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OBJECTIVE Elevated glycolytic enzymes in renal glomeruli correlated with preservation of renal function in the Medalist Study, individuals with ≥50 years of type 1 diabetes. Specifically, pyruvate kinase M2 (PKM2) activation protected insulin-deficient diabetic mice from hyperglycemia-induced glomerular pathology. This study aims to extend these findings in a separate cohort of individuals with type 1 and type 2 diabetes and discover new circulatory biomarkers for renal protection through proteomics and metabolomics of Medalists’ plasma. We hypothesize that increased glycolytic flux and improved mitochondrial biogenesis will halt the progression of diabetic nephropathy. RESEARCH DESIGN AND METHODS Immunoblots analyzed selected glycolytic and mitochondrial enzymes in postmortem glomeruli of non-Medalists with type 1 diabetes (n = 15), type 2 diabetes (n = 19), and no diabetes (n = 5). Plasma proteomic (SOMAscan) (n = 180) and metabolomic screens (n = 214) of Medalists with and without stage 3b chronic kidney disease (CKD) were conducted and significant markers validated by ELISA. RESULTS Glycolytic (PKM1, PKM2, and ENO1) and mitochondrial (MTCO2) enzymes were significantly elevated in glomeruli of CKD− versus CKD+ individuals with type 2 diabetes. Medalists’ plasma PKM2 correlated with estimated glomerular filtration rate (r2 = 0.077; P = 0.0002). Several glucose and mitochondrial enzymes in circulation were upregulated with corresponding downregulation of toxic metabolites in CKD-protected Medalists. Amyloid precursor protein was also significantly upregulated, tumor necrosis factor receptors downregulated, and both confirmed by ELISA. CONCLUSIONS Elevation of enzymes involved in the metabolism of intracellular free glucose and its metabolites in renal glomeruli is connected to preserving kidney function in both type 1 and type 2 diabetes. The renal profile of elevated glycolytic enzymes and reduced toxic glucose metabolites is reflected in the circulation, supporting their use as biomarkers for endogenous renal protective factors in people with diabetes.

Clinical studies have suggested that loss of insulin’s action and excessive IGF-1 levels in the glomeruli elevate the risk for diabetic kidney disease (DKD). To define the potential pathogenic role of IGF-1 and insulin signaling in DKD, we generated mice with selective loss of insulin receptor (IR) or IGF-1 receptor (IGF-1R) in the mesangial cells (MCs) by cross breeding IRflox/flox mice with Sm22α-Cre mice (SMIRKO) or IGF-1Rflox/flox mice with Myh11-Cre mice (SMIGF1RKO), and examined urinary albumin excretion (ACR) and mesangial expansion with 6 month duration of streptozotocin (STZ)-induced diabetes. IGF-1 levels were significantly increased by 1.5 fold in the renal cortex, but not in the plasma of all three types of diabetic mice. Diabetes induced elevation of ACR (2.0 fold), glomerular size and mesangial expansion (2.1 fold), in parallel with increased glomerular expressions of fibrosis-related (Tgfβ1, fibronectin and Col3a) and matrix (Has2) genes by 2-3 fold, significantly, in Wt and SMIRKO mice, but were prevented in diabetic SMIGF1RKO mice. In cultured MCs, IGF-1, at physiological levels, and insulin, only at supra-physiological levels, induced the expressions of Has2 (3.8 fold) and Tgfβ1 (1.7 fold). Deletion of IGF-1R, but not IR, in MCs inhibited both IGF-1 and insulin-induced expressions of Has2. Inhibitor of MAP kinase pathway, but not inhibitor of PI3 kinase reduced insulin or IGF-1-induced expressions of Has2 in MCs. IGF-1-induced phosphorylations of IGF-1R (39 fold), Akt (34 fold) and Erk/MAPK (8 fold) were significantly inhibited in both IR or IGF-1R-deleted MCs. Insulin-induced phosphorylations of IR-β (11 fold) and Akt (27 fold) were also decreased in IR-deleted MCs, but surprisingly increased in IGF-1R-deleted MCs. Thus, MAPK activation by elevated glomerular IGF-1 levels via IGF-1R, but not IR, on MCs, may contribute to the pathogenesis of DKD through induction of Has2 and fibrotic genes. Disclosure A. Ishikado: Employee; Self; Sunstar Group. Employee; Spouse/Partner; Sunstar Group. T. Shinjo: None. H. Yokomizo: None. Y. Maeda: None. K. Park: None. W. Qi: None. R. St-Louis: None. Q. Li: None. G.L. King: Research Support; Self; Janssen Pharmaceuticals, Inc.

