik, for all discussions regarding everything but research.
Finally, Johanna and Isak, for your love, support and happiness you give
me every day. This thesis is dedicated to you.
Johan Sällström
Uppsala, January 2010
60
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Dilley JR, Arendshorst WJ. Enhanced tubuloglomerular feedback activity in
rats developing spontaneous hypertension. Am J Physiol. 1984;247:F672-679.
Boberg U, Persson AE. Increased tubuloglomerular feedback activity in Milan
hypertensive rats. Am J Physiol. 1986;250:F967-974.
Deckert T, Poulsen JE, Larsen M. Prognosis of diabetics with diabetes onset
before the age of thirty-one. I. Survival, causes of death, and complications.
Diabetologia. 1978;14:363-370.
Ditzel J, Schwartz M. Abnormally increased glomerular filtration rate in
short-term insulin-treated diabetic subjects. Diabetes. 1967;16:264-267.
Mogensen CE. Kidney function and glomerular permeability to macromolecules in early juvenile diabetes. Scand J Clin Lab Invest. 1971;28:79-90.
Rudberg S, Persson B, Dahlquist G. Increased glomerular filtration rate as a
predictor of diabetic nephropathy - an 8-year prospective study. Kidney Int.
1992;41:822-828.
Amin R, Turner C, van Aken S, Bahu TK, Watts A, Lindsell DR, Dalton
RN, Dunger DB. The relationship between microalbuminuria and glomerular
filtration rate in young type 1 diabetic subjects: The Oxford Regional Prospective Study. Kidney Int. 2005;68:1740-1749.
Mogensen CE, Christensen CK. Predicting diabetic nephropathy in insulindependent patients. N Engl J Med. 1984;311:89-93.
Thomson SC, Deng A, Bao D, Satriano J, Blantz RC, Vallon V. Ornithine
decarboxylase, kidney size, and the tubular hypothesis of glomerular hyperfiltration in experimental diabetes. J Clin Invest. 2001;107:217-224.
Maddox DA, Brenner BM. Glomerular Ultrafiltration. In: Brenner BM, Levine SA, eds. The Kidney. Vol 1. 7th ed. Philadelphia: Saunders; 2003:353-412.
Brenner BM, Troy JL, Daugharty TM. The dynamics of glomerular ultrafiltration in the rat. J Clin Invest. 1971;50:1776-1780.
Källskog Ö, Lindbom LO, Ulfendahl HR, Wolgast M. Kinetics of the
glomerular ultrafiltration in the rat kidney. An experimental study. Acta
Physiol Scand. 1975;95:293-300.
Karlsen FM, Holstein-Rathlou NH, Leyssac PP. A re-evaluation of the determinants of glomerular filtration rate. Acta Physiol Scand. 1995;155:335-350.
Kon V, Ichikawa I. Effector loci for renal nerve control of cortical microcirculation. Am J Physiol. 1983;245:F545-553.
Edwards RM, Trizna W. Characterization of alpha-adrenoceptors on isolated
rabbit renal arterioles. Am J Physiol. 1988;254:F178-183.
Kornfeld M, Salomonsson M, Gutierrez A, Persson AE. The influence of
beta-adrenergic activation on noradrenergic alpha1 activation of rabbit afferent
arterioles. Pflugers Arch. 2000;441:25-31.
61
17. Baylis C, Brenner BM. Modulation by prostaglandin synthesis inhibitors of
the action of exogenous angiotensin II on glomerular ultrafiltration in the rat.
Circ Res. 1978;43:889-898.
18. Murray RD, Churchill PC. Effects of adenosine receptor agonists in the isolated, perfused rat kidney. Am J Physiol. 1984;247:H343-348.
19. Schubert R, Mulvany MJ. The myogenic response: established facts and
attractive hypotheses. Clin Sci (Lond). 1999;96:313-326.
20. Loutzenhiser R, Griffin KA, Bidani AK. Systolic blood pressure as the trigger for the renal myogenic response: protective or autoregulatory? Curr Opin
Nephrol Hypertens. 2006;15:41-49.
21. Schnermann J, Wright FS, Davis JM, von Stackelberg W, Grill G. Regulation of superficial nephron filtration rate by tubulo-glomerular feedback. Pflugers Arch. 1970;318:147-175.
22. Schnermann J, Persson AE, Agerup B. Tubuloglomerular feedback. Nonlinear relation between glomerular hydrostatic pressure and loop of henle perfusion rate. J Clin Invest. 1973;52:862-869.
23. Briggs JP, Schubert G, Schnermann J. Quantitative characterization of the
tubuloglomerular feedback response: effect of growth. Am J Physiol.
1984;247:F808-815.
24. Osswald H, Nabakowski G, Hermes H. Adenosine as a possible mediator of
metabolic control of glomerular filtration rate. Int J Biochem. 1980;12:263-267.
