Polyol Pathway Mediates High Glucose-Induced Collagen Synthesis in Proximal Tubule

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Kidney International, Vol. 45 (1994), pp. 659—666

Polyol pathway mediates high glucose-induced collagen synthesis in proximal tubule

ANTHONY J. BLEYER, PETER FUMO, EDWARD R. SNIPES, STANLEY GOLDFARB, DAVID A. SIMMONS, and FUAD N. ZIYADEH

The Renal-Electrolyte and Hypertension Division, the Diabetes Section, and the Penn Center for Molecular Studies of Kidney Diseases, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA

Polyol pathway mediates high glucose-induced collagen synthesis in conclusion also supported by evidence from tissue culture proximal tubule. The poiyoi pathway in diabetes is activated in tissues that are not dependent on insulin for glucose uptake. To examine the [6-8]. role of the polyol pathway in renal extracellular matrix accumulation, Recent studies in streptozotocin-induced diabetes mellitus in we incubated murine proximal tubule cells in either normal or high the rat have provided convincing evidence that the early phase glucose concentration in the presence or absence of the aldose reduc- of kidney involvement is characterized by increased expression tase inhibitor sorbinil. Raising medium glucose from 100 to 450 mg/dl of collagen type IV in the proximal tubular epithelium [9]. We for 72 hours increased cell sorbitol levels sevenfold. Addition of 0.4mM sorbinil reduced sorbitol content to virtually undetectable levels ashave previously used proximal tubule epithelial cells main- measured by gas chromatography. Sorbinil (0.1 to 0.2 mM) also reduced tained in continuous cell culture in order to study the direct the secretion of collagens types IV and I in the high glucose concen- effects of high glucose concentration on growth and collagen tration after 48 to 72 hours but had no appreciable effect in the normal synthesis [10—12]. These cells elaborated increased amounts of glucose concentration. Concordantly, 0.1 mrt sorbinil inhibited the high glucose-induced stimulation of al(IV) and a2(I) mRNA levels without collagen types IV and I under the influence of high ambient affecting levels in normal glucose concentration. To study the role ofglucose [10, 11]. Concordantly, increased steady-state mRNA transcriptional activation of collagen genes, we transfected proximal levels for the al chain of type IV collagen and the a2 chain of tubule cells with a chioramphenicol acetyltransferase (CAT) reporter type I collagen were also demonstrated. Using nuclear run-off gene linked to the promoter and regulatory elements of ol(IV) gene. CAT activity increased several-fold in the cells grown in the high versus assays to measure in vitro gene transcription rate [10], and the normal glucose concentration; this transcriptional activation in culture chloramphenicol acetyltransferase (CAT) reporter gene assay media containing high glucose concentration was reduced by treatment [8, 10], collagen gene transcription rates were also enhanced of the cells with 0.1 m sorbinil. Thus, high ambient glucose activates under the influence of high ambient glucose. Thus, elevated the polyol pathway in proximal tubule cells, and may mediate the high glucose concentration stimulated collagen gene transcription in glucose-induced stimulation of gene expression for collagens types IV and 1. For type IV collagen, activation of the polyol pathway may alsoproximal tubule cells, and this effect contributed to increased involve, at least in part, transcriptional activation of cis-acting gene collagen synthesis and secretion. regulatory elements. The cellular mechanisms involved in the high glucose-in- duced transcriptional activation of collagen genes are unclear, but one such mechanism may involve the increased activity of the polyol pathway by high ambient glucose levels. Since its Diabetic nephropathy is characterized by cellular hypertro-identification, the role of the polyol pathway in the myriad phy in both glomerular and tubular compartments, progressivecomplications of diabetes mellitus has been the subject of accumulation of extracellular matrix components in the glomer-intense investigation [13, 14]. Aldose reductase, the first and ular mesangium, and thickening of the glomerular and tubular rate-limiting enzyme in this pathway, catalyzes the NADPH- basement membranes [1—SI. Albeit less well-recognized, pro-dependent reduction of hexose or pentose sugars to their gressive tubulointerstitial fibrosis is also a typical feature of the corresponding sugar alcohols, or polyols [1]. This metabolic disease, particularly when reduction in the glomerular filtrationconversion is of great functional significance in tissues which do rate (GFR) becomes apparent [2, 5]. Although capillary hyper-not require insulin for glucose uptake: the kidney, lens, retina, tension, an abnormal metabolic environment, and genetic pre-and peripheral nerves. Importantly, these organs are also the disposition may all participate in the pathogenesis of diabeticsites of significant diabetic complications. nephropathy, hyperglycemia per se remains an essential factor The role of the poiyol pathway in diabetic nephropathy has that is necessary for the occurrence of the kidney disease, a been the focus of increased attention. Goldfarb et al [15] have shown that sorbinil, an aldose reductase inhibitor, greatly diminished the elevated GFR in short-term streptozotocin- Received for publication July 8, 1993 induced diabetes in rats, presumably by increasing afferent and in revised form October 22, 1993 arteriolar resistance to normal levels. In a six-month random- Accepted for publication October 25, 1993 ized trial of 20 type I normo-albuminuric diabetic subjects, © 1994 by the International Society of Nephrology ponalrestat reduced renal hyperfiltration [16]. Ponalrestat also

