Nephrol Dial Transplant (2003) 18: 2255–2261
DOI: 10.1093/ndt/gfg397
Original Article
Osmoregulation of aldose reductase and sorbitol dehydrogenase in
cultivated interstitial cells of rat renal inner medulla
Jürgen Steffgen1,*, Katrin Kampfer1, Clemens Grupp1, Christoph Langenberg1, Gerhard A. Müller1
and R. Willi Grunewald1
1
Abteilung Nephrologie und Rheumatologie, Georg-August-Universität Göttingen, Göttingen, Germany
Abstract
Background. Little is known about sorbitol metabolism in renal papillary interstitial cells. For characterization we studied regulation of sorbitol synthesis by
aldose reductase (AR) and degradation by sorbitol
dehydrogenase (SDH) in papillary interstitial cells.
Methods. Interstitial cells were isolated from rat renal
inner medulla to a pure cell fraction. mRNA was
isolated from cultivated cells and sorbitol, AR and
SDH activity were determined enzymatically in homogenates.
Results. Sorbitol concentration in these cells at
300 mosmol/l was 4.4±0.3 vs 78±3.6 mmol/g protein at 600 mosmol/l. At steady-state conditions at
300 mosmol/l, AR activity was nearly the same as SDH
activity (15.1±1.6 vs 16.6±2.0 U/g protein). At
600 mosmol/l, AR activity increased to 82.5±11.4 U/g
protein and SDH activity to 31.5±6.0 U/g protein.
Studying the time course of enzyme activity after
changing osmolarity from 300 to 600 mosmol/l, we
found half maximal stimulation after 2–3 (AR) or 3
(SDH) days. The amount of AR-mRNA preceded the
rise of enzyme activity, whereas SDH-mRNA was not
significantly influenced. Lowering osmolarity from 600
to 300 mosmol/l, enzyme activity decreased to less than
half within 2 (AR) or 1 (SDH) day(s).
Conclusions. The results suggest that sorbitol metabolism contributes to handling of osmotic stress in rat
renal papillary interstitial cells.
Keywords: aldose reductase; enzyme activity; osmoregulation; sorbitol; sorbitol dehydrogenase
Correspondence and offprint requests to: Dr Jürgen Steffgen,
Abteilung Klinische Forschung, Boehringer-Ingelheim Pharma
GmBH & Co KG, D-88397 Biberach an der Riss, Germany.
Email: [email protected].
*Present address: Abteilung Klinische Forschung, BoehringerIngelheim Pharma GmBH & Co KG, Biberach an der Riss,
Germany
Introduction
Diuresis and anti-diuresis confronts kidney cells and
especially inner medullary cells with extreme changes in
urine osmolarity. In rat kidney, urine osmolarity can be
increased by up to 10-fold to 3000 mosmol/l [1].
It has been established that the main osmotic
response in the kidney involves more transmembrane
movements of organic than of inorganic osmolytes [2].
As high concentrations of polyols or some amino acids
do not significantly perturb protein function, they
allow renal cells to adapt to wide changes in osmolarity
without endangering cellular function.
Within organic osmolytes sorbitol deserves special
interest because disturbance of sorbitol metabolism
is discussed together with complications of diabetes
mellitus (for example [3]). Intracellular sorbitol synthesis is regulated by aldose reductase (AR, EC 1.1.1.21)
and degradation by sorbitol dehydrogenase (SDH, EC
1.1.1.14).
High sorbitol and AR concentrations were found in
rat inner medulla [4] with highest enzyme activity
in inner medullary collecting duct (IMCD) cells [5].
In mammalian inner medulla, the content of sorbitol as
well as myo-inositol, betaine, taurine and glycerophosphorylcholine varied in parallel with extracellular
osmolarity [6]. Nearly all studies concentrated on
IMCD cells or cells derived from renal papillary
epithelia (PAP-HT25 cells) [7]. In these cells a rise of
sorbitol, AR protein, AR activity and AR mRNA
could be observed in the presence of higher osmolarity
[5,8–10].
On the other hand interstitial cells of the inner
medulla are exposed to the same osmotic stress as
IMCD cells. Until now little has been known about
sorbitol metabolism in interstitial cells. So far it has
been speculated that SDH activity is dominating in
these cells [5].
A model of intrapapillary interaction between
IMCD and interstitial (IS) cells was proposed [11].