25. Smith JA, Sivaprasadarao A, Munsey TS, Bowmer CJ, Yates MS. Immunolocalisation of adenosine A(1) receptors in the rat kidney. Biochem Pharmacol. 2001;61:237-244.
26. Weaver DR, Reppert SM. Adenosine receptor gene expression in rat kidney.
Am J Physiol. 1992;263:F991-995.
27. Weihprecht H, Lorenz JN, Briggs JP, Schnermann J. Vasomotor effects of
purinergic agonists in isolated rabbit afferent arterioles. Am J Physiol.
1992;263:F1026-1033.
28. Hansen PB, Castrop H, Briggs J, Schnermann J. Adenosine induces vasoconstriction through Gi-dependent activation of phospholipase C in isolated
perfused afferent arterioles of mice. J Am Soc Nephrol. 2003;14:2457-2465.
29. Gutierrez AM, Kornfeld M, Persson AE. Calcium response to adenosine and
ATP in rabbit afferent arterioles. Acta Physiol Scand. 1999;166:175-181.
30. Brown R, Ollerstam A, Johansson B, Skott O, Gebre-Medhin S, Fredholm
B, Persson AE. Abolished tubuloglomerular feedback and increased plasma
renin in adenosine A1 receptor-deficient mice. Am J Physiol Regul Integr
Comp Physiol. 2001;281:R1362-1367.
31. Sun D, Samuelson LC, Yang T, Huang Y, Paliege A, Saunders T, Briggs J,
Schnermann J. Mediation of tubuloglomerular feedback by adenosine: evidence from mice lacking adenosine 1 receptors. Proc Natl Acad Sci U S A.
2001;98:9983-9988.
32. Persson AE, Wright FS. Evidence for feedback mediated reduction of glomerular filtration rate during infusion of acetazolamide. Acta Physiol Scand.
1982;114:1-7.
33. Leyssac PP, Karlsen FM, Skott O. Dynamics of intrarenal pressures and
glomerular filtration rate after acetazolamide. Am J Physiol. 1991;261:F169-178.
62
34. Hashimoto S, Huang YG, Castrop H, Hansen PB, Mizel D, Briggs J,
Schnermann J. Effect of carbonic anhydrase inhibition on GFR and renal
hemodynamics in adenosine-1 receptor-deficient mice. Pflugers Arch.
2004;448:621-628.
35. Jensen PK, Christiansen JS, Steven K, Parving HH. Strict metabolic control
and renal function in the streptozotocin diabetic rat. Kidney Int. 1987;31:47-51.
36. Jensen PK, Christiansen JS, Steven K, Parving HH. Renal function in streptozotocin-diabetic rats. Diabetologia. 1981;21:409-414.
37. Jensen PK, Kristensen KS, Rasch R, Persson AEG. Decreased sensitivity of
the tubuloglomerular feedback mechanism in experimental diabetic rats. Paper
presented at: 11th Eric K. Fernström Symposium, 1988; Örenäs Castle, Lund,
Sweden.
38. Vallon V, Richter K, Blantz RC, Thomson S, Osswald H. Glomerular hyperfiltration in experimental diabetes mellitus: potential role of tubular reabsorption. J Am Soc Nephrol. 1999;10:2569-2576.
39. Rapoport RM, Murad F. Agonist-induced endothelium-dependent relaxation in
rat thoracic aorta may be mediated through cGMP. Circ Res. 1983;52:352-357.
40. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the
relaxation of arterial smooth muscle by acetylcholine. Nature. 1980;288:373-376.
41. Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G. Endotheliumderived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci U S A. 1987;84:9265-9269.
42. Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the
biological activity of endothelium-derived relaxing factor. Nature.
1987;327:524-526.
43. Alderton WK, Cooper CE, Knowles RG. Nitric oxide synthases: structure,
function and inhibition. Biochem J. 2001;357:593-615.
44. Van Vliet BN, Chafe LL, Montani JP. Characteristics of 24 h telemetered
blood pressure in eNOS-knockout and C57Bl/6J control mice. J Physiol.
2003;549:313-325.
45. Shesely EG, Maeda N, Kim HS, Desai KM, Krege JH, Laubach VE,
Sherman PA, Sessa WC, Smithies O. Elevated blood pressures in mice lacking endothelial nitric oxide synthase. Proc Natl Acad Sci U S A.
1996;93:13176-13181.
46. Wattanapitayakul SK, Mihm MJ, Young AP, Bauer JA. Therapeutic implications of human endothelial nitric oxide synthase gene polymorphism. Trends
Pharmacol Sci. 2001;22:361-368.
47. Bredt DS, Hwang PM, Glatt CE, Lowenstein C, Reed RR, Snyder SH.
Cloned and expressed nitric oxide synthase structurally resembles cytochrome
P-450 reductase. Nature. 1991;351:714-718.