659 660 Bleyeret a!: Sorbinil and renal collagen reduced glomerular prostaglandin production and urinary albu-sisted of Dulbecco's modified Eagle medium (DMEM: GIBCO min excretion in diabetic rats without altering GFR [17]. TheLaboratories, Grand Island, New York, USA) containing a long-term effects of polyol pathway inhibition on renal extra-physiologically normal D-glucose concentration of 100 mgldl cellular matrix accumulation have not been uniformly favor-(5.5 mM) supplemented with 10% inactivated fetal calf serum, able. The aldose reductase inhibitor Statil did not reduce the100 U/mI penicillin, 100 g/ml streptomycin and 2 m addi- magnitude of glomerulosclerosis in diabetic rats after seventional L-glutamine. months despite normalization of erythrocyte sorbitol levels with this drug [18]. Neither tolrestat [191 nor ponalrestat [20] Growth conditions for poiyoi measurements and collagen reduced the thickening of the glomerular basement membrane mRNA extraction in six-month old severely diabetic rats. Ponalrestat [21] also had For each experiment, 2 X 106 MCT cells were plated into no effect on whole kidney mRNA encoding the a! chain of typeeach of four 75 cm2 flasks, and the cells were maintained in the IV collagen in severely diabetic rats. However, it should begrowth media for several days until a confluent monolayer was noted that the mRNA for this chain decreased with age in bothachieved. The cells were rested for 24 hours in serum-free normal and untreated diabetic animals [21]. On the other hand,DMEM containing 100 mg/dl D-glucose. The media were then Patel and Boot-Handford [22] showed diminished al(IV)changed to serum-free DMEM containing either 100 or 450 mRNA levels in diabetic animals treated with sorbinil for onemgldl glucose concentration, without or with 0.1 to 0.4 ifiM month. A decrease in width of the glomerular basement mem-sorbinil, and the cells were harvested after two to three days. brane has been reported in long-term diabetic rats fed sorbinilThese relatively high concentrations of sorbinil were chosen in [23]. These varying results may relate to the difficulty inorder to achieve effective high levels of the drug for up to 72 examining the effects of the polyol pathway in whole kidney,hours in culture and to assure maximal inhibition of aldose the likelihood that total inhibition of aldose reductase activityreductase. These doses did not affect cell viability as assessed was not achieved, the differences in duration and severity of grossly by phase-contrast microscopy nor was there significant diabetes, and the characteristics of various animal models. inhibition of cellular proliferation. To further examine the possible role of the polyol pathway in collagen biosynthesis in cultured proximal tubule cells, we have Fructose and sorbitol measurements undertaken a series of experiments showing that high glucose stimulates the activity of the polyol pathway as reflected by Polyols were measured as previously reported [14]. Cell marked intracellular sorbitol accumulation. Both the increasedmonolayers were washed three times with phosphate-buffered sorbitol production and the accompanying increased collagensaline (PBS), and the cells were then lysed with ice-cold biosynthesis were abrogated by treatment with sorbinil. Thusdeionized water. A known amount of alpha-methyl mannose these findings are consistent with the hypothesis that increasedpyranoside was added to each flask before lysis to be used as an activity of the polyol pathway may be involved in mediatinginternal standard for subsequent chromatographic assays. After some of the early distinctive changes of hyperglycemia in thelysis, the cells were centrifuged at 4°C for 20 minutes, and the supernatants were collected and stored at —20°C prior to kidney. chromatographic measurement. Barium zinc filtrates of lysed Methods cells were prepared by the method of Somogyi [281 and lyoph- ilized. The trimethylsilyl derivatives were extracted with hex- Cell culture ane and injected on packed columns of 3% SE-30 on 80/100 The mouse cortical tubule (MCT) cell line was prepared fromGasChrom Q(Supelco)in a Hewlett-Packard 5890 gas chro- kidney cortex of normal SJL mice [24]. After tubule microdis-matograph by using flame ionization detectors and a model 3392 section by one of us (S.G.), proximal tubule cells in primaryintegrator. For each experimental condition duplicate flasks culture were immunoselected using specific antibodies againstwere maintained in order to determine the number of cells. the 3M-i glycoprotein, a tubular target antigen for autoimmunePolyol levels per 106 cells were reported. nephritis. Transformation with a non-replicating, non-capsid forming strain of SV-40 resulted in immortalization of the cell Radioimmunoassay of collagen secretion line. We [10—12, 25, 26] and others have extensively character- Details of the tissue culture procedure were described else- ized this cell line as recently reviewed [27]. In summary, thewhere [10]. To measure collagen secretion in the conditioned characteristics of these cells indicating their proximal tubulemedia, the cells were grown in 24-well plates in the presence of origin include: positive staining for alkaline phosphatase and25 sg/ml L-ascorbic acid and 25 g/ml f3-aminopropionitrile, a cytokeratin; surface projections on electron microscopy consis-cross-linking inhibitor. After 48 or 72 hours, supernatants were tent with brush border villi; high affinity binding sites forremoved from each plate, transferred into several U-bottom angiotensin II, parathyroid hormone, insulin, insulin-likepolyvinyl chloride 96-microtiter wells (Costar, Cambridge, growth factor, and epidermal growth factor; and specific trans-Massachusetts, USA), and frozen at —20°C for subsequent port systems such as sodium-proton antiport and sodium-solid-phase radioimmunoassay of soluble collagens. Standard phosphate symport. Whether MCT cells resemble more thecollagens types IV and I were dispensed into polyvinyl chloride convoluted or the straight segment of the proximal tubule haswells at various concentrations for comparison with superna- not been established. tants from MCT cells. The wells were dried overnight at 4°C, MCT cells were maintained in a humidified atmosphere of 5%blocked with 4% bovine serum albumin (BSA), and incubated CO2 at 37°C and were passaged every 72 hours. For thewith 1:100 dilution of affinity-purified polyclonal rabbit anti- purposes of these studies the growth media of the cells con-type IV and anti-type I collagen antibodies (gifts of Dr. Antonio Bleyer et a!: Sorbinil and renal collagen 661

