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.
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Abstract |
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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 23 (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
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Introduction |
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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.
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Subjects and methods |
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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 1.0521.093 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 1.0811.093 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 Dulbeccos modified Eagles Medium (DMEM) and Nutrient Mix Hams 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 Hams 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, RTPCR
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 manufacturers 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 RTPCR 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 Students 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.
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Results |
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Discussion |
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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 RTPCR. 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 23 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 34 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 2448 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.
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Notes |
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References |
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