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Glucose-mediated induction of TGF-β1 and MCP-1 in mesothelial cells in vitro is osmolality and polyol pathway dependent.BackgroundGlucose is converted to sorbitol and then to fructose via the polyol pathway that has been implicated in the pathogenesis of organ damage. The contribution of the polyol pathway to mesothelial cell activation has, however, not been fully determined.MethodsThe effect of increasing glucose concentrations on transforming growth factor-β1 (TGF-β1) and monocyte chemoattractant protein-1 (MCP-1) secretion by human peritoneal mesothelial cells (HPMC) was examined. The importance of the polyol pathway was identified by its specific inhibition with an aldose reductase inhibitor.ResultsIncubation of HPMC with 5 to 100 mmol/L glucose resulted in an induction of aldose reductase mRNA and intracellular sorbitol accumulation accompanied by the induction of TGF-β1 and MCP-1 mRNA expression and protein secretion. Mannitol at the same concentrations also induced aldose reductase, TGF-β1 and MCP-1 mRNA and protein expression but at a lower level than glucose. Sorbinil dose-dependently reduced both intracellular sorbitol levels (79.8% reduction of 60 mmol/L d-glucose induced intracellular sorbitol with 100 μmol/L sorbinil (N = 3, P < 0.01) and glucose-induced TGF-β1 and MCP-1 secretion. Mannitol induced TGF-β1 and MCP-1 secretion was not reduced by sorbinil. The addition of 15 to 40 mmol/L sodium lactate, either alone or in the presence of d-glucose enhanced TGF-β1 and MCP-1 secretion, which was inhibited by sorbinil. In contrast, sodium pyruvate appeared to antagonize d-glucose–induced TGF-β1 and MCP-1 secretion.ConclusionThese data suggest that the polyol pathway and osmolality contribute to the regulation of HPMC function by glucose. Control of polyol pathway activation might reduce glucose-mediated damage to the peritoneal membrane and promote its long-term survival.

Gene expression and identification of gene therapy targets in diabetic nephropathy. A number of novel genes that are up-regulated in diabetic kidneys have been identified. Recently, transforming growth factor-βbgr; (TGF-βbgr;)–driven secreted proteins, i.e., connective tissue growth factor (CTGF) and gremlin, were identified. They are up-regulated in kidneys of diabetic animals and modulate the biology of mesangial cells. CTGF mediates TGF-βbgr;–induced matrix overproduction by the mesangial cells. Gremlin is a putative antagonist of bone morphogenetic protein-2 that blocks mesangial cell proliferation. Thus, gremlin may modulate the biology of mesangium by stimulating mesangial cell proliferation and in turn production of matrix. In addition, transcriptionally regulated kinases, serum glucocorticoid-regulated kinase and munc-13 have been identified. The former stimulates renal tubular Na+ transport and is involved in hyperfiltraion of diabetic kidneys by a Na+ transport feedback mechanism. Munc-13 has been shown to induce apoptosis in hyperglycemic state via diacylglycerol-activated, PKC-independent signaling pathway. Another pathway relevant to diabetic nephropathy is polyol pathway, where glucose is reduced to sorbitol by aldose reductase. Recently, a renal-specific reductase of the aldo-keto reductase family was isolated. It is up-regulated in diabetic mice, and this could serve as a suitable target for gene therapy in renal complications of diabetes. Several mitochondrial genome-encoded genes, such as, cytochrome oxidase and NADH dehydrogenase, are up-regulated in diabetic kidneys. A novel nuclear-encoded mitochondrial gene, i.e., translocase inner mitochondrial membrane 44 (Tim44), is up-regulated in diabetic kidneys, and it may also serve as another target for molecular therapeutic intervention at the core storage energy sites, i.e., mitochondria. In this review, these novel differentially regulated genes that respond to hyperglycemic stress are described, and they may serve as possible targets for gene therapy in the treatment of diabetic nephropathy.