48. Wilcox CS, Welch WJ, Murad F, Gross SS, Taylor G, Levi R, Schmidt
HH. Nitric oxide synthase in macula densa regulates glomerular capillary pressure. Proc Natl Acad Sci U S A. 1992;89:11993-11997.
49. Mundel P, Bachmann S, Bader M, Fischer A, Kummer W, Mayer B, Kriz
W. Expression of nitric oxide synthase in kidney macula densa cells. Kidney
Int. 1992;42:1017-1019.
63
50. Thorup C, Sundler F, Ekblad E, Persson AE. Resetting of the tubuloglomerular feedback mechanism by blockade of NO-synthase. Acta Physiol Scand.
1993;148:359-360.
51. Ito S, Ren Y. Evidence for the role of nitric oxide in macula densa control of
glomerular hemodynamics. J Clin Invest. 1993;92:1093-1098.
52. Thorup C, Persson AE. Inhibition of locally produced nitric oxide resets
tubuloglomerular feedback mechanism. Am J Physiol. 1994;267:F606-611.
53. Vallon V, Thomson S. Inhibition of local nitric oxide synthase increases homeostatic efficiency of tubuloglomerular feedback. Am J Physiol.
1995;269:F892-899.
54. Thorup C, Erik A, Persson G. Macula densa derived nitric oxide in regulation of glomerular capillary pressure. Kidney Int. 1996;49:430-436.
55. Vallon V, Traynor T, Barajas L, Huang YG, Briggs JP, Schnermann J.
Feedback control of glomerular vascular tone in neuronal nitric oxide synthase
knockout mice. J Am Soc Nephrol. 2001;12:1599-1606.
56. Thomson SC, Deng A. Cyclic GMP mediates influence of macula densa nitric
oxide over tubuloglomerular feedback. Kidney Blood Press Res. 2003;26:10-18.
57. Ren YL, Garvin JL, Carretero OA. Role of macula densa nitric oxide and
cGMP in the regulation of tubuloglomerular feedback. Kidney Int.
2000;58:2053-2060.
58. Brown R, Ollerstam A, Persson AE. Neuronal nitric oxide synthase inhibition sensitizes the tubuloglomerular feedback mechanism after volume expansion. Kidney Int. 2004;65:1349-1356.
59. Seddon MD, Chowienczyk PJ, Brett SE, Casadei B, Shah AM. Neuronal
nitric oxide synthase regulates basal microvascular tone in humans in vivo.
Circulation. 2008;117:1991-1996.
60. Ollerstam A, Pittner J, Persson AE, Thorup C. Increased blood pressure in
rats after long-term inhibition of the neuronal isoform of nitric oxide synthase.
J Clin Invest. 1997;99:2212-2218.
61. Beierwaltes WH, Arendshorst WJ, Klemmer PJ. Electrolyte and water balance in young spontaneously hypertensive rats. Hypertension. 1982;4:908-915.
62. Bianchi G, Baer PG, Fox U, Duzzi L, Pagetti D, Giovannetti AM. Changes
in renin, water balance, and sodium balance during development of high blood
pressure in genetically hypertensive rats. Circ Res. 1975;36:153-161.
63. Kawabe K, Watanabe TX, Shiono K, Sokabe H. Influence on blood pressure
of renal isografts between spontaneously hypertensive and normotensive rats,
utilizing the F1 hybrids. Jpn Heart J. 1978;19:886-894.
64. Bianchi G, Fox U, Di Francesco GF, Giovanetti AM, Pagetti D. Blood pressure changes produced by kidney cross-transplantation between spontaneously
hypertensive rats and normotensive rats. Clin Sci Mol Med. 1974;47:435-448.
65. Jumrussirikul P, Dinerman J, Dawson TM, Dawson VL, Ekelund U,
Georgakopoulos D, Schramm LP, Calkins H, Snyder SH, Hare JM, Berger
RD. Interaction between neuronal nitric oxide synthase and inhibitory G protein activity in heart rate regulation in conscious mice. J Clin Invest.
1998;102:1279-1285.
66. Barouch LA, Harrison RW, Skaf MW, Rosas GO, Cappola TP, Kobeissi
ZA, Hobai IA, Lemmon CA, Burnett AL, O'Rourke B, Rodriguez ER,
64
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
Huang PL, Lima JA, Berkowitz DE, Hare JM. Nitric oxide regulates the
heart by spatial confinement of nitric oxide synthase isoforms. Nature.
2002;416:337-339.
Tigerstedt R, Bergman PG. Niere und Kreislauf. Skand Arch Physiol.
1898;8:223-271.
Marks LS, Maxwell MH. Tigerstedt and the discovery of renin. An historical
note. Hypertension. 1979;1:384-388.
Candido R, Burrell LM, Jandeleit-Dahm KAM, Cooper ME. Vasoactive
Peptides and the Kidney. In: Brenner BM, Levine SA, eds. The Kidney. Vol 1.
8th ed. Philadelphia: Saunders; 2008:333-362.