Diabetic nephropathy (DN) is a major cause of end-stage renal disease, and therapeutic options for preventing its progression are limited. To identify novel therapeutic strategies, we studied protective factors for DN using proteomics on glomeruli from individuals with extreme duration of diabetes (ł50 years) without DN and those with histologic signs of DN. Enzymes in the glycolytic, sorbitol, methylglyoxal and mitochondrial pathways were elevated in individuals without DN. In particular, pyruvate kinase M2 (PKM2) expression and activity were upregulated. Mechanistically, we showed that hyperglycemia and diabetes decreased PKM2 tetramer formation and activity by sulfenylation in mouse glomeruli and cultured podocytes. Pkm-knockdown immortalized mouse podocytes had higher levels of toxic glucose metabolites, mitochondrial dysfunction and apoptosis. Podocyte-specific Pkm2-knockout (KO) mice with diabetes developed worse albuminuria and glomerular pathology. Conversely, we found that pharmacological activation of PKM2 by a small-molecule PKM2 activator, TEPP-46, reversed hyperglycemia-induced elevation in toxic glucose metabolites and mitochondrial dysfunction, partially by increasing glycolytic flux and PGC-1α mRNA in cultured podocytes. In intervention studies using DBA2/J and Nos3 (eNos) KO mouse models of diabetes, TEPP-46 treatment reversed metabolic abnormalities, mitochondrial dysfunction and kidney pathology. Thus, PKM2 activation may protect against DN by increasing glucose metabolic flux, inhibiting the production of toxic glucose metabolites and inducing mitochondrial biogenesis to restore mitochondrial function.

AIMS/HYPOTHESIS: Accelerated migration and proliferation of vascular smooth muscle cells (VSMCs) enhances arterial restenosis after angioplasty in insulin resistance and diabetes. Elevation of Src homology 2-containing protein tyrosine phosphatase 1 (SHP-1) induces apoptosis in the microvasculature. However, the role of SHP-1 in intimal hyperplasia and restenosis has not been clarified in insulin resistance and diabetes.METHODS: We used a femoral artery wire injury mouse model, rodent models with insulin resistance and diabetes, and patients with type 2 diabetes. Further, we modulated SHP-1 expression using a transgenic mouse that overexpresses SHP-1 in VSMCs (Shp-1-Tg). SHP-1 agonists were also employed to study the molecular mechanisms underlying the regulation of SHP-1 by oxidised lipids.RESULTS: Mice fed a high-fat diet (HFD) exhibited increased femoral artery intimal hyperplasia and decreased arterial SHP-1 expression compared with mice fed a regular diet. Arterial SHP-1 expression was also decreased in Zucker fatty rats, Zucker diabetic fatty rats and in patients with type 2 diabetes. In primary cultured VSMCs, oxidised LDL suppressed SHP-1 expression by activating Mek-1 (also known as Map2k1) and increased DNA methylation of the Shp-1 promoter. VSMCs from Shp-1-Tg mice exhibited impaired platelet-derived growth factor (PDGF)-stimulated tyrosine phosphorylation with a concomitant decrease in PDGF-stimulated VSMC proliferation and migration. Similarly, HFD-fed Shp-1-Tg mice and mice treated with the SHP-1 inducer, Icariside II, were protected from the development of intimal hyperplasia following wire injury.CONCLUSIONS/INTERPRETATION: Suppression of SHP-1 by oxidised lipids may contribute to the excessive VSMC proliferation, inflammatory cytokine production and intimal hyperplasia observed in arteries from diabetes and insulin resistance. Augmenting SHP-1 levels is a potential therapeutic strategy to maintain stent patency in patients with insulin resistance and diabetes.

Insulin and IGF-1 actions in vascular smooth muscle cells (VSMC) are associated with accelerated arterial intima hyperplasia and restenosis after angioplasty, especially in diabetes. To distinguish their relative roles, we delete insulin receptor (SMIRKO) or IGF-1 receptor (SMIGF1RKO) in VSMC and in mice. Here we report that intima hyperplasia is attenuated in SMIRKO mice, but not in SMIGF1RKO mice. In VSMC, deleting IGF1R increases homodimers of IR, enhances insulin binding, stimulates p-Akt and proliferation, but deleting IR decreases responses to insulin and IGF-1. Studies using chimeras of IR(extracellular domain)/IGF1R(intracellular-domain) or IGF1R(extracellular domain)/IR(intracellular-domain) demonstrate homodimer IRα enhances insulin binding and signaling which is inhibited by IGF1Rα. RNA-seq identifies hyaluronan synthase2 as a target of homo-IR, with its expression increases by IR activation in SMIGF1RKO mice and decreases in SMIRKO mice. Enhanced intima hyperplasia in diabetes is mainly due to insulin signaling via homo-IR, associated with increased Has2 expression., Both insulin and IGF-1 signaling have been implicated in arterial intimal hyperplasia. Here the authors dissect the relative contributions of insulin and IGF-1 receptors, showing that homodimers of insulin receptors, but not IGF-1 receptor, induce proliferation of vascular smooth muscle cells.

