Role of 12-lipoxygenase in the stimulation of p38 mitogen-activated protein kinase and collagen alpha5(IV) in experimental diabetic nephropathy and in glucose-stimulated podocytes

The 12-lipoxygenase (12-LO) pathway of arachidonic acid metabolism is implicated in extracellular matrix (ECM) synthesis, but its role in podocytes has not been studied. This study tested whether 12-LO induction by diabetes or by high glucose (HG) in cultured podocytes alters glomerular basement membrane by activating signal transduction pathways culminating in ECM synthesis. Sprague-Dawley rats received an injection of diluent (control [C]) or streptozotocin 65 mg/kg (DM) and were killed at 1 or 4 mo. Glomerular 12-LO mRNA and protein levels were higher in DM than in C glomeruli at 1 and 4 mo, and 12-LO localized predominantly in podocytes. Glomerular p38 mRNA and protein were higher in DM at months 1 and 4, but phospho-p38 mitogen-activated protein (MAPK) was increased only at month 1. Glomerular collagen alpha5(IV)/glutaraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA ratio was increased in DM at month 1 but not at month 4, whereas collagen alpha5(IV) protein was higher at both 1 and 4 mo. Mouse podocytes were cultured in media with 25 mM glucose (HG) with or without the 12-LO inhibitor cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC) or with 5.5 mM glucose + 19.5 mM mannitol (low glucose [LG+M]) for 10 d at 37 degrees C. 12-LO mRNA and protein levels were higher in HG than in LG+M as was the p38 MAPK/GAPDH mRNA ratio. Phospho-p38 MAPK protein but not total p38 MAPK was higher in HG compared with LG+M. Collagen alpha5(IV)/GAPDH mRNA ratio and protein were higher in HG than in LG+M. 12-LO inhibition by CDC decreased HG-induced phospho-p38 MAPK and the phospho-p38/total p38 MAPK ratio, collagen alpha5(IV)/GAPDH mRNA ratio, and collagen alpha5(IV) protein expression. In summary, diabetes in vivo and exposure of podocytes to HG in vitro stimulated 12-LO, p38 MAPK, and collagen alpha5(IV) mRNA and (activated) protein. 12-LO inhibition by CDC diminished the expression of podocyte phospho-p38 MAPK and collagen alpha5(IV) mRNA and protein. These findings implicate 12-LO and the p38 MAPK signaling pathway in the mediation of ECM synthesis by podocytes in diabetes.     

Mechanisms of mitogen-activated protein kinase activation in experimental diabetes

Various growth factors and vasoactive substances are implicated in the pathogenesis of renal growth seen in early diabetes mellitus (DM). Mitogen-activated protein kinase (MAPK) is an important mediator of these extracellular stimuli. Protein kinase C (PKC), an enzyme known to be stimulated in DM, also activates MAPK. Thus, MAPK activity was examined in glomeruli from streptozotocin-induced DM rats. MAPK activity, measured as myelin basic protein kinase, was elevated by approximately 50% in DM versus controls (CON). Increased protein contents of p42mapk and p44mapk, as well as increased tyrosine phosphorylation and mobility shift of p42mapk, were also observed in DM. Tyrosine dephosphorylation of pp42mapk, on the other hand, assessed by incubating glomerular membrane with or without sodium orthovanadate (vanadate), was significantly diminished in DM. Protein expression of MAPK phosphatase-1 (MKP-1), a dual specificity phosphatase that inactivates MAPK, was approximately 60% of CON. Reduction in MKP-1 was reproduced in cultured mesangial cells grown under high glucose (30 mM; HG). The suppression of MKP-1 was PKC-dependent since incubation of HG cells with phorbol 12-myristate 13-acetate for 24 h abolished it. Furthermore, calcium ionophore A23187 reversed the suppression, suggesting that blunted Ca2+ signalling, characteristic of HG cells secondary to PKC stimulation, may be the cause. These results demonstrate that glomerular MAPK is activated in DM by multiple mechanisms i.e., increases in protein contents, increased phosphorylation, and decreased dephosphorylation of the enzyme due to suppression of MKP-1. These alterations may have an implication in the pathogenesis of diabetic nephropathy.     

Mechanical stress and glucose concentration modulate glucose transport in cultured rat podocytes.