In this model at hypotonic conditions intracellular
Nephrol Dial Transplant 18(11) ß ERA–EDTA 2003; all rights reserved
2256
accumulated sorbitol is released from IMCD cells at the
basolateral side, taken up by IS cells and converted
within these cells into fructose. Fructose might be
recycled by uptake into IMCD cells and subsequent
reconverting fructose into sorbitol [12].
Recently, a rise in sorbitol concentration as well as
mRNA encoding for AR has been demonstrated in
isolated rat papillary IS cells by changing osmolarity
from 300 to 600 mosmol/l [13]. However enzymatic
activity of AR and SDH has not been studied in renal
papillary IS cells so far. Therefore, we studied activity
of these two enzymes at a steady state of 300 or
600 mosmol/l in cultivated rat renal papillary IS cells.
We also characterized changes in their mRNA levels
and the time course of AR and SDH activity after
elevating osmolarity from 300 to 600 mosmol/l as well
as after lowering osmolarity from 600 to 300 mosmol/l.
Subjects and methods
J. Steffgen et al.
medullary renal IS cells [15]. For instance, they were
extensively branched and contained lipid droplets. Purity of
this cell culture could be demonstrated by positive staining
for the lectin BSL-1 and negative staining for factor VIII
(for endothelial cells), cytokeratine (for thin limb of Henle
cells and IMCD cells) and DBA (for IMCD-cells). For
experiments IS cells after about 15 culture passages were
used which were incubated either at 300 or 600 mosmol/l
until steady state was reached.
To quantify the amount of sorbitol synthesized by
interstitial cells in comparison with IMCD cells, the IMCD
cells were isolated by the above-mentioned three-step
centrifugation procedure in the pellet as reported. Freshly
isolated IMCD cells were cultivated in 6-well plates at 37 C
in 5% CO2 atmosphere in a 600 mosmol/l DMEM containing
D-glucose (1000 mg/l) mixed with equal amounts of Ham’s
F-12, 10% fetal calf serum, 1% (v/v) non-essential amino
acids, 1 mM sodium pyruvate, 2 mM glutamine, penicillin
(50 U/ml) and streptomycin (50 U/ml) (all substrates obtained
from Gibco-BRL, Eggenstein, Germany). The osmolarity was
adjusted to 300 and 600 mosmol/l by addition of appropriate
amounts of NaCl.
Cell isolation
Detailed procedures for the isolation and culture of rat inner
medullary fibroblasts have been published previously [14].
In short, male Wistar rats (Harlan-Winkelmann, Borchen,
Germany, body weight 200–250 g) were fed with a standard
diet and received water ad libitum. All rats were killed by
cervical dislocation. Kidneys were immediately removed, and
the inner two-thirds of the inner medulla exactly excised.
Tissue was placed in 290 mosmol/l ice-cold HEPES-Ringer’s
buffer (composition in mmol/l: 118 NaCl, 16 H-HEPES, 16
Na-HEPES, 14 glucose, 3.2 KCl, 2.5 CaCl2, 1.8 MgSO4, 1.8
KH2PO4, pH 7.4), minced with a razor blade and subsequently incubated for 75 min at 37 C in the HEPES-Ringer’s
buffer containing in addition 0.2%(w/v) collagenase (CLS II,
Cooper, Frankfurt, Germany) and 0.2%(w/v) hyaluronidase
(Roche Diagnostics, Mannheim, Germany).
After completing the incubation procedure, the majority of
the collecting duct cells in suspension were removed through
pelleting them by low-speed centrifugation at 28 g for 2 min.
This centrifugation step was repeated twice.
The supernatants of the first two low-speed centrifugations containing the majority of IS cells were then
completely purified from IMCD cells by the use of beads
coated with the Dolichos Biflorus Agglutinin (DBA) as
reported previously [14]. The cell suspension was then placed
on the top of a continuous Nycodenz [5-(N-2,3-dihydroxypropylacetamide)-2,4,6,-triiodine-N0 -bis(2,3-dihydroxypropyl)-isophtalamide, Nycomed A J, Oslo, Norway]-gradient
with a density of 1.052–1.093 g/cm3 and spun at 1500 g for
45 min at 4 C. After centrifugation IS cells were mostly
enriched in that fraction with a density of 1.081–1.093 g/cm3.