3-Deoxyglucosone and AGEs in uremic complications: Inactivation of glutathione peroxidase by 3-deoxyglucosone. 3-Deoxyglucosone (3-DG) is accumulated not only in uremic serum but also in uremic erythrocytes. 3-DG rapidly reacts with protein amino groups to form advanced glycation end products (AGEs) such as imidazolone, pyrraline, and Nε-(carboxymethyl)lysine, among which imidazolone is the AGE that is most specific for 3-DG. In diabetes, hyperglycemia enhances the synthesis of 3-DG via the Maillard reaction and the polyol pathway and thereby leads to its high plasma and erythrocyte levels. In uremia, however, the decreased catabolism of 3-DG that may be due to the loss of 3-DG reductase activity in the end-stage kidneys may lead to a high plasma 3-DG level. The elevated 3-DG levels in uremic patients may promote the formation of AGEs such as imidazolone in erythrocytes, aortas, and dialysis-related amyloid deposits. Treatment with an aldose reductase inhibitor reduced the erythrocyte levels of 3-DG and AGEs such as imidazolone in diabetic uremic patients. This finding demonstrates an important role of the polyol pathway in the formation of erythrocyte 3-DG and AGEs. The erythrocyte levels of 3-DG are elevated in not only diabetic uremic but also nondiabetic uremic patients. 3-DG showed some cytotoxicities by inducing intracellular oxidative stress. In contrast, oxidative stress was demonstrated to cause accumulation of intracellular 3-DG. Recently, we have demonstrated that 3-DG inactivates intracellular enzymes such as glutathione peroxidase, a key enzyme in the detoxification of hydrogen peroxide. Thus, intracellular accumulation of 3-DG may enhance oxidative stress by inactivating the antioxidant enzymes. In conclusion, 3-DG may play a principal role in the development of uremic complications, such as dialysis-related amyloidosis, atherosclerosis, and enhanced oxidative stress.

of the earliest renal pathological changes in diabetes is Background. Thickening and reduplication of the tubular basement an increase in tubular basement membrane mass that membrane have been reported as early events in diabetic nephropathy. In this study, we have examined the polar requirements of proximal accompanies the development of renal hypertrophy [2]. tubular cells for the d-glucose‐stimulated accumulation of fibronectin These changes are strongly correlated with glycemic conand the mechanism by which this occurred, with particular emphasis trol and occur independently of the pathological changes on the polyol pathway. Methods. To determine the polarity of fibronectin generation in associated with progressive diabetic nephropathy [3]. It response to glucose, LLC-PK1 cells were grown on porous tissue culture suggests that alterations in renal structure may occur inserts. Monolayer confluence was determined by serial measurement of transepithelial resistance. Confluent cells were growth arrested by that are not specific to nephropathy but reflect a conseserum deprivation, and all experiments were performed under serum- quence of long-standing diabetes/hyperglycemia. We free conditions. and others have demonstrated that in vitro exposure of Results. Application of 25 m md -glucose to either the apical or basolateral aspect of LLC-PK1 cells led to fibronectin accumulation in human renal proximal tubular cells to 25 m md -glucose the basolateral compartment. This reached statistical significance 24 increased generation of type IV collagen and fibronectin hours following apical addition of glucose (2.6-fold increase compared [4‐6]. Our observations suggested that the accumulation with 5 m md -glucose, P 5 0.0025, N 5 6 vs. N 5 4 controls) and 12 hours after the basolateral addition of glucose (2.5-fold increase of these basement membrane constituents was related compared with 5 m md -glucose, P 5 0.03, N 5 6 vs. N 5 4 controls). to alterations in matrix degradation [5]. Exposure of cells to glucose at either their apical or basolateral aspect leads to accumulation of intracellular glucose and polyol pathway acti- In diabetes, proximal tubular cells may be exposed to vation, as assessed by sorbitol accumulation. The increase in fibronectin conditions of elevated glucose either apically as a result concentration in response to glucose was inhibited by the aldose reducof glycosuria or basely as a result of elevated interstitial tase inhibitor sorbinil. At a dose of 100 mm sorbinil, there was a 59% inhibition of fibronectin accumulation in response to apical glucose (P 5 tissue concentrations of glucose. Our studies on the mech0.004, N 5 3 sorbinil vs. N 5 4 controls) and a 66% inhibition in anism of glucose induction of transforming growth factor response to basolateral glucose (P 5 0.008, N 5 3 sorbinil vs. N 5 4 controls) 48 hours after the addition of the inhibitor. Furthermore, b1 (TGF-b1) in proximal tubular cells demonstrated that fibronectin accumulation was also demonstrated following both the induction of TGF-b1 mRNA was a polar phenomenon, apical and basolateral addition of 1 mm sorbitol, but not following the occurring only following the addition of glucose to the addition of 25 mm galactose to either aspect of the cells. Following the addition of sorbitol, there was a 2.8-fold increase in fibronectin 48 basolateral aspect of the cells [7]. hours after apical stimulation (P 5 0.01, N 5 3 treated vs. N 5 4 In this study, we have examined the polar requirements control) and a 2.27-fold increase following basolateral stimulation (P 5 0.04, N 5 3 treated vs. N 5 4 control) at 24 hours. of proximal tubular cells for the d-glucose‐stimulated Conclusions. In summary, these data demonstrate that fibronectin accumulation of fibronectin using the porcine LLC-PK1 generation in response to glucose was nonpolar in terms of application tubular cell line, grown in polarized culture on tissue of glucose but was polar in terms of fibronectin accumulation. The mechanisms of glucose-induced modulation of fibronectin were medi- culture inserts. We also examined the mechanism by ated by polyol pathway activation and were more specifically related which glucose led to the accumulation of fibronectin in to the metabolism of sorbitol to fructose.