Martinez-Hernandez, Thomas Jefferson University, Philadel-under different culture conditions as described above. The next phia, Pennsylvania, USA). Binding of these primary antibodiesday, the cells were passed into several sterile petri dishes and was measured using '251-labeled anti-rabbit antibodies (10were allowed to adhere and grow in 100 mg/dl glucose over- cpmlwell) in 4% BSA. Binding of supernatants is comparednight. This maneuver assured that the transfection efficiency with known standards as described previously [10, 24]. Colla-was equivalent among the different petri dishes before the gen secretion was expressed per 106 cells. experimental manipulation. The media were then changed to fresh serum-free DMEM containing 100 or 450 mgldl D-glucose, RNA isolation and collagen mRNA measurements without or with 0.1 m sorbinil, and after 48 hours the cells After growth in tissue culture flasks, the cells were washedwere washed several times with ice-cold PBS. Two ml of a with PBS then lysed with 0.5% NP-40 in Tris-EDTA buffer.solution of 40 mrvi Tris. HCI (pH 7.5), 10 mrvi EDTA, and 150 Replicates of 0.2 x 106 cell-equivalents of cytoplasmic RNAmM NaCl were applied to the monolayer, which was scraped off were denatured into a solution of 40% formalin in l2x SSCthe flask, pelleted, and subjected to three cycles of freeze-thaw (SSC is 0.15 M sodium chloride and 0.015 M sodium citrate, pHlysis. Equal amounts of cytosolic protein were mixed in a 7.0) at 65°C for 15 minutes, then centrifuged, and supernatantsreaction mixture of 180 jsl containing 20 sg of acetyl-Co-A, 1 spotted onto ZetaBind filters (CUNO Laboratory Products,Ci '4C-chloramphenicol (54 pCilmmol, Amersham, Arlington Meriden, Connecticut, USA) by use of a Hybridot apparatusHeights, Illinois, USA), and 250 mri Tris. HCI (pH 8.0). The (Bethesda Research Laboratories, Bethesda, Maryland, USA).reaction was run at 37°C for 60 minutes. Acetylated chioram- The dried blots were then pre-hybridized at 42°C for 16 hours inphenicol derivatives were extracted in ethyl acetate by thin fluid containing 0.5 M phosphate buffer (pH 7.2), 150 g/m1 Polylayer chromatography (TLC) using 25-mm silica gel plates and A, 150 g/ml denatured, sheared salmon sperm DNA, 7% SDS,chloroform:methanol (95:5) as carrier. TLC plates were then 1% BSA, and 1 msi EDTA. The blots were then labeled inautoradiographed and bands were cut out and counted for hybridization fluid containing 32P-labeled cDNA probes forradioactivity. CAT activity was expressed as percent '4C chlor- collagens al(IV) and a2(I), and for glyceraldehyde-3-phosphateamphenicol acetylated per hour per unit protein. dehydrogenase (GAPDH) (specific activity, 0.5 to 1.0 x l0 cpm/sg) for 16 hours at 42°C. The cDNA inserts used as probes Statistics were isolated by endonuclease digestion from their respective For each individual experiment, the mean of replicate deter- plasmids [10, 11], which were: a 0.66kb PstI fragment encodingminations was calculated. The data were presented as means (± murine al(IV) collagen gene from pcIS, a 0.85 kb XhoI fragmentSE) with N indicating the number of different experiments. containing the promoter-proximal exon region encoding murineStatistical analysis between two groups was performed by 2(I) collagen from pAZ1002 