The regeneration of ureteric function and
pathophysiology of ureteric obstruction - what
do Hedgehogs and EPO have to do with it?
Dr. Hilary Brotherhood, PGY-3
Dr. Dirk Lange, Associate Professor, The Stone Centre, VGH
Department of Urologic Sciences
Grand Rounds June 2016
Objectives
•
To review ureteric functional physiology and the
impact of obstruction on ureteric contractile properties
•
To review the impact of obstruction on ureteric
hemodynamics and pressure profiles
•
To discuss functional recovery of the obstructed ureter
•
To learn about research into pharmacological
intervention to accelerate functional recovery of ureter
following obstruction removal
Introduction
•
ureteric obstruction affects both
pediatric and adult population
•
can result in permanent renal damage
•
degree of renal injury depends on
severity of obstruction, chronicity,
baseline renal function, and presence
of other mitigating factors (UTI)
•
results in multiple histologic and
functional derangements in ureteric
physiology
Causes of Ureteric Obstruction
Congenital
-ureterocele
-obstructing
megaureter
-retrocaval
ureter
-prune belly
syndrome
Neoplastic
-urothelial
carcinoma
-metastases
Inflammation
-abscess
-ureteritis
cystica
-endometriosis
-amyloidosis
-tuberculosis
Miscellaneous
-trauma
-urinoma
-lymphocele
-pregnancy
-RPF
-pelvic
lipomatosis
Clinical Presentation of ureteric obstruction
•
acute - classic flank pain, nausea,
vomiting due to distension of renal
pelvis and visceral nerve activation.
Radiates to lower abdomen,
testicles, or labia
•
chronic - usually relatively painless,
can be asymptotic
Ureteric Contractile
Physiology
Propulsion of a Urinary Bolus
Griffiths, 1983. Griffiths DJ: The mechanics of urine transport in the upper urinary tract: 2. The discharge of the bolus into the bladder and
dynamics at high rates of flow. Neurourol Urodyn 1983; 2: pp. 167
Obstruction and Ureteric
Function
Obstruction and Ureteric Function
•
transient increase in amplitude and
frequency in peristaltic contraction,
then decerased
•
peristaltic contraction unable to coapt
ureteric wall
•
urine transport now dependent on
hydrostatic forces generated by
kidney
•
at 8 days, ureter diameter had
increased by 170% and length by 25%
Biancani, Zabinski, Weiss, 1973. Biancani P, Zabinski MP, and Weiss RM: Bidimensional deformation of acutely obstructed in vitro rabbit ureter. Am J Physiol 1973; 225: pp. 671
Obstruction and Ureteric
Contractile Properties
Obstruction and Ureteric Contraction
•
rabbit model, complete ureter
obstruction for 2 weeks
•
250% cross sectional muscle
area
•
24% ureteral length
•
100% ureteral outer diameter
Longitudinal
Circumferential
From Hausman M, Biancani P, Weiss RM. Obstruction induced changes in longitudinal force-length relations of rabbit ureter. Invest Urol 1979;17:223. Copyright Williams & Wilkins, 1979; B, from
Biancani P, Hausman, M, Weiss RM. Effect of obstruction on ureteral circumferential force-length relation. Am J Physiol 1982;243:F204.)
Obstruction and ureteric contraction
stress = force / unit
area muscle
Longitudinal
Circumferential
From Weiss RM, Biancani P. A rationale for ureteral tapering. Urology 1982;20:482.
Obstruction and ureteric Contraction
Laplace Equation
P = stress x wall thickness
radius
Pathophysiology of ureteric
obstruction - Unilateral vs
Bilateral
Pathophysiology of unilateral ureteric
obstruction
Moody TE, Vaughan ED Jr, Gillenwater JY. Relationship between RBF and ureteral pressure during 18 hours of total ureteral occlusion: implications for changing sites of increased renal resistance.
Invest Urol 1975;13:246–51.)
Obstruction induced increased
Rafferent arteriole
•
Initially: upregulation of RAAS and increased
thromboxane A2 expression, but Vaughan et al (2004)
did not reveal a significant effect of these mediators on
renal hemodynamic response to obstruction
Vaughan et al, 2004. Vaughan ED, Marion D, Poppas DP, et al: Pathophysiology of unilateral ureteral obstruction: studies from Charlottesville to New York. J Urol 2004; 172: pp. 2563-2569
Felsen et al, 2003. Felsen D, Schulsinger D, Gross SS, et al: Renal hemodynamic and ureteral pressure changes in response to ureteral obstruction: the role of nitric oxide. J Urol 2003; 169: pp. 373-376
Obstruction induced increased
Rafferent arteriole
Obstructed, no L-Arginine
Obstructed, given L-Arginine
Not obstructed, given L-Arginine
Felsen et al, 2003. Felsen D, Schulsinger D, Gross SS, et al: Renal hemodynamic and ureteral pressure changes in response to ureteral obstruction: the role of nitric
oxide. J Urol 2003; 169: pp. 373-376
Pathophysiology of Bilateral Ureteric
Obstruction
Bilateral ureteric obstruction
Intravascular Volume
Atrial Natriuretic Peptide (ANP)
Pathophysiology of Bilateral Ureteric
Obstruction
Nitric
oxide
Atrial
Natriuretic
Peptide
Pathophysiology of Bilateral
Ureteric Obstruction
Kim et al, 2001b. Kim SW, Lee J, Park JW, et al: Increased expression of atrial natriuretic peptide in the kidney of rats with bilateral ureteral obstruction.