Polyol pathway mediates high glucose-induced collagen synthesis in proximal tubule. The polyol pathway in diabetes is activated in tissues that are not dependent on insulin for glucose uptake. To examine the role of the polyol pathway in renal extracellular matrix accumulation, we incubated murine proximal tubule cells in either normal or high glucose concentration in the presence or absence of the aldose reductase inhibitor sorbinil. Raising medium glucose from 100 to 450 mg/dl for 72 hours increased cell sorbitol levels sevenfold. Addition of 0.4 m M sorbinil reduced sorbitol content to virtually undetectable levels as measured by gas chromatography. Sorbinil (0.1 to 0.2 m M ) also reduced the secretion of collagens types IV and I in the high glucose concentration after 48 to 72 hours but had no appreciable effect in the normal glucose concentration. Concordantly, 0.1 m M sorbinil inhibited the high glucose-induced stimulation of α1(IV) and α2(I) mRNA levels without affecting levels in normal glucose concentration. To study the role of transcriptional activation of collagen genes, we transfected proximal tubule cells with a chloramphenicol acetyltransferase (CAT) reporter gene linked to the promoter and regulatory elements of α1(IV) gene. CAT activity increased several-fold in the cells grown in the high versus normal glucose concentration; this transcriptional activation in culture media containing high glucose concentration was reduced by treatment of the cells with 0.1 m M sorbinil. Thus, high ambient glucose activates the polyol pathway in proximal tubule cells, and may mediate the high glucose-induced stimulation of gene expression for collagens types IV and I. For type IV collagen, activation of the polyol pathway may also involve, at least in part, transcriptional activation of cis -acting gene regulatory elements.