and a 1.3 kb PstI fragmentpaired or unpaired t-tests, as appropriate. Differences among encoding rat GAPDH gene from pBR322. The probes wereseveral groups were tested by analysis of variance. P < 0.05 labeled with 32P-dCTP (>3,000 Ci/mmol) by the random hex-was considered a significant difference. amer priming technique [10, 11]. The filters were washed twice for 15 minutes in 3x SCC, 0.5% SDS at 55°C and then Results autoradiographed (Kodak XAR-5 film) with intensifying screens at —70°C for one to three days (GAPDH) or 10 to 20 days Polyol measurements (collagens). After probing with the collagen cDNA, the filters Cells exposed to 100 mg/dl D-glucose contained very low but were washed for one to two hours at 65°C with a solutiondetectable sorbitol levels (Fig. lA). However, when cells were containing 5 mM Tris, 0.2 mM EDTA (pH 8.0), and 5% sodiumexposed to 450 mgldl D-glucose for 72 hours, there was more pyrophosphate to remove signal, and then were also rehybrid-than sevenfold increase in cell sorbitol levels. Importantly, this ized with GAPDH cDNA as an internal standard to control forincrease in cellular sorbitol content which was induced by the the amount of RNA loading. Relative mRNA levels wereelevated glucose concentration was abolished when the culture quantitated by densitometric scanning (Hoefer Instruments,medium contained 0.4 ms sorbinil (Fig. lA). Sorbinil treatment San Carlos, California, USA) followed by area integration. reduced sorbitol content to barely detectable levels in both normal and high glucose concentration. In other experiments Chioramphenicol acetyltransferase (CAT) reporter gene assay(not shown), 0.1 mst sorbinil reduced the high glucose-stimu- The transient transfection method and CAT enzyme assaylated sorbitol content by approximately 90%. were essentially as previously described [29]. The plasmids In contrast, Figure lb shows that the fructose content, used were: pAoCAT, containing the promoterless CAT gene;measured concomitantly with sorbitol, was only moderately pSV2CAT, containing the CAT gene linked to the SV-40higher in the cells cultured in 450 mgldl D-glucose. Moreover, promoter and enhancer; and p184 (gift of Dr. P. Killen, Univer-there was no significant effect on fructose content when the sity of Michigan), containing the CAT gene linked to a largecells were grown in 0.4 m sorbinil in either normal or high genomic region which includes the promoter common to theglucose concentration. al(IV) and a2(IV) genes, and a regulatory segment in the first intron of the al(IV) gene [10, 30]. Ten micrograms of plasmid Collagen secretion DNA were mixed with 500 d of 124 mvt CaCl2. This solution Results after 48 or 72 hours of culture were similar and was then added dropwise to 500 pJ of 50 mrvi HEPES (pH 7.1),therefore were pooled; also, the similar results of sorbinil 280 msx NaCl, and 1.5 mr.i Na2HPO4 with precipitation occur-treatment at 0.1 or 0.2 mri were pooled (Fig. 2). The secretion ring over 30 minutes at 22°C. The DNA-calcium phosphaterates for collagens types IV and I in cells grown for 48 to 72 precipitate was then applied dropwise to cultured MCT cellshours in 450 mgldl D-glucose were both significantly stimulated 662 Bleyeret al: Sorbinil and renal collagen * A * A Cell sorbitol 30 6 .9