The Nycodenz was removed by two centrifugation steps with
culture medium (composition see below) and the cells plated
in culture wells. Isolated cells were kept in Dulbecco’s
modified Eagle’s Medium (DMEM) and Nutrient Mix
Ham’s F12 (1:1), supplemented with glutamine (2 mM),
sodium pyruvate (1 mM), non-essential amino acids
(1% v/v), penicillin (50 U/ml), streptomycin (50 U/ml) and
10% fetal calf serum (all components from Gibco, Eggenstein,
Germany). Cells spontaneously immortalized while cultivated in the medium but kept typical characteristics of inner
Determination of sorbitol, AR and SDH
enzyme activity
Sorbitol was determined in homogenates of cultured IS or
IMCD cells by a commercially available test kit (Boehringer
Mannheim, Germany) as described earlier [16]. In this test,
sorbitol is oxidized in the presence of sorbitol dehydrogenase
and NAD to fructose and NADH þ Hþ. In a following
reaction NADH is oxidized by iodonitrotetrazolium chloride
in the presence of diaphorase to formazan. The samples were
incubated for 45 min at room temperature in the dark. Afterwards the increase of absorbance was measured at 492 nm.
Sorbitol from Fluka (Neu-Ulm, Germany) was used as
external and internal standard.
Aldose reductase activity was determined in homogenates
of cultured IS cells. In the presence of AR, DL-glyceraldehyde
is reduced by NADPH to glycerol and NADP. The assay
contained (in mM) 50 phosphate buffer (pH 6.0), 400 Li2SO4,
10 DL-glyceraldehyde and 0.1 NADPH. The decrease of
absorbance at 340 nm was measured at 37 C in the presence
and absence of DL-glyceraldehyde to correct for unspecific
NADPH reductase activity [10].
SDH activity was determined in homogenates of cultured
IS cells. In the presence of SDH fructose is reduced by NADH
to sorbitol and NAD. The assay contained 106 mmol/l
triethanolamine buffer (adjusted to pH 7.4 with 2 mmol/l
NaOH), 1.2 mmol/l NADH and 1.19 mmol/l fructose. The
decrease of absorbance at 340 nm was monitored for 6 min
at 37 C in the presence and absence of fructose in order to
correct for unspecific oxidation of NADH [5].
Protein was measured in triplicate according to Lowry et al.
[17] after precipitation of the protein with 10 % w/v ice-cold
trichloroacetic acid. Concentrations of bovine serum albumin
(Boehringer Mannheim, Germany) between 0.2 and 1.0 g/l
were used as standards.
RNA preparation, RT–PCR
Equal amounts of cultivated IS cells were harvested after
trypsination, cell pellet was spun out (3500 r.p.m.) and
Osmoregulation of aldose reductase and sorbitol dehydrogenase
resuspended. The quantity of protein in each sample was
determined. Total RNA was prepared by lysing cells in
guanidinium isothiocyanate containing solution and further
isolation by a silica gel based technique using RNeasy Kit
(Qiagen) according to the manufacturer’s description.
First strand cDNA was synthesised using the oligo-(dT)primer and the Superscript II DNA polymerase (Gibco-BRL).
For the detection of AR and SDH mRNA, primers AR
sense (50 -ACTGCCATTGCAAAGGCATCGTGGT-30 ), AR
antisense (50 -CCCCCATAGGACTGGAGTTCTAAG C-30 ),
SDH sense (50 -GGTGGAAAGTGTGCTGGGGA-30 ) and
SDH antisense (50 -GGGGTTCTGGGTCATTGGGG-30 )
were used, identifying a 668 bp (AR) or 367 bp (SDH)
PCR-product as recently described [10].
PCR was performed in the presence of 2.4 mmol/l MgCl2
for 30 cycles in a Perkin Elmer Thermocycler (Gene Amp
2400) with 30 s at 94 C for denaturing, 30 s at 60 C for
annealing and 50 s at 72 C for amplification with a final
elongation of 7 min at 72 C. Each amplification was
performed in duplicate.
No data exist regarding internal standards like -actin at
different osmolarities. On preliminary experiments, raising
osmolarity from 300 to 600 mosmol/l increased the amount of
-actin mRNA identified by RT–PCR 1.6-fold, indicating
that -actin could not be used as an internal standard.
Therefore, protein content was used as an external standard,
thus having the same standard for enzyme activity determinations and PCR. Using this method we found reproducible
changes in AR expression when the osmolarity was changed
as reported earlier [10]. Semi-quantitative assessment of
optical density of the PCR products on agarose gel was
performed using the Fluor-STM Multilmager with MultiAnalyst Software (Bio-Rad, CA).
2257
Fig. 1. Sorbitol content in rat renal IS cells (A) and IMCD cells (B)
at 300 or 600 mosmol/l at steady-state conditions. Data are
mean ± SEM from eight independent experiments.