BACKGROUND: Recent studies show that mechanical stress modifies both morphology and protein expression in podocytes. Ambient glucose is another factor modulating protein synthesis in these cells. In diabetes, podocytes experience elevated glucose concentrations as well as mechanical strain generated by high intracapillary pressures. Both these factors are responsible for podocyte injury, leading to impairment of kidney glomerular function. In the present study, we examined the effects of glucose concentration and mechanical stress on glucose uptake in podocytes. METHODS: Following a 24 h pre-incubation in low (2.5 mM, LG), normal (5.6 mM, NG) or high (30 mM, HG) glucose media, cultured rat podocytes were exposed to 4 h mechanical stress. We used the labelled glucose analogue, [3H]2-deoxy-D-glucose, to measure glucose uptake. The distribution of facilitative glucose transporters GLUT2 and GLUT4 was assessed by flow cytometry. RESULTS: In the control (static) cells, glucose uptake was similar in the three glucose groups. In mechanically stressed podocytes, glucose uptake increased 2-fold in the LG and NG groups but increased 3-fold in the HG group. In the NG cells, mechanical load increased the membrane expression of GLUT2 and reduced the membrane-bound GLUT4. In stretched HG cells, the membrane expression of both GLUT2 and GLUT4 was decreased. High glucose decreased the plasma membrane GLUT2 content in the stretched cells, whereas both static and stretched podocytes showed an elevation in GLUT4. CONCLUSION: Mechanical stress potentiated glucose uptake in podocytes and this effect was enhanced by high ambient glucose. The decreased expression of GLUT2 and GLUT4 on the surface of stretched cells suggests that the activity of other glucose transporters may be regulated by mechanical stress in podocytes.     

Differential expression of nephrin according to glomerular size in early diabetic kidney disease

Diabetic nephropathy (DN) is clinically characterized by proteinuria. Many studies tried to demonstrate a relationship between proteinuria and changes in nephrin in various forms of glomerular diseases including DN, but the results are not consistent. Glomerular hypertrophy occurs in DN, yet hypertrophy does not develop in all glomeruli concurrently. For investigation of the differences in nephrin expression according to glomerular size, glomeruli were isolated from 10 control and 10 streptozotocin-induced diabetic rats at 6 wk after the induction of diabetes by a sieving technique using sieves with pore sizes of 250, 150, 125, and 75 microm. Glomeruli then were classified into large glomeruli (LG; on the 125-microm sieve) and small glomeruli (SG; on the 75-microm sieve) groups. Glomerular volumes were determined using an image analyzer, and mRNA and protein expression was determined by real-time PCR and Western blot, respectively. The mean volumes of diabetic LG (1.51 +/- 0.06 x 10(6) microm(3)) and control LG (1.37 +/- 0.05 x 10(6) microm(3)) were significantly higher than those of diabetic SG (0.94 +/- 0.03 x 10(6) microm(3)) and control SG (0.87 +/- 0.03 x 10(6) microm(3); P < 0.01). Nephrin mRNA expression was significantly reduced in the diabetic LG group compared with the diabetic SG and control glomeruli groups (P < 0.05). In contrast, nephrin mRNA expression was significantly higher in the diabetic SG group compared with the diabetic LG and control glomeruli groups (P < 0.05). Even after correction for 18s rRNA and Wilms' tumor-1 mRNA expression, the differences in nephrin mRNA expression remained significant. The expression of nephrin protein showed a similar pattern to the mRNA expression. In conclusion, these data suggest that the nephrin gene is differentially expressed according to glomerular size. Furthermore, more hypertrophied glomeruli with lesser nephrin expression may be responsible for albuminuria in the early stage of DN.     

High glucose decreases collagenase expression and increases TIMP expression in cultured human peritoneal mesothelial cells.