C) Cl)4a) 20 O.)cDQ) T

> 10 Cl) >,0. I— 0 100 450 0 100 450 Glucose, mg/dl Glucose, mg/dl B

210 600 180 0 500 150 a) 400 120 I - 300 90 o 60 0 200 U- 0.I) 30 100

0 II, 0 100 450 100 450

Glucose, mg/dl Glucose, mg/dl Fig. 1. Effects of high glucose and sorbinil on cell polyol content in Fig.2. Effects of high glucose and sorbinil on collagen secretion in proximal tubule cells in culture. Cell sorbitol (A) and fructose (B) were proximal tubule cells. Cells were cultured for 48 or 72 hours in media measured in cells cultured for 72 hours in serum-free DMEM containing containing 100 or 450 mg/dl glucose, without () or with addition of 0.1 either 100 or 450 mgldl glucose, without () or with addition of 0.4mM or 0.2 mi sorbinil (U). Radioimmunoassay of collagens (type IV in A, sorbinil (U). Data (mean SE) are expressed per 106cells.*p < 0.001 type I in B) was performed on conditioned supernatants. Data were by ANOVA. pooled (mean SE, N =11)and expressed per 106 cells. *P < 0.05 by ANOVA.

(Fig. 2) as previously reported [10, 11]. High glucose increased Collagen gene expression type IV collagen by approximately 80%, and type I collagen by Figure 3A demonstrates that the mRNA expression of the al approximately 50%. When 0.1 to 0.2 mai sorbinil was added tochain of type IV collagen relative to that of GAPDH mRNA was the media, there were significant reductions in the high-glucose-more than 70% higher in the cells grown for 48 hours in 450 induced stimulation of collagen secretion rates to levels whichmgldl D-glucose than in 100 mg/dl D-glucose (N =3,each in were comparable to those in 100 mgldl glucose. On the otherduplicate). This response to high glucose in this series of studies hand, collagen secretion rates in cells exposed to 100 mgldlwas somewhat less robust than in our previous studies [10, 11]. glucose were not significantly altered by the addition of sorbinil. Nevertheless, and as seen in Figure 3A, the glucose-induced As expected, the production of type IV collagen in proximalincrement in the level of al(IV) mRNA was abrogated when the tubule epithelial cells was quantitatively much greater than ofcells were grown in the presence of 0.1 mt.s sorbinil; the level type I collagen [10, 11]. attained was similar to that in cells cultured in 100 mgldl Bleyer et a!: Sorbinil and renal collagen 663