Statistical analysis
For statistical analysis the unpaired Student’s t-test and
analysis of variance were employed. A difference was
considered statistically significant at P < 0.05. Mean values
with their respective standard errors are given throughout.
Results
IS cells were isolated from rat renal inner medulla
as described in the Subjects and methods. At steadystate conditions (14 days incubation) at 300 mosmol/l
sorbitol concentration in these IS cells was
4.4 ± 0.3 mmol/g protein. After raising osmolarity
with NaCl to 600 mosmol/l (Figure 1A) the steadystate sorbitol concentration increased to 78 ± 3.6 mmol/
g protein (P < 0.0001). Sorbitol concentration measured for comparison in isolated IMCD cells (Figure
1B) was 49 ± 3.5 at 300 mosmol/l and 216 ± 24.7 at
600 mosmol/l (P < 0.0001). The data indicate that IS
cells like IMCD cells respond to osmotic stress with
an increase in sorbitol synthesis.
Analysis of the time course of sorbitol concentrations
in IS cells (Figure 2) demonstrated that 6 days after
the increase in osmolarity from 300 to 600 mosmol/l
sorbitol has nearly reached state-state level (Figure 2A).
Furthermore, 6 days after decreasing osmolarity from
Fig. 2. Time course of sorbitol concentration in rat renal papillary IS
cells after raising extracellular osmolarity from 300 to 600 mosmol/l
(A) or decreasing osmolarity from 600 to 300 mosmol/l (B). Data are
mean ± SEM from four independent experiments, ss, steady state
after 2 weeks.
600 to 300 mosmol/l sorbitol concentration was also
close to steady-state level.
To characterize sorbitol metabolism we tested the
activity of AR (Figure 3A) and SDH (Figure 3B) in IS
cells. In steady-state conditions at 300 mosmol/l in vitro
AR activity was nearly the same as SDH activity
(15.1 ± 1.6 vs 16.6 ± 2.0 U/g protein). In steady state
at 600 mosmol/l, AR activity increased more than
four times to 82.5 ± 11.4 U/g protein (P < 0.0001).
However, even SDH activity increased by 2-fold in
steady state conditions at 600 mosmol/l to
31.5 ± 6.0 U/g protein (P < 0.02). As reported earlier
[10] AR activity in IMCD cells was 119 ± 33 U/g
protein at 300 mosmol/l and 410 ± 76 U/g protein
2258
J. Steffgen et al.
Fig. 3. Activity of AR (A) and SDH (B) in homogenates of rat renal
IS cells at 300 or 600 mosmol/l at steady-state conditions. Data are
mean ± SEM from 12–14 independent experiments.
Fig. 5. Time course of AR (A) and SDH (B) enzyme activity after
raising extracellular osmolarity from 300 to 600 mosmol/l. Data are
mean ± SEM from five to eight independent experiments. ss, steady
state.
Fig. 4. Expression of AR-mRNA detected by RT–PCR in homogenates of rat renal IS cells at 300, 450 and 600 mosmol/l. Semiquantitative intensity analysis of optical density by using the
Fluor-STM Multilmager with Multi-Analyst Software. Intensity at
300 mosmol/l was set to 1. Data are mean ± SD from three
independent experiments.
at 600 mosmol/l, whereas SDH activity could not be
detected in cultivated IMCD cells.
To elucidate whether the osmotic regulation of AR in
IS cells also takes place at the mRNA level like in
IMCD cells, we determined the amount of mRNA
encoding for AR at different osmolarities by RT–PCR.
As shown in Figure 4, the amount of mRNA encoding
for AR increased from 300 through 450 to 600
mosmol/l. At 600 mosmol/l AR-mRNA detected by
RT–PCR was 2.1-fold higher than at 300 mosmol/l.
mRNA encoding for SDH however did not change
significantly with osmolarity (1.25-fold, not shown).
In further experiments we studied the time course of
enzyme activity after changing osmolarity from 300 to
600 mosmol/l (Figure 5) by measuring enzyme activity
between 0 and 14 days. We found half maximal
stimulation after 2–3 days for AR (Figure 5A) or 2
days for SDH (Figure 5B). After 6 days, steady-state
level was reached for both enzymes, and there was no
significant further increase after 14 days.