BACKGROUND: Peritoneal fibrosis (PF), a serious problem in long-term continuous ambulatory peritoneal dialysis (CAPD) patients, is characterized by extracellular matrix (ECM) accumulation which results from an imbalance between the synthesis and the degradation of ECM components. Previous studies have demonstrated that ECM synthesis is increased in human peritoneal mesothelial cells (HPMCs) under high glucose conditions, but the effects of high glucose on degradative pathways have not been fully explored. This study was undertaken to elucidate the effects of high glucose on these proteolytic processes in cultured HMPCs. METHODS: HPMCs were isolated from human omentum and were exposed to 5.6 mM glucose (NG), 5.6 mM glucose +34.4 mM mannitol (NG + M), or 40 mM glucose (HG) with or without PKC inhibitor (PKCi). Real-time PCR and western blot were performed to determine collagenases (MMP-1, -8 and -13) and TIMPs (TIMP-1 and -2) mRNA and protein expression, respectively. The individual activities of collagenases in culture media were determined by ELISA. RESULTS: Types I and III collagen protein expression were significantly increased in HG-conditioned media compared to NG media (P < 0.05). The MMP-1, -8 and -13/GAPDH mRNA ratios were significantly lower in HPMCs exposed to HG medium compared to NG cells by 64, 52 and 37%, respectively, and their protein expression by 76, 42 and 49%, respectively, in HG- vs NG-conditioned media. The activities of collagenases in HG-conditioned media were also significantly lower than those in NG media (P < 0.05). In contrast, HG significantly increased TIMPs mRNA ratios and protein expression in HPMCs. These changes in collagenase and TIMP expression induced by HG were abrogated upon pre-treatment with PKCi. CONCLUSION: In conclusion, impaired matrix degradation may contribute to ECM accumulation in PF.     

Activation of the renin-angiotensin system within podocytes in diabetes

The autocrine and paracrine activation of the renin-angiotensin system (RAS) within cells of the kidney plays a role in the overall pathophysiology of the renal disease due to diabetes. In this study, we focus on components of the RAS in the podocyte as these cells are important in the pathogenesis of glomerulosclerosis and proteinuria. Immortalized mouse podocytes were exposed to media containing normal glucose (NG) or high glucose (HG) for in vitro studies. In vivo studies utilized kidney tissue obtained from rats treated for 3 months with streptozotocin to induce diabetes. Angiotensinogen (AGT) and the angiotensin II (AII) type 1 receptor mRNA and protein were significantly increased in the podocytes cultured under the high glucose conditions. Both angiotensins I and II levels were significantly higher in cell lysates and the conditioned media of cells grown in high glucose. There were no differences in renin activity, angiotensin-converting enzyme level, or AII type 2 receptor level. Glomerular AGT and AII type 1 receptor assessed by means of immunohistochemistry were increased in diabetic rats compared with the control rats. Other measured components of the RAS within the glomeruli were not different. We suggest that increased AGT, an attendant increase in AII and increased AII type 1 receptor in podocytes experiencing diabetic conditions play an important role in the pathogenesis of diabetic nephropathy.     

Effect of angiotensinⅡ type 1 receptor blocker on the 12-lipoxygenase activity and P-cadherin expression in type 2 diabetic rat glomeruli

Objective To investigate the effect of angiotensinⅡ(AngⅡ) type 1 receptor blocker (ARB) on 12-lipoxygenase (12-LO) activity and P-cadherin expression in type 2 diabetic rat glomeruli. Methods Podocytes were stimulated by 10-7 mol/L AngⅡ for 24 hours. 12(S)-HETE (1 mg•kg-1•d-1) and AngⅡ (400 ng•kg-1•min-1) were infused to rats by osmotic mini-pump for 1 week and 2 weeks respectively. Rats fed with high fat diet received low dose streptozotocin (STZ) to make type 2 diabetes and divided into 2 groups: low dose STZ (DN group), low dose STZ＋ARB treatment (Losartan group). Rats fed with regular chow were used as control group. All the rats were sacrificed after 6 weeks. Urine, blood, kidney cortical tissue and isolated glomeruli by sieving method were collected at the end of study respectively. ELISA, RT-PCR and Western blotting for related target were performed respectively. Results AngⅡincreased 12(S)-HETE levels in podocytes and glomeruli (all P＜0.01). AngⅡ levels in the glomeruli were significantly increased by 12(S)-HETE stimulation (P＜0.01). Blood glucose, kidney/body weight and 24 hour urinary protein were increased in DN group compared with that in control group (all P＜0.01). However, urine protein, Kidney/body weight were decreased in Losartan group compared with DN group (all P＜0.05). Increment of 12(S)-HETE content and decrement of P-cadherin expression were observed in DN glomeruli compared with that in control group(all P＜0.01). These abnormalities were prevented by administration of the losartan (all P＜0.05). Conclusions AngⅡ can down-regulate glomerular P-cadherin expression via activation of 12-LO.ARB can ameliorate the progression of DN via up-regulation of glomerular P-cadherin through inhibition of 12-LO activation in type 2 DN rats.     