A al(IV) B a2(l) 2 2

1.5 C) 2C cc E >C)

CC C) cc

0 0 100 450 100 450

Glucose C mq/dI Glucose, my/dI

Glucose Sorbinil (mg/dl) (l mM) 100 - S. Fig. 3. Effects of high glucose and sorbinil on steady-state collagen 450 mRNA levels in proximal tubule cells. Cells were cultured for 48 hours in media containing 100 or 450 mg/dl glucose without () or with addition of 0.1 ms sorbinil (U. Dot-blot RNA hybridization followed by densitometric scanning of autoradiographs were performed as described in Methods. The data are from three experiments each performed in duplicate for nl(IV) collagen (A) and +...*.. a2(I) collagen (B). Autoradiographs of representative blots from one 450 experiment are provided in C. The collagen mRNA level relative to + •• •ft the corresponding GAPDH mRNA level in control cells (100 mgldl a1(IV) ;(I) GAPDH glucose, no sorbinil added) was assigned a value of unity. D-glucose. Sorbinil did not affect the mRNA levels in normalto 1.5-fold the level in 100 mgldl D-glucose. These results glucose concentration. indicate that high glucose concentration leads to transcriptional Figure 3B depicts the results of studies on a2(I) collagenactivation of the al(IV) gene in proximal tubule cells, likely mRNA determination. Cultures for 48 hours in high glucosethrough increased activity of the polyol pathway. Production of increased a2(I) mRNA levels relative to GAPDH mRNA bylarge amounts of acetylated chioramphenicol by pSV2-trans- more than twofold, and 0.1 m sorbinil normalized these levelsfected cells in normal glucose concentration is shown in lane 1 (N =3,each in duplicate). as the positive control (Fig. 4). As the negative control, Representative mRNA dot-blot hybridizations from one ex-pAo-transfected cells in normal glucose concentration yielded periment are shown in Figure 3C. virtually no acetylated products (lane 2, Fig. 4). As was demonstrated previously [10], the high glucose concentration in CAT reporter gene assay the culture medium had no significant effect on the CAT Increased collagen gene transcription may account, at least inreporter gene assay of cells transfected with either the positive part, for increased mRNA levels at steady state. We have(pSV2) or negative (pAo) control plasmids (data not shown). previously reported that 450 mg/dl D-glucose increased the transcription rates for collagens types IV and I as determined Discussion by nuclear run-off assay in MCT cells [10]. Using CAT reporter To begin to assess the importance of the polyol pathway in gene assays, we also demonstrated transcriptional activation bythe tubulointerstitial changes of diabetes mellitus we focused in the high glucose concentration [8, 10]. That this effect of highthis study on the high glucose-induced stimulation of collagen glucose may also be mediated by increased activity of thebiosynthesis in cultured proximal tubule cells, and the effects polyol pathway is supported by the observation of an inhibitorythereon of the aldose reductase inhibitor sorbinil. We first effect of sorbinil on transcriptional activation (Fig. 4). Produc-showed a sevenfold increase in sorbitol content in cells grown tion of acetylated chloramphenicol was increased by 3.5-fold inin high glucose that was totally abrogated by the addition of p184-transfected cells grown in 450 mgldl D-glucose comparedsorbinil, indicating marked activation of the polyol pathway in with cells grown in 100 mgldl (N =3).The addition of 0.1 mMproximal tubule cells exposed to elevated ambient glucose. We sorbinil to the media containing 450 mgldl D-glucose resulted inalso noted that increasing the glucose concentration in the a marked decrease in the production of the acetylated product,culture medium resulted in significant increases in the rate of 664 Bleyeret al: Sorbinil and renal collagen