At the mRNA level (Figure 6) there was a rapid
increase of AR-mRNA detected by RT–PCR starting
6 h after changing osmolarity from 300 to 600 mosmol/l
and reaching maximal activity after 24–48 h. After 4
and 6 days (steady state of enzyme activity) a
decrease in the amount of AR-mRNA could be
Fig. 6. Time course of AR-mRNA expression detected by RT–PCR
in homogenates of rat renal IS cells 0–48 h, 4 and 6 days after
changing osmolarity from 300 to 600 mosmol/l. Semi-quantitative
intensity analysis of optical density by using the Fluor-STM Multilmager with Multi-Analyst Software. Intensity at 300 mosmol/l was
set to 1. Data are mean ± SD from three independent experiments.
observed. Therefore, it can be concluded, that the rise
of AR-mRNA shows an overshoot with a maximum
after 24–48 h. The data also indicate that after raising
osmolarity, the increase of AR-mRNA in IS cells
precedes the increase of enzyme activity.
Lowering osmolarity from 600 to 300 mosmol/l
(Figure 7), we found a rapid decrease of enzyme activity
to less than half of the activity at 600 mosmol/l
within 2 (AR, Figure 7A) or 1 (SDH, Figure 7B)
day(s), indicating rapid down-regulation of enzyme
activity.
Osmoregulation of aldose reductase and sorbitol dehydrogenase
Fig. 7. Time course of AR (A) and SDH (B) enzyme activity after
lowering extracellular osmolarity from 600 to 300 mosmol/l. Data are
mean ± SEM from five to eight independent experiments. ss, steady
state.
Discussion
Until now, little has been known about sorbitol
metabolism in papillary IS cells, although these cells
are exposed to the same osmotic stress as papillary
collecting duct cells. Because conditions can be better
controlled in tissue culture than in vivo, we studied
isolated and cultivated papillary IS cells. As reported
recently [14], a cell suspension from rat renal inner
medulla could be completely purified of IMCD cells by
low-speed centrifugation and subsequent use of
DBA-coated beads. After centrifugation on a continuous Nycodenz gradient and about five culture
passages, these cell cultures contained only IS cells,
which could be demonstrated by positive staining
with the lectin BSL-1 and negative staining with
factor VIII (for endothelial cells), cytokeratine (for
thin limb of Henle cells and IMCD cells) and DBA (for
IMCD-cells) [15].
As IMCD cells, IS cells accumulate sorbitol during
hypertonic conditions. The lower amount of sorbitol
in IS cells in comparison with IMCD cells at 300
and 600 mosmol/l can be explained by a relatively
high activity of SDH in IS cells, which degrades part
of the synthesized sorbitol, whereas in IMCD cells
no SDH enzyme activity could be detected [10].
Recently Burger-Kentischer et al. reported on an
increase in glycerophosphorylcholine, betaine, myoinositol and sorbitol content in isolated rat IMCD as
well as IS cells [13] after changing osmolarity from
300 to 600 mosmol/l. The absolute amount of
sorbitol in these experiments differed somewhat
2259
from our results, which might be due to differences
in cell isolation. The authors isolated IS cells with a
200 g centrifugation step. From our own experimental experience with such a kind of isolation it cannot
be excluded that this cell culture still contained other
cell types. Additionally, these authors did not report
on negative staining for other cell markers in their
culture. Nevertheless, both studies demonstrated that
IS cells like IMCD cells increase intracellular sorbitol
in response to higher osmolarity.
It could be expected that this increase in sorbitol
content is due to higher synthesis, however, enzyme
activity of AR and SDH in IS cells was not studied
before. We could demonstrate significantly higher
activity of AR and of SDH in IS cells incubated at
600 mosmol/l than in cells incubated at 300 mosmol/l.
At both osmolarities the absolute amount of AR
activity was much higher in IMCD cells than in IS
cells, however in both cell types AR-activity was
about four times higher at 600 mosmol/l than at
300 mosmol/l. Also, in rabbit PAP-HT25 cells, AR
activity was 4-fold higher at 500 mosmol/l than at
300 mosmol/l [18].
The most surprising result, however, was the
significantly higher activity of SDH in IS cells at
600 than at 300 mosmol/l. One explanation for this
higher SDH activity might be that higher sorbitol
levels at 600 mosmol/l induce SDH activity. As AR
activity is stimulated more than two times stronger
than SDH activity by increasing extracellular
osmolarity, osmotic regulation is still possible in
IS cells.