Ito M, Oliverio MI, Mannon PJ, Best CF, Maeda N, Smithies O, Coffman
TM. Regulation of blood pressure by the type 1A angiotensin II receptor gene.
Proc Natl Acad Sci U S A. 1995;92:3521-3525.
Gross V, Milia AF, Plehm R, Inagami T, Luft FC. Long-term blood pressure
telemetry in AT2 receptor-disrupted mice. J Hypertens. 2000;18:955-961.
Siragy HM, Inagami T, Ichiki T, Carey RM. Sustained hypersensitivity to
angiotensin II and its mechanism in mice lacking the subtype-2 (AT2) angiotensin receptor. Proc Natl Acad Sci U S A. 1999;96:6506-6510.
Sigmund CD, Gross KW. Structure, expression, and regulation of the murine
renin genes. Hypertension. 1991;18:446-457.
Hansen PB, Yang T, Huang Y, Mizel D, Briggs J, Schnermann J. Plasma
renin in mice with one or two renin genes. Acta Physiol Scand. 2004;181:431-437.
Skinner SL, McCubbin JW, Page IH. Renal Baroceptor Control of Renin
Secretion. Science. 1963;141:814-816.
Fray JC. Stretch receptor control of renin release in perfused rat kidney: effect
of high perfusate potassium. J Physiol. 1978;282:207-217.
Blaine EH, Davis JO, Witty RT. Renin release after hemorrhage and after
suprarenal aortic constriction in dogs without sodium delivery to the macula
densa. Circ Res. 1970;27:1081-1089.
Scholz H, Vogel U, Kurtz A. Interrelation between baroreceptor and macula
densa mechanisms in the control of renin secretion. J Physiol. 1993;469:511-524.
Bock HA, Hermle M, Brunner FP, Thiel G. Pressure dependent modulation
of renin release in isolated perfused glomeruli. Kidney Int. 1992;41:275-280.
Salomonsson M, Skott O, Persson AE. Lack of effect of intraluminal pressure on renin release from isolated afferent arterioles. Pflugers Arch.
1992;421:466-468.
Taugner R, Kirchheim H, Forssmann WG. Myoendothelial contacts in
glomerular arterioles and in renal interlobular arteries of rat, mouse and Tupaia
belangeri. Cell Tissue Res. 1984;235:319-325.
Wagner C, de Wit C, Kurtz L, Grunberger C, Kurtz A, Schweda F. Connexin40 is essential for the pressure control of renin synthesis and secretion.
Circ Res. 2007;100:556-563.
Skott O, Briggs JP. Direct demonstration of macula densa-mediated renin
secretion. Science. 1987;237:1618-1620.
Biava C, West M. Fine structure of normal human juxtaglomerular cells. I. General structure and intercellular relationships. Am J Pathol. 1966;49:679-721.
65
85. Albinus M, Finkbeiner E, Sosath B, Osswald H. Isolated superfused juxtaglomerular cells from rat kidney: a model for study of renin secretion. Am J
Physiol. 1998;275:F991-997.
86. Kurtz A, Della Bruna R, Pfeilschifter J, Bauer C. Role of cGMP as second
messenger of adenosine in the inhibition of renin release. Kidney Int.
1988;33:798-803.
87. Laske-Ernst J, Stehle A, Vallon V, Quast U, Russ U. Effect of adenosine on
membrane potential and Ca2+ in juxtaglomerular cells. Comparison with angiotensin II. Kidney Blood Press Res. 2008;31:94-103.
88. Fredholm BB, AP IJ, Jacobson KA, Klotz KN, Linden J. International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacol Rev. 2001;53:527-552.
89. Oppermann M, Mizel D, Kim SM, Chen L, Faulhaber-Walter R, Huang
Y, Li C, Deng C, Briggs J, Schnermann J, Castrop H. Renal function in
mice with targeted disruption of the A isoform of the Na-K-2Cl co-transporter.
J Am Soc Nephrol. 2007;18:440-448.
90. Brown RD, Thoren P, Steege A, Mrowka R, Sallstrom J, Skott O, Fredholm BB, Persson AE. Influence of the adenosine A1 receptor on blood pressure regulation and renin release. Am J Physiol Regul Integr Comp Physiol.
2006;290:R1324-1329.
91. Schweda F, Segerer F, Castrop H, Schnermann J, Kurtz A. Blood pressuredependent inhibition of Renin secretion requires A1 adenosine receptors. Hypertension. 2005;46:780-786.
92. Kim SM, Mizel D, Huang YG, Briggs JP, Schnermann J. Adenosine as a
mediator of macula densa-dependent inhibition of renin secretion. Am J Physiol
Renal Physiol. 2006;290:F1016-1023.
93. Castrop H, Kammerl M, Mann B, Jensen BL, Kramer BK, Kurtz A.
Cyclooxygenase 2 and neuronal nitric oxide synthase expression in the renal
cortex are not interdependent in states of salt deficiency. Pflugers Arch.