Fig. 4. Effects of high glucose and sorbinil on transcriptional activation of type IV collagen in proximal tubule cells. Details of transient transfection and CAT enzyme activity are given in Methods. Cells were cultured for 48 hours in media containing 100 or 450 mg/dl glucose without or with addition of 0.1 mrs sorbinil (SN). The autoradiographs are for one representative experiment showing TLC- separated acetylated (that is, CAT activity) and unacetylated '4C-chloramphenicol. Plasmids used for transfection, indicated at bottom of lanes, were pSV2, a positive- control plasmid containing CAT gene linked to SV-40 promoter and enhancer elements; pAo, a negative-control plasmid containing CAT gene without regulatory elements; and p184, containing the CAT gene linked to a genomic region that includes the promoter common to the al(IV) and a2(IV) collagen genes, and a regulatory segment in the first intron of the al(IV) gene. Cells transfected with pSV2 and pAo were grown in 100 mgldl pSV2 pA0 100 450 100 450 glucose, and CAT activity for either plasmid + + was not affected by the high glucose SN SN concentration (not shown). secretion of collagen types IV and I, accompanied by elevateda nonspecific effect of sorbinil. Sorbinil also had no inhibitory levels of the respective mRNAs encoding al(IV) and a2(1).effect on the expression of the control GAPDH mRNA. A Importantly, the increments in collagen secretion and messagesurprising finding in our study, which has also been noted levels were prevented by the addition of sorbinil. Furthermore,before [33], was that sorbinil did not reduce fructose content, as using the CAT reporter gene assay in transient transfectionwould be expected if fructose was synthesized directly from experiments, we showed transcriptional activation of the colla-sorbitol. This may indicate that fructose production could also gen type IV gene under the influence of high glucose and thisbe governed by metabolic processes other than the polyol response was also largely prevented by the addition of thepathway. aldose reductase inhibitor. These findings indicate that the In normal kidneys, most aldose reductase activity resides in observed stimulation by high glucose of collagen biosynthesis inthe inner medulla and papilla, where aldose reductase-derived proximal tubule cells is likely to be mediated by increasedsorbitol synthesis probably serves an osmoprotective function activity of the polyol pathway. to maintain cell volume in a hyperosmolar environment [34—36]. The sorbinil-induced decrease in cellular sorbitol content inSignificant levels of aldose reductase are present in mesangial media with high glucose concentration reflects a markedlycells and epithelial podocytes in the glomerulus, and in the enhanced flux of glucose through the polyol pathway, likelydistal convoluted tubule [31, 37]. The presence of aldose catalyzed by aldose reductase. Although not directly measuredreductase in the proximal tubule under normal conditions is in these studies, the relatively low basal enzymatic activity ofunclear. Corder et al [35] noted aldose reductase activity in aldose reductase in proximal tubule cells under control condi-proximal convoluted tubules derived from nondiabetic kidney, tions [31] was probably markedly stimulated when the cellsbut most other studies have not borne this out [31, 32, 37, 38]. were grown in high glucose concentration. However, it shouldHowever, ample evidence exists for the presence of aldose be noted that the activity of other enzymes (such as aldehydereductase in cultured proximal tubular epithelium. Renal corti- reductase) could also have been stimulated. Aldehyde reduc-cal epithelial cells [39] and proximal tubule cells [40] accumu- tase, an enzyme normally present in high amounts in proximallated sorbitol upon exposure in culture to high ambient glucose, tubule epithelium [32], can also reduce glucose to sorbitol andand this effect was prevented by aldose reductase inhibition. its activity can be inhibited by relatively high concentrations ofPetrash et al [41] also demonstrated that both high glucose and sorbinil. Nevertheless, and irrespective of the relative contri-hypertonic media increased sorbitol accumulation in human bution of either enzyme system to the observed effects of highproximal tubule cells. Aldose reductase gene expression was glucose in the proximal tubule, the response to sorbinil in thedetected in these cells by Northern analysis using a probe which cells exposed to high glucose provide evidence for increaseddid not cross react with aldehyde reductase. Interestingly, flux of glucose through the polyol pathway. Furthermore, thealthough high ambient glucose increased aldose reductase bio- absence of any significant effect of sorbinil in cells grown inactivity [41], it did not change the amount of enzyme or its media with physiologic concentrations of glucose indicate thatmessage level. the observed changes in collagen biosynthesis were not due to Although the glomerulus has been the focus of attention