Kidney Int 2001; 59: pp. 1274-1282
Molecular Response to Ureteral Obstruction
•
Focus of Stone Centre Research Program is to
develop novel approaches to improve the treatment of
kidney stone disease
•
Includes ureteral stent biomaterial design and most
recently how the ureter responds to indwelling stents
and obstruction
Primary Focus On Indwelling Stents
•
Purpose of ureteral stents is to drain the kidney in the
presence of an obstruction or post-op following stone
removal
•
Stents associated with significant patient discomfort/
severe pain
•
Known Causes: Irritation of urothelium as stent moves
inside ureter and bladder
•
Unknown Causes: ?
Stent-Induced Hydronephrosis and
Peristalsis
despite stents maintaining drainage past an
obstruction they themselves trigger hydronephrosis
A
**
**
**
**
3
Peristalticactivityscore
•
2
1
0
Controls
Contralateral
Stented
Poststent
removal
J Urol. 2010 Feb;183(2):765-71
5301
Development 129, 5301-5312 (2002)
Printed in Great Britain © The Company of Biologists Limited 2002
DEV20016
Targeting Hedgehog Signaling
DEVELOPMENT AND DISEASE
Shh signaling is required for mesenchymal cell
proliferation
The mutant kidneys in newborn pups were 52% smaller than
those in their wild-type littermates (Fig. 5A; wild type, n=3;
mutant, n=4; P=0.002), and the glomerular number was
reduced by 40% (P=0.004). However, the glomerular density
in the mutant kidneys increased by 26% (Fig. 4B, P=0.03). To
Sonic hedgehog regulates proliferation and differentiation of mesenchymal
cells in the mouse metanephric kidney
Jing Yu, Thomas J. Carroll and Andrew P. McMahon*
Department of Molecular and Cellular Biology, 16 Divinity Avenue, Harvard University, Cambridge MA 02138, USA
*Author for correspondence (e-mail: [email protected])
Shh signaling in kidney development 5305
determine if this increase in glomerular density is due to
differential effects of Shh on cortical and medullary regions of
the kidney (Shh expression is primarily in the medulla), we
further quantified the cortical and medullary volume. The
reduction of the cortical and the medullary volume of Shh
mutants is similar, 51% (P=0.003) and 46% (P=0.002),
respectively. The cortical glomerular density in mutant kidneys
increased by 24% (P=0.02), similar to that of the whole mutant
kidneys. These data suggest that the higher glomerular density
in the entire mutant kidney is not due to the underdevelopment
of the medullary region relative to the rest of the kidney. No
gross size differences were seen between the glomerulus of the
mutant kidneys and that of the wild type.
Accepted 12 August 2002
Inactivation of
Hedgehog results inanalyses demonstrate that sonic hedgehog promotes
Signaling by the ureteric bud epithelium is essential
for survival, proliferation and differentiation of the
mesenchymal cell proliferation, regulates the timing of
hydrometer
and
metanephric mesenchyme
during kidney development.
differentiation of smooth muscle progenitor cells, and sets
Most studies that have addressed ureteric signaling have
the pattern of mesenchymal differentiation through its
hydronephrosis
focused on the proximal,
branching, ureteric epithelium.
dose-dependent inhibition of smooth muscle formation. In
•
SUMMARY
We demonstrate that sonic hedgehog is expressed in the
ureteric epithelium of the distal, non-branching medullary
collecting ducts and continues into the epithelium of the
ureter – the urinary
• outflow tract that connects the kidney
with the bladder. Upregulation of patched 1, the sonic
hedgehog receptor and a downstream target gene of the
signaling pathway in the mesenchyme surrounding the
distal collecting ducts and the ureter suggests that sonic
hedgehog acts as a paracrine signal. In vivo and in vitro
addition, we also show that bone morphogenetic protein 4
is a downstream target gene of sonic hedgehog signaling in
kidney stroma and ureteral mesenchyme, but does not
mediate the effects of sonic hedgehog in the control of
mesenchymal proliferation.