2000;441:235-240.
94. Pieruzzi F, Munforti C, Di Blasio A, Busca G, Dadone V, Zanchetti A,
Golin R. Neuronal nitric oxide synthase and renin stimulation by sodium deprivation are dependent on the renal nerves. J Hypertens. 2002;20:2039-2045.
95. Singh I, Grams M, Wang W, Yang T, Killen P, Smart A, Schnermann J,
Briggs J. Coordinate regulation of renal expression of nitric oxide synthase,
renin, and angiotensinogen mRNA by dietary salt. Am J Physiol.
1996;270:F1027-1037.
96. Liu R, Persson AE. Simultaneous changes of cell volume and cytosolic calcium concentration in macula densa cells caused by alterations of luminal NaCl
concentration. J Physiol. 2005;563:895-901.
97. Bolger PM, Eisner GM, Shea PT, Ramwell PW, Slotkoff LM. Effects of
PGD2 on canine renal function. Nature. 1977;267:628-630.
98. Schweda F, Klar J, Narumiya S, Nusing RM, Kurtz A. Stimulation of renin
release by prostaglandin E2 is mediated by EP2 and EP4 receptors in mouse
kidneys. Am J Physiol Renal Physiol. 2004;287:F427-433.
66
99. Bonventre JV, Gronich JH, Nemenoff RA. Epidermal growth factor enhances glomerular mesangial cell soluble phospholipase A2 activity. J Biol
Chem. 1990;265:4934-4938.
100. Peti-Peterdi J, Komlosi P, Fuson AL, Guan Y, Schneider A, Qi Z, Redha
R, Rosivall L, Breyer MD, Bell PD. Luminal NaCl delivery regulates basolateral PGE2 release from macula densa cells. J Clin Invest. 2003;112:76-82.
101. Yang T, Park JM, Arend L, Huang Y, Topaloglu R, Pasumarthy A, Praetorius H, Spring K, Briggs JP, Schnermann J. Low chloride stimulation of
prostaglandin E2 release and cyclooxygenase-2 expression in a mouse macula
densa cell line. J Biol Chem. 2000;275:37922-37929.
102. Salvemini D, Misko TP, Masferrer JL, Seibert K, Currie MG, Needleman
P. Nitric oxide activates cyclooxygenase enzymes. Proc Natl Acad Sci U S A.
1993;90:7240-7244.
103. DiBona GF, Kopp UC. Neural control of renal function. Physiol Rev.
1997;77:75-197.
104. La Grange RG, Sloop CH, Schmid HE. Selective stimulation of renal nerves
in the anesthetized dog. Effect on renin release during controlled changes in renal hemodynamics. Circ Res. 1973;33:704-712.
105. Golin R, Pieruzzi F, Munforti C, Busca G, Di Blasio A, Zanchetti A. Role
of the renal nerves in the control of renin synthesis during different sodium intakes in the rat. J Hypertens. 2001;19:1271-1277.
106. Johnson JA, Davis JO, Gotshall RW, Lohmeier TE, Davis JL, Braverman
B, Tempel GE. Evidence for an intrarenal beta receptor in control of renin release. Am J Physiol. 1976;230:410-418.
107. Johns EJ, Kopp UC. Neuronal Control of Renal Function. In: Alpern RJ,
Hebert SC, eds. Seldin and Giebisch's The Kidney: Physiology & Pathophysiology. 4 ed: Elsevier Inc.; 2008:925-946.
108. Kim SM, Chen L, Faulhaber-Walter R, Oppermann M, Huang Y, Mizel
D, Briggs JP, Schnermann J. Regulation of Renin Secretion and Expression
in Mice Deficient in {beta}1- and {beta}2-Adrenergic Receptors. Hypertension. 2007.
109. Grandjean B, Annat G, Vincent M, Sassard J. Influence of renal nerves on
renin secretion in the conscious dog. Pflugers Arch. 1978;373:161-165.
110. Holdaas H, DiBona GF. The role of left atrial receptors in the regulation of
renin release in anesthetized dogs. Acta Physiol. Scand. 1981;111:497-500.
111. Kurtz A, Penner R. Angiotensin II induces oscillations of intracellular calcium and blocks anomalous inward rectifying potassium current in mouse renal
juxtaglomerular cells. Proc Natl Acad Sci U S A. 1989;86:3423-3427.
112. Fridman KU, Wysocki M, Friberg PR, Andersson OK. Candesartan
cilexetil in hypertension: effects of six weeks' treatment on haemodynamics,
baroreceptor sensitivity and the renin-angiotensin-aldosterone system. Blood
Press. 1999;8:242-247.
113. Lang RE, Tholken H, Ganten D, Luft FC, Ruskoaho H, Unger T. Atrial
natriuretic factor--a circulating hormone stimulated by volume loading. Nature.