Molecular mechanisms of glucose action on angiotensinogen gene expression in rat proximal tubular cells. Background Clinical studies have shown that the angiotensin-converting enzyme (ACE) inhibitors or angiotensin II (Ang II) receptor antagonists decrease proteinuria and slow the progression of nephropathy in diabetes, indicating that Ang II plays an important role in the development of nephropathy. We have previously reported that high levels of glucose stimulate the expression of rat angiotensinogen (ANG) gene in opossum kidney (OK) proximal tubular cells. We hypothesized that the stimulatory effect of d(+)-glucose on the expression of the ANG gene in kidney proximal tubular cells is mediated via de novo synthesis of diacylglycerol (DAG) and the protein kinase C (PKC) signal transduction pathway. Methods Immortalized rat proximal tubular cells (IRPTCs) were cultured in monolayer. The stimulatory effect of glucose on the activation of polyol pathway and PKC signal transduction pathway in IRPTCs was determined. The immunoreactive rat ANG (IR-rANG) in the culture medium and the cellular ANG mRNA were measured with a specific radioimmunoassay and a reverse transcription-polymerase chain reaction assay, respectively. Results d(+)-glucose (25mm) markedly increased the intracellular levels of sorbitol, fructose, DAG, and PKC activity as well as the expression of IR-rANG and ANG mRNA in IRPTCs. These stimulatory effects of d(+)-glucose (25mm) were blocked by an inhibitor of aldose reductase, Tolrestat. PKC inhibitors also inhibited the stimulatory effect of d(+)-glucose (25mm) on the expression of the IR-rANG in IRPTCs. The addition of phorbol 12-myristate 13-acetate further enhanced the stimulatory effect of d(+)-glucose (25mm) on the expression of the IR-rANG in IRPTCs and blocked the inhibitory effect of Tolrestat. Conclusion These studies suggest that the stimulatory effect of a high level of d(+)-glucose (25mm) on the expression of the ANG gene in IRPTCs is mediated, at least in part, via the de novo synthesis of DAG, an activator of PKC signal transduction pathway.

Mesangial cell actin disassembly in high glucose mediated by protein kinase C and the polyol pathway. High glucose alters mesangial cell cytoskeletal structure and function. We postulated that high glucose causes mesangial cell filamentous (F) actin disassembly through a protein kinase C (PKC) mechanism involving the polyol pathway. Rat mesangial cells (passage < 10, N = 60/group) were growth-arrested and then cultured in glucose 5.6mM (NG), 15mM (MG) or 30mM (HG) for 48 hours, with or without the aldose reductase inhibitor Tolrestat 0.3mM. F and globular (G) actin were labeled with rhodamine-phalloidin and FITC-DNase-I, respectively. Both fluorescence probes were imaged simultaneously in each cell using dual-channel confocal laser microscopy. In HG, F-actin disassembly was observed and measured by a 40% decrease in F-/G-actin fluorescence intensity ratio (no change in NG + mannitol 24.4 mM). In separate experiments, cells were labeled with BODIPY FL-bisindolylmaleimide, specific for most PKC isoforms, and fluorescence intensity/cell was measured. In NG, exposure to phorbol 12-myristate 13-acetate (PMA) 0.1 µM for 15 minutes caused perinuclear and nuclear translocation of PKC, and F-actin disassembly identical to observations in HG alone. In HG, total PKC fluorescence increased by 50% and PMA exposure for 24 hours normalized both the total PKC and F-/G-actin fluorescence ratio. In NG and HG, exposure (15 min) to PMA 0.1 µM increased PKC activity three to four times, measured by in situ32P-phosphorylation of EGF-receptor substrate. By immunofluorecence and confocal imaging, diacylglycerol-sensitive PKC-δ was localized to the cytosol in NG, and after 15 minutes exposure to PMA, translocated to the perinuclear region and plasma membrane. In HG, PKC-δ immunofluorescence was significantly increased/cell, distributed in a cytoskeletal pattern and the intensity was glucose-concentration dependent (30 > 15 > 5.6 mM). In HG, exposure to PMA for 24 hours returned the PKC-δ fluorescence to the intensity and cytosolic pattern observed in NG, and simultaneously prevented F-actin disassembly. Tolrestat significantly reduced the total PKC and PKC-δ fluorescence intensity and F-actin disassembly observed in HG. Immunoblot confirmed increased PKC-δ in HG, which was normalized by Tolrestat. The immunofluorescence pattern of diacylglycerol-insensitive PKC-ζ was unchanged in HG, with PMA or Tolrestat. We conclude that mesangial cell F-actin disassembly in high glucose is likely mediated through diacylglycerol-sensitive PKC isoforms, including PKC-δ, and involves the polyol pathway.