We have reported that up-regulation of PKM2 in renal glomeruli isolated from patients with more than 50 years of type 1 diabetes (Joslin Medalist Study) correlated with preservation of renal function even in the presence of chronic hyperglycemia. Further, pharmacological activation of PKM2 in T1D mice and podocyte-specific deletion of PKM2 in cultured cells and in vivo strongly showed that PKM2 activation protects renal glomeruli from hyperglycemia/diabetes induced damage (Nat. Med 2017). To confirm these findings in type 1 and type 2 diabetes, we performed the following: 1. We showed the protein expressions of glycolytic (PKM1, PKM2, ENO1) and mitochondrial (MTCO2) enzymes to be significantly elevated in glomeruli of post mortem kidneys in people with T2D without DN compared to those with DN, or nondiabetic controls. 2. Plasma PKM2 levels (SomaLogic aptamer assay) in the Medalists (n=180) correlated with albumin-to-creatinine ratio (ACR;p=0.03) and eGFR (p=0.0002) at baseline. In a replication cohort (n=219) of patients with T1D in the longitudinal Joslin Kidney Study (JKS) baseline plasma PKM2 also correlated with ACR (p=0.0001). High baseline plasma PKM2 in JKS was independently associated with a slower decline in eGFR (p=0.004) and a slower progression to end-stage renal disease (p=0.0002). 3. TEPP-46, a PKM2 activator, prevented diabetes induced increases in mRNA expression of pro-fibrotic (Col3a1, Fn1, TGFb1) and inflammatory (Ccl2) genes, and enhanced key mitochondrial protein (Ppargc1a) in uninephrectomized T2D db/db mice. No differences in ACR were observed between TEPP-46 treated and untreated diabetic mice. These findings support the hypothesis that PKM2 activation may protect or even reverse the initiation and progression of diabetic glomerular dysfunction and pathology in both T1D and T2D, suggesting that activating PKM2 could be a clinically effective therapeutic target to stop the progression of DN. Disclosure D. Gordin: None. T. Shinjo: None. R. St-Louis: None. W. Qi: None. L.J. Tinsley: None. D.M. Pober: None. M.J. Brissett: None. M. Niewczas: None. J. Dreyfuss: None. H. Pan: None. T. Sadowski: Employee; Self; Sanofi. A. Kannt: Employee; Self; Sanofi. Stock/Shareholder; Self; Sanofi. H. Shah: None. H.A. Keenan: Research Support; Self; Sanofi. Employee; Self; Sanofi Genzyme. A. Krolewski: None. G.L. King: Research Support; Self; Sanofi-Aventis.

Diabetic nephropathy (DN) affects approximately 30–40% of patients with type 1 (T1DM) and type 2 diabetes (T2DM). It is a major cause of end-stage renal disease (ESRD) for the developed world. Hyperglycemia and genetics are major causal factors for the initiation and progression of DN. Multiple abnormalities in glucose and mitochondrial metabolism induced by diabetes likely contribute to the severity of DN. Recent clinical studies in people with extreme duration of T1DM (> 50 years, Joslin Medalist Study) have supported the importance of endogenous protective factors to neutralize the toxic effects of hyperglycemia on renal and other vascular tissues. Using renal glomeruli from these patients (namely Medalists) with and without DN, we have shown the importance of increased glycolytic flux in decreasing the accumulation of glucose toxic metabolites, improving mitochondrial function, survival of glomerular podocytes, and reducing glomerular pathology. Activation of a key glycolytic enzyme, pyruvate kinase M2 (PKM2), resulted in the normalization of renal hemodynamics and mitochondrial and glomerular dysfunction, leading to the mitigation of glomerular pathologies in several mouse models of DN.