INTRODUCTION
Grobstein, 1955), and the metanephric mesenchyme is required
for the growth and branching of the ureteric bud (Ekblom,
1992; Erickson, 1968; Grobstein, 1953; Grobstein, 1955). The
developmental roles (if any) of the distal collecting ducts and
the ureteric epithelium are unknown. Because smooth muscle
Fig. 3. Conditional removal of Shh activity from the urothelium with
forms adjacent to
theseresults
regions,
it seems
that these
HoxB7/Cre
in hypoplasia,
hydroureterlikely
and hydronephrosis.
(A,B) Whole-mount
of the newborn
kidney and ureter of wildtissues might regulate
smoothview
muscle
development.
type (WT) and HoxB7/Cre, Shhc/n mice. (C-J) Hematoxylin and
Sonic hedgehog
(Shh),
a Drosophila
Hedgehog
Eosin staining
of coronal
sections (C,D) and parasagittal
sections (Hh)
Caused by ureteral
aperistalsis
The metanephric kidney forms through reciprocal inductive
interactions between the ureteric bud epithelium and the
metanephric mesenchyme. The ureteric bud, an outgrowth of
the Wolffian (or mesonephric) duct, invades the metanephric
blastema and arborizes to form the collecting duct system
within the kidney and the ureter (the latter connects the
Key words: Kidney, Sonic hedgehog, Shh, Proliferation, Smooth
muscle, Mouse, Ureter, Hydroureter
Fig. 4. Ptch and Bmp4 expression in mutant kidneys. (A-H) In situ
hybridization of Ptch and Bmp4 probes to E14.5 kidney (A,B,E,F)
and ureter (C,D,G,H). The insets show Bmp4 expression in
glomeruli. Scale bars: 100 µm in A,B,E,F; 50 µm in C,D,G,H.
(I) RT-PCR of Bmp4 transcripts from E12.5 mesenchymal cells
dissected from the ureter (lane 1) and those cultured without (lane 2)
Specific Research Questions
•
Considering that stents are supposed to facilitate
urinary flow, why do they stop peristalsis?
•
What are the molecular mechanisms that regulate
ureteral peristalsis?
Studied Gli-1 Expression in Stented
and Unstented Ureters
D
3
Stent
insertion
Stentremoval
2
Recovery
Gli1
Score
1
0
-1
Inflammation
baseline 48hr.
1wk.
2wk.
4wk.
7wk.
10wk.
-2
-3
•
•
Gli-1 expression in ureteral smooth muscle decreases
with stent indwelling time
Recovers following Removal of stent
Functional Consequences
Unstented
48 hr stented
7 day stented
Ureteraldiameter(mm)
25
Timetopeakforce
Controls
20
15
Contralateral
10
Stented
5
0
0
30
60
90
Force/SMarea(mN/mm²)
120
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Time(sec)
Summary of Findings
•
Indwelling stents dilate the ureter (desirable)
•
Stretches ureteral smooth muscle resulting in overall
dysfunction and peristalsis (undesirable)
•
Decreased peristalsis = lower urine flow and increased
reflux resulting in hydronephrosis
•
Can we target molecular pathways (i.e. Gli-1) to
maintain peristalsis with a stent in place = improved
urine flow and less patient discomfort
How Does Ureter Respond To Ureteral
Obstruction ?
•
Disruption of ureteral peristalsis
• Reduction
and/or cessation of urine flow from kidney to bladder
• Increased
pressure on the kidneys and hydroureteronephrosis
• Renal
injury, dysfunction and eventual failure
• Common
Research Focus:
Effect of ureteral obstruction on kidney function
• What
we don’t know:
How does the ureter respond to obstruction?
How does the ureter recover following obstruction?
Ureteral Recovery Following
Obstruction Reversal
obstructed ureters of mice for 24 hrs, 48 hrs, and 72
hrs, followed hydronephrosis via ultrasound and
quantified peristalsis
Peristaltic activity( %)
100
80
60
40
r= -0.88
n= 66
20
0
0
1
2
3
4
5
6
7
8
9
Intrapelvic diameter (mm)
100
80
Group I
120
Group II
100
Group III
60
*
40
***
*
20
Peristaltic activity ( %)
Peristaltic activity ( %)
•
Obstruction
80
60
*
40
Pre-obstruction
Day hydronephrosis had resolved
*
*
*
20
0
0
0
Hydronephrosis
resolved
1d
2d
3d
Time
8d
11d
14d
Summary of Findings
Refutes hypothesis that resolution of
hydronephrosis normal ureteral function
Ureteral dysfunction = decreased urine flow
Potentially prolonged negative effects on kidney
function
CAN WE ACCELERATE RECOVERY
FOLLOWING OBSTRUCTION REVERSAL?
Can Recovery Be Enhanced Via
Pharmacological Intervention ?