Comparison of Glomerular and Podocyte mRNA Profiles in Streptozotocin-Induced Diabetes

Evaluating the mRNA profile of podocytes in the diabetic kidney may indicate genes involved in the pathogenesis of diabetic nephropathy. To determine if the podocyte-specific gene information contained in mRNA profiles of the whole glomerulus of the diabetic kidney accurately reflects gene expression in the isolated podocytes, we crossed Nos3^−/− IRG mice with podocin-rtTA and TetON-Cre mice for enhanced green fluorescent protein labeling of podocytes before diabetic injury. Diabetes was induced by streptozotocin, and mRNA profiles of isolated glomeruli and sorted podocytes from diabetic and control mice were examined 10 weeks later. Expression of podocyte-specific markers in glomeruli was downregulated in diabetic mice compared with controls. However, expression of these markers was not altered in sorted podocytes from diabetic mice. When mRNA levels of glomeruli were corrected for podocyte number per glomerulus, the differences in podocyte marker expression disappeared. Analysis of the differentially expressed genes in diabetic mice also revealed distinct upregulated pathways in the glomeruli (mitochondrial function, oxidative stress) and in podocytes (actin organization). In conclusion, our data suggest reduced expression of podocyte markers in glomeruli is a secondary effect of reduced podocyte number, thus podocyte-specific gene expression detected in the whole glomerulus may not represent that in podocytes in the diabetic kidney.     

血管紧张素Ⅱ诱导足细胞c-Abl的表达改变

目的 探讨血管紧张素Ⅱ( AngⅡ)诱导下大鼠肾脏及条件永生性小鼠足细胞c-Abl的表达变化.方法 采用AngⅡ泵(400ng·kg-1·min-1)植入SD大鼠皮下的方法建立AngⅡ输注模型,24只大鼠成模后被随机分为AngⅡ输注2周组、AngⅡ输注4周组、AngⅡ输注+替米沙坦(ARB,3 mg·kg-1·min-1)干预2周组及4周组,同时设生理盐水输注组和健康对照组,每组6只.分别于成模后2周末、4周末处死大鼠取肾.电镜下观察肾脏足细胞超微结构的改变;免疫荧光法检测肾脏c-Abl表达;实时PCR及Western印迹检测c-Abl mRNA及蛋白水平的改变.对于体外培养条件永生性小鼠足细胞,免疫荧光法检测足细胞肾脏c-Abl的表达,实时PCR及Western印迹法检测足细胞在AngⅡ不同作用浓度(10-9 mol/L～10-6 mol/L)及不同作用时间点(0h、3h、6h、12 h、24 h)c-Abl mRNA和蛋白水平的变化.结果 (1)免疫荧光检测结果显示肾脏足细胞有c-Abl表达;实时PCR及Western印迹结果显示AngⅡ输注2周组和4周组大鼠肾脏c-Abl表达增加(P＜0.05),ARB干预组大鼠肾脏c-Abl表达较同期AngⅡ输注组均有降低(P＜0.05).(2)体外培养的条件永生性小鼠足细胞胞质及胞核有c-Abl表达.AngⅡ可诱导培养的小鼠足细胞c-Abl mRNA及蛋白表达增加(P＜0.05),且呈时间和剂量依赖性.结论 c-Abl在肾足细胞及体外培养足细胞均有表达,AngⅡ可诱导c-Abl表达上调.c-Abl可能参与了AngⅡ诱导的足细胞损伤.     

表没食子儿茶素没食子酸酯对糖尿病大鼠肾脏细胞外调节蛋白激酶活性的影响

目的 观察表没食子儿茶素没食子酸酯（EGCG）对糖尿病大鼠肾脏细胞外调节蛋白激酶（ERK）活性的影响。方法 采用链脲佐菌素腹腔注射建立糖尿病大鼠模型，实验分3组：正常对照组、糖尿病模型组和EGCG干预组。EGCG干预组于模型成功后1周给予EGCG5mg·kg^-1·d^-1腹腔注射。以免疫组织化学方法检测肾组织磷酸化ERK（p-ERK）的表达。大鼠肾系膜细胞株分组：正常对照组（NG，葡萄糖5mmol／L），高糖组（HG，葡萄糖30mmol／L），HG＋EGCG1组（100μg／L），HG＋EGCG2组（200μg／L），HG＋EGCG4组（400μg／L），甘露醇组（5mmol／L葡萄糖＋25mmol／L甘露醇）。用四甲基偶氮唑蓝比色（MTT）法检测细胞增殖活性；常规生化分析系膜细胞氧化应激状态；Western印迹检测ERK、p-ERK和p27蛋白表达水平。结果 EGCG呈剂量和时间依赖方式抑制高糖时系膜细胞的增殖活性及抗氧化作用。EGCG干预后糖尿病大鼠肾脏p-ERK蛋白免疫组化染色明显减弱。高糖时系膜细胞p-ERK及p27蛋白表达上调，EGCG呈剂量和时间依赖方式下调高糖时p-ERK蛋白的表达；呈剂量依赖方式下调p27蛋白的表达。结论 EGCG能有效改善糖尿病肾损伤程度，其作用机制可能是通过调节ERK活性，抑制p27蛋白表达而减少糖尿病肾病损害。     