1985;314:264-266.
67
114. Edwards BS, Zimmerman RS, Schwab TR, Heublein DM, Burnett JC, Jr.
Atrial stretch, not pressure, is the principal determinant controlling the acute release of atrial natriuretic factor. Circ Res. 1988;62:191-195.
115. Burnett JC, Jr., Granger JP, Opgenorth TJ. Effects of synthetic atrial natriuretic
factor on renal function and renin release. Am J Physiol. 1984;247:F863-866.
116. Shi SJ, Nguyen HT, Sharma GD, Navar LG, Pandey KN. Genetic disruption of atrial natriuretic peptide receptor-A alters renin and angiotensin II levels. Am J Physiol Renal Physiol. 2001;281:F665-673.
117. Kurtz A, Della Bruna R, Pfeilschifter J, Taugner R, Bauer C. Atrial natriuretic peptide inhibits renin release from juxtaglomerular cells by a cGMPmediated process. Proc Natl Acad Sci U S A. 1986;83:4769-4773.
118. Taugner R, Rosivall L, Buhrle CP, Groschel-Stewart U. Myosin content
and vasoconstrictive ability of the proximal and distal (renin-positive) segments of the preglomerular arteriole. Cell Tissue Res. 1987;248:579-588.
119. Chiu T, Reid IA. Role of cyclic GMP-inhibitable phosphodiesterase and nitric
oxide in the beta adrenoceptor control of renin secretion. J Pharmacol Exp
Ther. 1996;278:793-799.
120. Shakur Y, Holst LS, Landstrom TR, Movsesian M, Degerman E, Manganiello V. Regulation and function of the cyclic nucleotide phosphodiesterase
(PDE3) gene family. Prog Nucleic Acid Res Mol Biol. 2001;66:241-277.
121. Grunberger C, Obermayer B, Klar J, Kurtz A, Schweda F. The calcium
paradoxon of renin release: calcium suppresses renin exocytosis by inhibition
of calcium-dependent adenylate cyclases AC5 and AC6. Circ Res.
2006;99:1197-1206.
122. Persson PB. Renin: origin, secretion and synthesis. J Physiol. 2003;552:667-671.
123. Kim HS, Maeda N, Oh GT, Fernandez LG, Gomez RA, Smithies O. Homeostasis in mice with genetically decreased angiotensinogen is primarily by an increased number of renin-producing cells. J Biol Chem. 1999;274:14210-14217.
124. Beierwaltes WH. Macula densa stimulation of renin is reversed by selective
inhibition of neuronal nitric oxide synthase. Am J Physiol. 1997;272:R1359-1364.
125. Persson PB, Baumann JE, Ehmke H, Hackenthal E, Kirchheim HR, Nafz
B. Endothelium-derived NO stimulates pressure-dependent renin release in
conscious dogs. Am J Physiol. 1993;264:F943-947.
126. Castrop H, Schweda F, Mizel D, Huang Y, Briggs J, Kurtz A, Schnermann
J. Permissive role of nitric oxide in macula densa control of renin secretion.
Am J Physiol Renal Physiol. 2004;286:F848-857.
127. Sigmon DH, Carretero OA, Beierwaltes WH. Endothelium-derived relaxing
factor regulates renin release in vivo. Am J Physiol. 1992;263:F256-261.
128. Ollerstam A, Skott O, Ek J, Persson AE, Thorup C. Effects of long-term
inhibition of neuronal nitric oxide synthase on blood pressure and renin release.
Acta Physiol Scand. 2001;173:351-358.
129. Zanchi A, Schaad NC, Osterheld MC, Grouzmann E, Nussberger J, Brunner HR, Waeber B. Effects of chronic NO synthase inhibition in rats on reninangiotensin system and sympathetic nervous system. Am J Physiol.
1995;268:H2267-2273.
130. Wangensteen R, Sainz J, Rodriguez-Gomez I, Moreno JM, Osuna A, Vargas F. Chronic blockade of neuronal nitric oxide synthase does not affect long-
68
131.
132.
133.
134.
135.
136.
137.
138.
139.
140.
141.
142.
143.
144.
145.
146.
term control of blood pressure in normal, saline-drinking or deoxycorticosterone-treated rats. Exp Physiol. 2003;88:243-250.
Vidal MJ, Romero JC, Vanhoutte PM. Endothelium-derived relaxing factor
inhibits renin release. Eur J Pharmacol. 1988;149:401-402.
Schricker K, Kurtz A. Liberators of NO exert a dual effect on renin secretion
from isolated mouse renal juxtaglomerular cells. Am J Physiol. 1993;265:
F180-186.
Kurtz A, Gotz KH, Hamann M, Kieninger M, Wagner C. Stimulation of
renin secretion by NO donors is related to the cAMP pathway. Am J Physiol.
1998;274:F709-717.