Renal tubular basement membrane and collagen type IV in diabetes mellitus. The pathogenesis of the multiple structural lesions in diabetic nephropathy remains debated, and likely is multifactorial. The uniform thickening of the renal basement membranes lining the glomerular and tubular elements appears to be a consequence of the metabolic perturbations which are directly related to hyperglycemia. While most investigations have focused on the increased accumulation of extracellular matrix in the glomerular basement membrane and the mesangium, and their relation to derangements in glomerular function, little is known regarding the pathogenesis and the significance Of the tubulointerstitial changes and the thickened tubular basement membrane (TBM). It is possible that these latter changes are causally related to the cellular hypertrophy of the renal tubular epithelium that lines the TBM. It has been postulated that in the earlier stages of the disease, hyperglycemia induces renal tubular hypertrophy and stimulates the synthesis of the various matrix components which are normal constituents of the TBM. Later, the structural composition of the TBM is susceptible to further modifications by non-enzymatic glycation, and this aberrant process may impart a relative resistance to matrix degradation leading to a slow turnover. In vitro investigations on murine proximal tubule cells in culture have provided evidence that elevated ambient glucose is a sufficient stimulus for cellular hypertrophy and increased biosynthesis of collagen type IV, the predominant constituent of TBM. High glucose levels increase steady-state collagen IV mRNA, partly due to transcriptional activation of cis -acting elements of the gene which are controlled by putative glucose-responsive trans -acting proteins. This effect of hyperglycemia may be the consequence of increased activity of the polyol pathway, with attendant alterations in cellular myo -inositol metabolism. Treatment with sorbinil, an aldose reductase inhibitor, or supplementation of the medium with supra-physiologic levels of myo -inositol, prevents the stimulation by high glucose of the increased secretion and biosynthesis of collagen IV. The glucose-induced cellular hypertrophy, however, is minimally aifected by these maneuvers, and may result from activation of humoral or local mediators including transforming growth factor-β.

Diabetes-induced glomerular dysfunction: Links to a more reduced cytosolic ratio of NADH/NAD + . These studies were undertaken to examine effects of elevated glucose levels on glycolysis, sorbitol pathway activity, and the cytosolic redox state of NADH/NAD + in isolated glomeruli. Blood-free glomeruli were isolated from kidneys of male, Sprague-Dawley rats using standard sieving techniques, then incubated for one hour at 37°C, pH 7.4, pO 2 ∼500 torr, in Krebs bicarbonate/Hepes buffer containing 5 or 30 mM glucose. Elevated glucose levels increased glucose 6-phosphate, fructose 6-phosphate, total triose phosphates, lactate, the lactate/pyruvate ratio, sorbitol, and fructose, but did not affect sn-glycerol 3-phosphate, pyruvate, or myo -inositol levels. The more reduced glomerular cytosolic redox state (manifested by the tissue lactate/pyruvate ratio) induced by 30 mM glucose was completely abrogated by aldose reductase inhibitors added to the diet two to seven days prior to glomerular isolation. These observations, coupled with evidence linking glucose- and diabetes-induced glomerular dysfunction to increased sorbitol pathway metabolism, support the hypothesis that metabolic imbalances associated with a more reduced ratio of cytosolic NADH/NAD + (resulting from increased glucose metabolism via the sorbitol pathway) play an important role in mediating glucose- and diabetes-induced glomerular dysfunction.

The epidemic of chronic kidney disease in Nicaragua (Mesoamerican nephropathy) has been linked with recurrent dehydration. Here we tested whether recurrent dehydration may cause renal injury by activation of the polyol pathway, resulting in the generation of endogenous fructose in the kidney that might subsequently induce renal injury via metabolism by fructokinase. Wild-type and fructokinase-deficient mice were subjected to recurrent heat-induced dehydration. One group of each genotype was provided water throughout the day and the other group was hydrated at night, after the dehydration. Both groups received the same total hydration in 24 h. Wild-type mice that received delayed hydration developed renal injury, with elevated serum creatinine, increased urinary NGAL, proximal tubular injury, and renal inflammation and fibrosis. This was associated with activation of the polyol pathway, with increased renal cortical sorbitol and fructose levels. Fructokinase-knockout mice with delayed hydration were protected from renal injury. Thus, recurrent dehydration can induce renal injury via a fructokinase-dependent mechanism, likely from the generation of endogenous fructose via the polyol pathway. Access to sufficient water during the dehydration period can protect mice from developing renal injury. These studies provide a potential mechanism for Mesoamerican nephropathy.