High glucose-enhanced mesangial cell extracellular signal-regulated protein kinase activation and alpha1(IV) collagen expression in response to endothelin-1: role of specific protein kinase C isozymes

High glucose (HG) stimulates glomerular mesangial cell (MC) expression of extracellular matrix, a process involving protein kinase C (PKC) isozymes and enhanced signaling by autocrine peptides such as endothelin-1 (ET-1). The purpose of this study was to identify the specific PKC isozymes mediating the effects of HG on MC extracellular signal-regulated protein kinase (ERK1/2) signaling and alpha1(IV) collagen expression in response to ET-1. HG (30 mmol/l for 72 h) enhanced ET-1-stimulated alpha1(IV) collagen mRNA expression from 1.2 +/- 0.1-fold to 1.9 +/- 0.2-fold (P < 0.05 vs. normal glucose [NG] + ET-1), and the effect was significantly reduced by Calphostin C or the MEK (mitogen-activated protein kinase kinase) inhibitor PD98059. In transiently transfected MCs, dominant-negative (DN)-PKC-delta, -epsilon, or -zeta inhibited ET-1 activation of ERK1/2. Likewise, downstream of ERK1/2, ET-1 stimulated Elk-1-driven GAL4 luciferase activity to 11 +/- 1-fold (P < 0.002 vs. NG + ET-1) in HG, and DN-PKC-delta, -epsilon, or -zeta attenuated this response to NG levels. HG enhanced ET-1-stimulated intracellular alpha1(IV) collagen protein expression, assessed by confocal immunofluorescence imaging, showed that individual DN-PKC-delta, -epsilon, -zeta, as well as DN-PKC-alpha and -beta, attenuated the response. Thus, HG-enhanced ET-1 stimulation of alpha1(IV) collagen expression requires PKC-delta, -epsilon, and -zeta to act through an ERK1/2-dependent pathway and via PKC-alpha and -beta, which are independent of ERK1/2.     

Antioxidants ameliorate the expression of vascular endothelial growth factor mediated by protein kinase C in diabetic podocytes.

BACKGROUND: The increased production of reactive oxygen species (ROS) may be involved in the onset or development of diabetic vascular complications. The release of ROS from podocytes plays a role in the pathogenesis of glomerular damage in various experimental glomerular diseases. Although it is assumed that the podocyte injury also plays an important role in diabetic nephropathy, the mechanism is still unknown. METHODS: Using a differentiated mouse podocyte cell line, we investigated: (1) whether a high level of ambient glucose increases the level of ROS, (2) whether the protein kinase C (PKC) pathway is involved in a high-glucose-induced generation of ROS and vascular endothelial growth factor (VEGF) and (3) whether antioxidants ameliorate PKC-mediated VEGF expression in diabetic milieu. RESULTS: Intracellular ROS generation was significantly higher in high glucose than in control conditions in cultured podocytes. High ambient glucose also increased VEGF mRNA and protein expression. The high-glucose-induced increases in ROS and VEGF mRNA and protein by podocytes were effectively inhibited by pretreatment with various antioxidants and were completely restored by PKC inhibition. The results show that cultured mouse podocytes produce ROS in response to high glucose, and that PKC is involved in high-glucose-induced ROS and VEGF production by podocyte. CONCLUSION: Increased ROS in podocytes may play a role in the pathogenesis of podocyte injury in diabetic nephropathy.     