He XR, Greenberg SG, Briggs JP, Schnermann JB. Effect of nitric oxide on
renin secretion. II. Studies in the perfused juxtaglomerular apparatus. Am J
Physiol. 1995;268:F953-959.
Reid IA. Role of nitric oxide in the regulation of renin and vasopressin secretion. Front Neuroendocrinol. 1994;15:351-383.
Parving HH, Mauer M, Ritz E. Diabetic nephropathy. In: Brenner BM, Levine
SA, eds. The Kidney. Vol 2. 7th ed. Philadelphia: Saunders; 2003:1777-1818.
Messent JW, Elliott TG, Hill RD, Jarrett RJ, Keen H, Viberti GC. Prognostic significance of microalbuminuria in insulin-dependent diabetes mellitus:
a twenty-three year follow-up study. Kidney Int. 1992;41:836-839.
Mogensen CE. Microalbuminuria predicts clinical proteinuria and early mortality in maturity-onset diabetes. N Engl J Med. 1984;310:356-360.
Nelson RG, Knowler WC, Pettitt DJ, Saad MF, Charles MA, Bennett PH.
Assessment of risk of overt nephropathy in diabetic patients from albumin excretion in untimed urine specimens. Arch Intern Med. 1991;151:1761-1765.
Parving HH, Oxenboll B, Svendsen PA, Christiansen JS, Andersen AR.
Early detection of patients at risk of developing diabetic nephropathy. A longitudinal study of urinary albumin excretion. Acta Endocrinol (Copenh).
1982;100:550-555.
Deckert T, Kofoed-Enevoldsen A, Vidal P, Norgaard K, Andreasen HB,
Feldt-Rasmussen B. Size- and charge selectivity of glomerular filtration in
Type 1 (insulin-dependent) diabetic patients with and without albuminuria.
Diabetologia. 1993;36:244-251.
Vernier RL, Steffes MW, Sisson-Ross S, Mauer SM. Heparan sulfate proteoglycan in the glomerular basement membrane in type 1 diabetes mellitus.
Kidney Int. 1992;41:1070-1080.
Christiansen JS, Gammelgaard J, Tronier B, Svendsen PA, Parving HH.
Kidney function and size in diabetics before and during initial insulin treatment. Kidney Int. 1982;21:683-688.
Moe OW, Wright SH, Palacín M. Renal Handling of Organic Solutes. In:
Brenner BM, Levine SA, eds. The Kidney. Vol 1. 8 ed. Philadelphia: Saunders;
2008:214-247.
Carney SL, Wong NL, Dirks JH. Acute effects of streptozotocin diabetes on
rat renal function. J Lab Clin Med. 1979;93:950-961.
Mogensen CE. Maximum tubular reabsorption capacity for glucose and renal
hemodynamcis during rapid hypertonic glucose infusion in normal and diabetic
subjects. Scand J Clin Lab Invest. 1971;28:101-109.
69
147. Vidotti DB, Arnoni CP, Maquigussa E, Boim MA. Effect of long-term type
1 diabetes on renal sodium and water transporters in rats. Am J Nephrol.
2008;28:107-114.
148. Freitas HS, Anhe GF, Melo KF, Okamoto MM, Oliveira-Souza M, Bordin
S, Machado UF. Na(+) -glucose transporter-2 messenger ribonucleic acid expression in kidney of diabetic rats correlates with glycemic levels: involvement
of hepatocyte nuclear factor-1alpha expression and activity. Endocrinology.
2008;149:717-724.
149. Bank N, Aynedjian HS. Progressive increases in luminal glucose stimulate
proximal sodium absorption in normal and diabetic rats. J Clin Invest.
1990;86:309-316.
150. Brochner-Mortensen J, Stockel M, Sorensen PJ, Nielsen AH, Ditzel J.
Proximal glomerulo-tubular balance in patients with type 1 (insulin-dependent)
diabetes mellitus. Diabetologia. 1984;27:189-192.
151. Tuttle KR, Bruton JL, Perusek MC, Lancaster JL, Kopp DT, DeFronzo
RA. Effect of strict glycemic control on renal hemodynamic response to amino
acids and renal enlargement in insulin-dependent diabetes mellitus. N Engl J
Med. 1991;324:1626-1632.
152. Hryciw DH, Lee EM, Pollock CA, Poronnik P. Molecular changes in proximal tubule function in diabetes mellitus. Clin Exp Pharmacol Physiol.
2004;31:372-379.
153. Rasch R, Dorup J. Quantitative morphology of the rat kidney during diabetes
mellitus and insulin treatment. Diabetologia. 1997;40:802-809.
154. Pollock CA, Lawrence JR, Field MJ. Tubular sodium handling and tubuloglomerular feedback in experimental diabetes mellitus. Am J Physiol.
1991;260:F946-952.
155. Leyssac PP, Karlsen FM, Holstein-Rathlou NH, Skott O. On determinants
of glomerular filtration rate after inhibition of proximal tubular reabsorption.