in Bleyer et a!: Sorbinil and renal collagen 665 diabetes mellitus, the tubulointerstitium has also been recog-itor may ameliorate this fibrogenic process. Our in vitro system nized as a target of the disease [2]. In fact, the degree ofallows us to look directly at the effects of the increased activity interstitial expansion is thought to correlate very well withof the polyol pathway on collagen synthesis independent of function and prognosis in many renal diseases including dia-hemodynamic or hormonal changes known to occur in diabetes betic nephropathy [4, 5, 42—44]. Moreover, diffuse tubularmellitus. However, additional in vivo studies are necessary to basement (IBM) thickening is a hallmark of the disease, andevaluate the efficacy and therapeutic potential of aldose reduc- this lesion is more likely to be due to the altered metabolictase inhibitors in diabetic nephropathy. milieu than to genetic predisposition, as suggested by a study of identical twins discordant for diabetes mellitus in which TBM Acknowledgments thickness was greater in the diabetic than the nondiabetic This work was supported in part by the National Institutes of Health sibling [45]. Animal studies have corroborated the finding of(NIH) Grants DK-39565, DK-45191, DK-44513, and Training Grant increased matrix accumulation in the tubular epithelium of theDK-07006, and by Pfizer, Inc. P. Fumo is a recipient of a National diabetic kidney. In situ nucleic acid hybridization studies inResearch Service Award from NIH. E. R. Snipes was supported by a Fellowship from the National Kidney Foundation. The authors grate- streptozotocin-diabetic rats [9] have localized the increasedfully acknowledge the support and advice of Dr. Peter J. Oates of collagen al(IV) mRNA levels to proximal tubular epithelium inPfizer, Inc. the deeper cortical and outer medullary portions of the kidney. It is also of interest to note that kidney expression of type I Reprint requests to Fuad N. Ziyadeh, M.D., 700 Clinical Research collagen mRNA was modestly, but not significantly, increasedBldg., Renal-Electrolyte and Hypertension Division, University of [9]. Desjardins et al [46] using protein A-gold immunocyto-Pennsylvania, 422 Curie Blvd., Philadelphia, Pennsylvania 19104-6144, chemical techniques have also demonstrated increased staining USA. for tsl(IV), a2(IV) and a3(IV) collagen chains along the TBM in References streptozotocin-diabetic rats. The mechanism of polyol pathway-mediated increase in 1. ZIYADEH FN, GOLDFARB S, KERN EFO: Diabetic nephropathy: collagen synthesis remains to be elucidated, but several possi- Metabolic and biochemical mechanisms, in Contemporary Issues in bilities can be entertained including stimulation of transforming Nephrology (vol 20), Churchill Livingstone, New York, 1989, pp growth factor-/3 [26], activation of protein kinase C (PKC), or 87—1 13 2. ZIYADEH FN, GOLDFARB 5: The renal tubulointerstitium in diabe- changes in myo-inositoi metabolism or cellular redox potential tes mellitus. Kidney mt 39:464-475, 1991 [1, 8]. We have not thus far been able to demonstrate an 3. STERNBERG M, COHEN-FORTERE L, PEYROUX J: Connective tissue inhibitory effect of sorbinil on glucose-induced stimulation of in diabetes mellitus: Biochemical alterations of the extra-cellular transforming growth factor-a mRNA in MCT cells (unpublished matrix with special reference to proteoglycans, collagens and basement membranes. Diab et Metab 11:27—50, 1985 observations). On the other hand, high glucose stimulates in 4. MAIJER SM, STEFFES MW, ELLIS EN, SUTHERLAND DER, glomeruli the de novo synthesis of the PKC activator diacyl- BROWN DM, GOETZ FC: Structural-functional relationships in glycerol (DAG) [47], which in turn leads to transcriptional diabetic nephropathy. J Clin Invest 74:1143—1155, 1984 activation of collagen genes [48]. High glucose also leads to 5. LANE PH, STEFFES MW, FIORETTO P, MAUER SM: Renal intersti- increased cytosolic NADH/NAD and lactate/pyruvate ratios tial expansion in insulin-dependent diabetes mellitus. Kidney mt 43:661—667, 1993 in glomeruli which can be prevented when the diabetic rats are 6. Avo SH, RADNIK RA, GLASS WF II, GARONI JA, RAMPT ER, fed the aldose reductase inhibitor toirestat [33]. Aldose reduc- APPLING DR, KREISBERG JI: Increased extracellular matrix synthe- tase utilizes NADPH to reduce glucose to sorbitol which is then sis and mRNA in mesangial cells grown in high-glucose medium. oxidized to fructose by sorbitol dehydrogenase, utilizing NAD Am J Physiol 260:F185—Fl91, 1991 7. ZIYADEH FN: The extracellular matrix in diabetic nephropathy. as a hydrogen acceptor. Altered cellular myo-inositol metabo- Am J Kidney Dis 22:736—744, 1993 lism may be playing a role as well. In a previous study on 8. ZIYADEH FN: Renal tubular basement membrane and collagen type cultured proximal tubule cells, we showed that elevating the IV in diabetes mellitus. Kidney mt 43:114—120, 1993 myo-inositol concentration in the high-glucose medium in- 9. 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