Global adiponectin suppress the high expression of monocyte chemotactic protein-1 induced by high glucose in NRK52E cells

Objective To investigate the effect of globular adiponectin on the high expression of monocyte chemotactic protein-1 (MCP-1) induced by high glucose in rat renal tubular epithelial cells(NRK52E), and its relationship with adiponectin receptors and p38MAPK. Methods NRK52E cells were cultured in vitro and divided into six groups: normal glucose group (NG, 5.6 mmol/L glucose), high glucose group(HG, 25 mmol/L glucose), gAd group1 (HG+gAd 2 mg/L), gAd group2 (HG+gAd 5 mg/L), gAd group3 (HG+gAd 10 mg/L), p38MAPK antagonist group：(SB, HG+SB203580 10 μmol/L). The protein expression of phosphorylated p38MAPK (p-p38MAPK), total p38MAPK (t-p38MAPK), MCP-1 and AdipoR1/AdipoR2 were examined by western blotting. The mRNA expression of MCP-1 and AdipoR1/AdipoR2 were detected by RT-PCR and real-time PCR respectively. Results Compared with NG group, the mRNA and protein expression of MCP-1 increased significantly in HG group (all P＜0.05). The phosphorylation of p38MAPK increased (P＜0.05) with no change in t-p38MAPK protein. The addition of gAd or SB203580 inhibited the unregulation of MCP-1 and p-p38MAPK induced by HG. Two kinds of adipoR,adipoR1 and adipoR2,were all detectable in NG group, and mRNA and protein expression of adipoR1 was higher than that of adipoR2 (P＜0.01). Compared with NG group, the expression of adipoR decreased in HG group, but the difference had no statistical significance(P＞0.05). Compared to HG group, the mRNA and protein expression of adipoR1 increased in gAd groups (all P＜0.01). Conclusion The gAd can dose-dependently attenuate the overexpression of MCP-1 induced by high glucose, and this protective effect may be mediated by adipoR1 and p38MAPK.     

Effect of high glucose on mesangial cell protein kinase C-delta and -epsilon is polyol pathway-dependent.

In diabetes mellitus, enhanced activity of mesangial cell protein kinase C (PKC) may contribute to nephropathy. The purpose of this study was to determine whether high glucose alters mesangial cell diacylglycerol-sensitive PKC-alpha, -beta2, -delta, and -epsilon content, cellular distribution, and activity through polyol pathway activation. Primary cultured rat mesangial cells (passage 10) were growth-arrested in 0.5% fetal bovine serum and cultured in 5.6 mM glucose (NG) or 30 mM glucose (HG) for 48 h, with or without the aldose reductase inhibitor tolrestat or ARI-509. PKC isoform content in total cell lysates, or cytosol, membrane (Triton X-soluble), and particulate (sodium dodecyl sulfate-soluble) fractions was analyzed by immunoblotting, and band density in HG was expressed as a percentage of corresponding NG values. In HG at 48 h, increased total PKC-alpha (222 +/- 17% of NG, P < 0.001), -beta2 (209 +/- 12%, P < 0.001), and -epsilon (195 +/- 19%, P < 0.001) were observed. L-Glucose had no effect on total PKC isoform content. HG caused increased membrane- and particulate-associated PKC-alpha (257 +/- 87 and 327 +/- 66%, respectively, P < 0.05), membrane-associated PKC-delta (143 +/- 10%, P < 0.05), and membrane-associated PKC-epsilon (186 +/- 11%, P < 0.001), with no change in cytosol contents. The HG effects were not mimicked by L-glucose. In NG or HG, PKC-beta2 was not detected in the cytosol fraction, and membrane and particulate association were unchanged with phorbol ester stimulation. Confocal immunofluorescence imaging revealed that in HG, PKC-alpha, -delta, and -epsilon translocate to the nucleus and plasma membrane. Total PKC activity measured by in situ 32P-phosphorylation of the epidermal growth factor receptor substrate increased from 18 +/- 1 pmol/min per mg cell protein in NG to 33 +/- 3 pmol/min per mg cell protein in HG (P < 0.002 versus NG). In NG, tolrestat and ARI-509 exposure caused increased PKC activity, enhanced accumulation of total PKC-alpha and -beta2, with no change in total or fractional recovery of PKC-delta or -epsilon. In HG, tolrestat and ARI-509 prevented the increase in total PKC-epsilon and membrane-associated PKC-delta and -epsilon. It is concluded that within 48 h of HG, enhanced mesangial cell PKC activity is associated with accumulation and cellular redistribution of diacylglycerol-sensitive PKC isoforms, and that increased PKC-epsilon content and membrane-associated PKC-delta and -epsilon are dependent on polyol pathway activation.