Am J Physiol. 1994;266:R1544-1550.
156. Johansson B, Halldner L, Dunwiddie TV, Masino SA, Poelchen W, Gimenez-Llort L, Escorihuela RM, Fernandez-Teruel A, Wiesenfeld-Hallin
Z, Xu XJ, Hardemark A, Betsholtz C, Herlenius E, Fredholm BB. Hyperalgesia, anxiety, and decreased hypoxic neuroprotection in mice lacking the
adenosine A1 receptor. Proc Natl Acad Sci U S A. 2001;98:9407-9412.
157. Lykkegard S, Poulsen K. Ultramicroassay for plasma renin concentration in
the rat using the antibody-trapping technique. Anal Biochem. 1976;75:250-259.
158. Goldblatt H, Katz YJ, Lewis HA, Richardson E. Studies on Experimental
Hypertension : Xx. the Bioassay of Renin. J Exp Med. 1943;77:309-313.
159. Casellas D, Dupont M, Kaskel FJ, Inagami T, Moore LC. Direct visualization of renin-cell distribution in preglomerular vascular trees dissected from rat
kidney. Am J Physiol. 1993;265:F151-156.
160. Qi Z, Whitt I, Mehta A, Jin J, Zhao M, Harris RC, Fogo AB, Breyer MD.
Serial determination of glomerular filtration rate in conscious mice using
FITC-inulin clearance. Am J Physiol Renal Physiol. 2004;286:F590-596.
161. Gabrielsson J, Weiner D. Pharmacokinetic concepts. Pharmacokinetic and
Pharmacodynamic Data Analysis: Concepts and Applications. 3rd ed. Stockholm: Swedish Pharmaceutical Press; 2000:45-174.
70
162. Bozzola JJ, Russell LD. Quantitative Electron Microscopy. Electron Microscopy Boston: Jones and Bartlett Publishers; 1992:287-303.
163. Friis UG, Jensen BL, Sethi S, Andreasen D, Hansen PB, Skott O. Control
of renin secretion from rat juxtaglomerular cells by cAMP-specific phosphodiesterases. Circ Res. 2002;90:996-1003.
164. Beierwaltes WH. cGMP stimulates renin secretion in vivo by inhibiting phosphodiesterase-3. Am J Physiol Renal Physiol. 2006;290:F1376-1381.
165. Sayago CM, Beierwaltes WH. Nitric oxide synthase and cGMP-mediated
stimulation of renin secretion. Am J Physiol Regul Integr Comp Physiol.
2001;281:R1146-1151.
166. Angeli P, Jimenez W, Veggian R, Fasolato S, Volpin R, MacHenzie HS,
Craighero R, Libera VD, Sticca A, Arroyo V, Gatta A. Increased activity of
guanosine 3'-5'-cyclic monophosphate phosphodiesterase in the renal tissue of
cirrhotic rats with ascites. Hepatology. 2000;31:304-310.
167. Klar J, Sandner P, Muller MW, Kurtz A. Cyclic AMP stimulates renin gene
transcription in juxtaglomerular cells. Pflugers Arch. 2002;444:335-344.
168. Schmid C, Castrop H, Reitbauer J, Della Bruna R, Kurtz A. Dietary salt
intake modulates angiotensin II type 1 receptor gene expression. Hypertension.
1997;29:923-929.
169. Bush MA, Pollack GM. Pharmacokinetics and pharmacodynamics of 7nitroindazole, a selective nitric oxide synthase inhibitor, in the rat hippocampus. Pharm Res. 2001;18:1607-1612.
170. Qi Z, Fujita H, Jin J, Davis LS, Wang Y, Fogo AB, Breyer MD. Characterization of susceptibility of inbred mouse strains to diabetic nephropathy. Diabetes. 2005;54:2628-2637.
171. Faulhaber-Walter R, Chen L, Oppermann M, Kim SM, Huang Y,
Hiramatsu N, Mizel D, Kajiyama H, Zerfas P, Briggs JP, Kopp JB,
Schnermann J. Lack of A1 Adenosine Receptors Augments Diabetic Hyperfiltration and Glomerular Injury. J Am Soc Nephrol. 2008.
172. Vallon V, Schroth J, Satriano J, Blantz RC, Thomson SC, Rieg T. Adenosine A(1) receptors determine glomerular hyperfiltration and the salt paradox in
early streptozotocin diabetes mellitus. Nephron Physiol. 2009;111:p30-38.
173. Slomowitz LA, Deng A, Hammes JS, Gabbai F, Thomson SC. Glomerulotubular balance, dietary protein, and the renal response to glycine in diabetic
rats. Am J Physiol Regul Integr Comp Physiol. 2002;282:R1096-1103.
174. Korner A, Eklof AC, Celsi G, Aperia A. Increased renal metabolism in diabetes. Mechanism and functional implications. Diabetes. 1994;43:629-633.
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