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

1Abteilung Nephrologie und Rheumatologie, Georg-August-Universität Göttingen, Göttingen, Germany

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: jsteffgen{at}gmx.de.



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
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 µmol/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



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
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 [5,810].

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 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
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
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-N'-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 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.

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 + H+. 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 µmol/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 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 (5'-ACTGCCATTGCAAAGGCATCGTGGT-3'), AR antisense (5'-CCCCCATAGGACTGGAGTTCTAAGC-3'), SDH sense (5'-GGTGGAAAGTGTGCTGGGGA-3') and SDH antisense (5'-GGGGTTCTGGGTCATTGGGG-3') 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 ß-actin at different osmolarities. On preliminary experiments, raising osmolarity from 300 to 600 mosmol/l increased the amount of ß-actin mRNA identified by RT–PCR 1.6-fold, indicating that ß-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 Multi-Analyst Software (Bio-Rad, CA).

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
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
IS cells were isolated from rat renal inner medulla as described in the Subjects and methods. At steady-state conditions (14 days incubation) at 300 mosmol/l sorbitol concentration in these IS cells was 4.4 ± 0.3 µmol/g protein. After raising osmolarity with NaCl to 600 mosmol/l (Figure 1A) the steady-state sorbitol concentration increased to 78 ± 3.6 µmol/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.



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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.

 
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 600 to 300 mosmol/l sorbitol concentration was also close to steady-state level.



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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.

 
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 at 600 mosmol/l, whereas SDH activity could not be detected in cultivated IMCD cells.



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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.

 
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).



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Fig. 4. Expression of AR-mRNA detected by RT–PCR in homogenates of rat renal IS cells at 300, 450 and 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.

 
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.



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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.

 
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 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.



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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.

 
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.



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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
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
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, myo-inositol 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 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 (dDAVP-treatment) 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 could demonstrate an increase of AR-mRNA with increasing osmolarity but no significant change of SDH-mRNA 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 AR-mRNA 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 AR-mRNA 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 AR-mRNA, up-regulation of SDH activity is independent from changes in SDH-mRNA. Rat IMCD as well as IS 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.



   Notes
 
*Present address: Abteilung Klinische Forschung, Boehringer-Ingelheim Pharma GmBH & Co KG, Biberach an der Riss, Germany Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 30. 8.01
Accepted in revised form: 28. 5.03





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