Erythropoietin (EPO): Protective Effects in other organs
Journal of Pediatric Surgery (2006) 41, 352 – 357
www.elsevier.com/locate/jpedsurg
Erythropoietin restores bowel damage and
hypoperistalsis in gastroschisis
Aykut Ozdamar a, Koray Topcu a, Mukaddes Gumustekinb, Duygu Gurelc,
Ayse Gelalb, Erdener Ozerc, Basak Ucana, Gunyuz Temir a, Aytac Karkiner a,
Irfan Karaca a, Munevver Hosgora,*
a
Journal Turkey
of Pediatric Surgery (2006) 41, 352 – 357
Department of Pediatric Surgery, Dr. Behcet Uz Children’s Hospital, Izmir,
Department of Pharmacology, Dokuz Eylul University, Izmir, Turkey
Department of Pathology, Dokuz Eylul University, Izmir, Turkey
b
c
Index words:
Gastroschisis;
Erythropoietin;
Gastrointestinal
contractility
Abstract
Background and Purpose: Despite the decreased mortality in gastroschisis (Gx), patients experience
postoperative intestinal hypoperistalsis, malabsorption, and shortened bowel length. The trophic effects
of recombinant human erythropoietin (rEpo) in the developing small bowel have been reported,
increasing the length and height of the villi, and villous surface area. This study investigated the effects
of rEpo on intestinal malfunction in the chick embryos with Gx.
Methods: Thirteen-day-old fertilized chicken eggs were used to create Gx model. Study groups
included the following: group 1, control; group 2, Gx-only; group 3, Gx + 0.075% saline exchange;
group 4, Gx + 10 IU rEpo exchange; group 5, Gx + 20 IU rEpo exchange. The bowels were evaluated
by in vitro muscle strip technique, and the response was expressed as a percentage of the maximum
carbachol-evoked contraction (E max). In addition, parasympathetic ganglion cells per 10 plexuses and
villi height were determined by light microscopy. Results were evaluated statistically by Mann-Whitney
U, v 2, and Fisher’s Exact test tests.
Results: Saline exchange had no effect on ganglion cell number ( P =a .63) and villi height ( Pa= .10). In
Koray
Topcu
, Mukaddes Gumustekinb, Duyg
Aykut( P Ozdamar
group 4, ganglion cell number was not increased
= .82), but villi, height
increase
was significant
b
c
( P = .03). In Gx + 20 IU rEpo group, both the
number
of ganglia
( P = .0001) and
villi height
( P = .002)
Ayse
Gelal
, Erdener
Ozer
, Basak
Ucana, Gunyuz Temir a, Ayta
were significantly increased. The decrease in contractility in group
2 ( P = .0121) was significantly
a
a,*
IrfannoKaraca
Munevver
Hosgor
reversed by rEpo 20 IU treatment ( P = .0216),
significant ,difference
was obtained
in groups 3
( P = .0809) and 4 ( P = .1516) compared with group 2.
Conclusion: These data suggest that rEpo has prokinetic effects on hypoperistalsis and restores bowel
a
Department of Pediatric Surgery, Dr. Behcet Uz Children’s Hospital, Izmir, Turkey
damage in Gx.
b
D 2006 Published by Elsevier Inc.
Department of Pharmacology, Dokuz Eylul University, Izmir, Turkey
Erythropoietin restores bowel damage and
hypoperistalsis in gastroschisis
EPO Treatment Accelerates Resolution
of Hydronephrosis
•
Gave 20IU EPO to mice on 4 consecutive days preobstruction and obstructed for 24 hrs, 48 hrs, and 72 hrs.
•
Followed recovery using ultrasound and quantifying
peristalsis
CON
EPO
72 hrs
obstruction
48 hrs postrelease
96 hrs postrelease
192 hrs postrelease
EPO Accelerates Recovery of Ureteral
Function
•
Time for ureteral
peristalsis to
return to normal
levels accelerated
in EPO treated
animals
Continued Research
•
Intention is not to promote use of EPO for patients with obstruction
(significant side-effect profile and costly)
•
Studying EPO-induced molecular mechanisms that regulate ureteral
protection and accelerated recovery
•
Potential targets for less expensive, safer and more effective future
therapeutic agents
•
Current studies in the lab aimed at understanding molecular mechanisms
regulating ureteral dysfunction following obstruction (including members of
Hedgehog pathway) and how they are changed by EPO.
•
Also obtaining ethics for human ureteral tissue from transplant donors
(unused tissue) to study EPO treatment on peristaltic activity using tissue
baths
Acknowledgements
•
Dr. Ben Chew
•
Dr. Chun Seow (Pathology & Laboratory Medicine)
•
Stone Centre Research Lab:
•
Joey Lo
•
Elliya Park
•
David Choy
•
Dr. Claudia Janssen (now in Mainz, Germany)