Increased erythrocyte 3-DG and AGEs in diabetic hemodialysis patients: Role of the polyol pathway. Background 3-Deoxyglucosone (3-DG) accumulating in uremic serum plays an important role in the formation of advanced glycation end products (AGEs). To determine if 3-DG is involved in the formation of intracellular AGEs, we measured the erythrocyte levels of 3-DG and AGEs such as imidazolone and Ne-carboxymethyllysine (CML) in hemodialysis (HD) patients with diabetes. Further, to determine if the polyol pathway is involved in the formation of erythrocyte 3-DG and AGEs, an aldose reductase inhibitor (ARI) was administered to these patients. Methods The erythrocyte levels of sorbitol, 3-DG, imidazolone, and CML were measured in ten diabetic HD patients before and after treatment with ARI (epalrestat) for eight weeks, and were compared with those in eleven healthy subjects. 3-DG was incubated in vitro with hemoglobin for two weeks to determine if imidazolone and CML are formed by reacting 3-DG with hemoglobin. Results The erythrocyte levels of sorbitol, 3-DG, imidazolone, and CML were significantly elevated in diabetic HD patients as compared with healthy subjects. The erythrocyte levels of 3-DG significantly decreased after HD, but sorbitol, imidazolone or CML did not. The administration of ARI significantly decreased the erythrocyte levels of sorbitol, 3-DG and imidazolone, and tended to decrease the CML level. Imidazolone was rapidly produced in vitro by incubating 3-DG with hemoglobin, and CML was also produced, but less markedly as compared with imidazolone. Conclusion The erythrocyte levels of 3-DG and AGEs are elevated in diabetic HD patients. The administration of ARI reduces the erythrocyte levels of 3-DG and AGEs, especially imidazolone, as well as sorbitol. Thus, 3-DG and AGEs, especially imidazolone, in the erythrocytes are produced mainly via the polyol pathway. ARI may prevent diabetic and uremic complications associated with AGEs.

Aldose reductase expression is induced by hyperglycemia in diabetic nephropathy.BackgroundDespite good metabolic control, many patients with type 1 diabetes still develop nephropathy, implicating a role for genetic factors. Recent studies examining the regulatory region of the aldose reductase (ALR2) gene, the rate-limiting enzyme of the polyol pathway, support its role as a candidate gene for nephropathy. Here we report the quantitation of ALR2, together with sorbitol dehydrogenase mRNA in the peripheral blood mononuclear cells (PBMCs) of type 1 diabetic patients with (N = 29) and without nephropathy (N = 11) following stimulation with high levels of d-glucose.MethodsPBMCs from patients and normal controls were cultured for five days with phytohemagglutinin in either normoglycemia (11mmol/L d-glucose) or supplemented with 10mmol/L d-glucose (moderate hyperglyemia) or 20mmol/L d-glucose (hyperglycemia). The RNA was extracted and analyzed by ribonuclease protection assay.ResultsALR2 mRNA levels were significantly elevated with increasing d-glucose concentration (normal to hyperglycemic) in those patients with nephropathy (P < 0.0001). In marked contrast, in those without nephropathy and in the normal healthy controls, there was no change in mRNA expression. Furthermore, those patients with nephropathy and the Z–2/X susceptibility genotype had the greatest increase in ALR2 mRNA compared with those with low-risk genotypes (P < 0.007).ConclusionThese results show that patients with nephropathy exhibit marked disturbances in the expression of the enzyme components of the polyol pathway. Ultimately this leads to tissue damage and ischemia.