In former experiments with homogenates of rat renal
inner medulla, activity of SDH increased from 0.84 to
1.26 U/g protein under diuretic conditions [5]. Our in
vitro data are in contrast to this rise in SDH activity at
lower osmotic conditions (diuresis). SDH activities in
these in vivo experiments were very low and nearly at the
detection limit; nevertheless, it cannot be excluded that
a different regulation of SDH activity in vitro from
in vivo exists.
Parallel significant increase of sorbitol synthesis
(increase of AR activity) and sorbitol degradation
(increase of SDH activity) has never been described
before. At first, elevated SDH activity seems to be
opposite to effective osmotic adaptation. However,
together with the lower absolute amount of AR
activity in IS cells and the missing activity of SDH
in IMCD cells, this increase of SDH activity in IS
cells may indicate different distribution of enzyme
activity of sorbitol metabolism as discussed before
[5,11].
Recently, an increase of mRNA encoding for AR has
been shown under anti-diuretic conditions (dDAVPtreatment) in rat kidney IS cells by in situ hybridization
[19]. In the same experiments there was no alteration of
SDH mRNA expression in these cells. Additionally,
these investigators demonstrated a significant rise of
AR-mRNA, but not SDH-mRNA in isolated IMCD or
isolated IS cells after increasing osmolarity from 300
to 600 mosmol/l [13]. In accordance with the data, we
2260
could demonstrate an increase of AR-mRNA with
increasing osmolarity but no significant change of SDHmRNA in our experiments using semi-quantitative
RT–PCR. Therefore, in IS cells as well as in IMCD cells
[10] or PAP-HT25 cells [2], higher osmolarity results in
higher levels of AR-mRNA.
When we characterized the time course of AR and
SDH activity after raising osmolarity from 300 to
600 mosmol/l, we noticed steady-state level for both
enzymes after 6 days. Half maximal increase of
enzyme activity could be observed after 2–3 days
for AR and 3 days for SDH. The time course for
AR activity is similar in IMCD cells (half maximal
activity 3 days [10]) or PAP-HT25 cells (half maximal
activity 2 days [18]). There are no data available on
time course of SDH activity in other renal medullary
cells. Steady state reached for AR and SDH after 6
days corresponded to steady state reached for sorbitol
concentration.
In our experiments with IS cells lowering osmolarity
from 600 to 300 mosmol/l resulted in a rapid decrease of
enzyme activity with half maximal reduction after 1
(SDH) or 2 days (AR). After reduction of osmolarity
from 600 to 300 mosmol/l, decrease of AR activity in
PAP-HT25 cells was slower with half maximal reduction lasting 3–4 days [8]. In these experiments SDH
activity was at the lower limit of detection and showed
a small decrease within 9 days.
In our experiments with IS cells the amount of
AR-mRNA reached a maximum after 24–48 h and
decreased after 4 and 6 days. The time course of ARmRNA is similar in IMCD-cells or PAP-HT25 cells
with a maximum within 24 h and a decrease at longer
incubation times [2,10]. In all these cell types the
increase of AR mRNA precedes the increase of
AR activity. Therefore, the concept of osmotic regulation of AR via activation of a so-called osmotic
response element [20], which induces synthesis of ARmRNA followed by increased AR protein synthesis and
activity [2] should also fit to osmoregulation of AR in IS
cells.
However, neither we nor others [13,19] could demonstrate osmotic up-regulation of mRNA encoding
for SDH. On the other hand we could demonstrate
significant up-regulation of SDH activity by increasing
osmolarity and down-regulation with decreasing osmolarity. Therefore, regulation of SDH activity seems to
be independent of regulation of SDH-mRNA. At the
moment there are no data available on the mechanism
of osmotic regulation of SDH, which remains to be
clarified.
In summary, we could demonstrate for the first time a
cell type in which both enzymes of sorbitol metabolism
are stimulated and decreased in parallel. As AR activity
is stimulated more than two times stronger than SDH
activity by increasing extracellular osmolarity, osmotic
regulation by changing concentrations of sorbitol is
possible in IS cells. Whereas up-regulation of AR
activity in these cells is preceded by an increase of ARmRNA, up-regulation of SDH activity is independent
from changes in SDH-mRNA. Rat IMCD as well as IS
J. Steffgen et al.
cells react to an increase in osmolarity from 300 to
600 mosmol/l by increasing concentrations of glycerophosphorylcholine, betaine, myo-inositol and sorbitol
[13]. Our results support the contribution of sorbitol
metabolism to handling of osmotic stress in rat renal
papillary IS cells.
Conflict of interest statement. None declared.
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Received for publication: 30.8.01
Accepted in revised form: 28.5.03