Rationale: Activation of monocytes/macrophages by hyperlipidemia associated with diabetes mellitus and obesity contributes to the development of atherosclerosis. PKCδ (protein kinase C δ) expression and activity in monocytes were increased by hyperlipidemia and diabetes mellitus with unknown consequences to atherosclerosis. Objective: To investigate the effect of PKCδ activation in macrophages on the severity of atherosclerosis. Methods and Results: PKCδ expression and activity were increased in Zucker diabetic rats. Mice with selective deletion of PKCδ in macrophages were generated by breeding PKCδ flox/flox mice with LyzM-Cre and ApoE −/ − mice (MPKCδKO/ApoE −/− mice) and studied in atherogenic (AD) and high-fat diet (HFD). Mice fed AD and HFD exhibited hyperlipidemia, but only HFD-fed mice had insulin resistance and mild diabetes mellitus. Surprisingly, MPKCδKO/ApoE −/− mice exhibited accelerated aortic atherosclerotic lesions by 2-fold versus ApoE −/− mice on AD or HFD. Splenomegaly was observed in MPKCδKO/ApoE −/− mice on AD and HFD but not on regular chow. Both the AD or HFD increased macrophage number in aortic plaques and spleen by 1.7- and 2-fold, respectively, in MPKCδKO/ApoE −/− versus ApoE −/− mice because of decreased apoptosis (62%) and increased proliferation (1.9-fold), and not because of uptake, with parallel increased expressions of inflammatory cytokines. Mechanisms for the increased macrophages in MPKCδKO/ApoE −/− were associated with elevated phosphorylation levels of prosurvival cell-signaling proteins, Akt and FoxO3a, with reduction of proapoptotic protein Bim associated with PKCδ induced inhibition of P85/PI3K. Conclusions: Accelerated development of atherosclerosis induced by insulin resistance and hyperlipidemia may be partially limited by PKCδ isoform activation in the monocytes, which decreased its number and inflammatory responses in the arterial wall.

Hyperglycemia and hypoxia have independent and convergent roles in the development of renal disease. Transforming growth factor-β1(TGF-β1) is a key cytokine promoting the production of extracellular matrix proteins. The cationic-independent mannose 6-phosphate receptor (CI-M6PR) is a membrane protein that binds M6P-containing proteins. A key role is to activate latent TGF-β1. PXS25, a novel CI-MPR inhibitor, has antifibrotic properties in skin fibroblasts, but its role in renal fibrosis is unclear. The aim was to study the role of PXS25 in matrix protein production under high glucose ± hypoxic conditions in human proximal tubule (HK-2) cells. HK-2 cells were exposed to high glucose (30 mM) ± 100 μM PXS25 in both normoxic (20% O2) and hypoxic (1% O2) conditions for 72 h. Cellular fibronectin, collagen IV, and matrix metalloproteinase-2 (MMP-2) and MMP-9 were assessed. Total and active TGF-β1were measured by ELISA. High glucose and hypoxia independently induced TGF-β1production. Active TGF-β1, but not total TGF-β1was reduced with concurrent PXS25 in the presence of high glucose, but not in hyperglycemia+hypoxia conditions. Hyperglycemia induced fibronectin and collagen IV production ( P < 0.05), as did hypoxia, but only hyperglycemia-induced increases in matrix proteins were suppressed by concurrent PXS25 exposure. High glucose induced MMP-2 and -9 in normoxic and hypoxic conditions, which was not modified in the presence of PXS25. High glucose and hypoxia can independently induce endogenous active TGF-β1production in human proximal tubular cells. PXS25 inhibits conversion of high glucose-induced release of active TGF-β1, only in the absence of hypoxia.

BACKGROUND AND PURPOSE Aortic valve stenosis (AVS) is associated with significant cardiovascular morbidity and mortality. To date, no therapeutic modality has been shown to be effective in retarding AVS progression. We evaluated the effect of angiotensin-converting enzyme inhibition with ramipril on disease progression in a recently developed rabbit model of AVS. EXPERIMENTAL APPROACH The effects of 8 weeks of treatment with either vitamin D2 at 25 000 IU for 4 days a week alone or in combination with ramipril (0.5 mg·kg−1) on aortic valve structure and function were examined in New Zealand white rabbits. Echocardiographic aortic valve backscatter (AVBS) and aortic valve : outflow tract flow velocity ratio were utilized to quantify changes in valve structure and function. KEY RESULTS Treatment with ramipril significantly reduced AVBS and improved aortic valve : outflow tract flow velocity ratio. The intravalvular content of the pro-oxidant thioredoxin-interacting protein was decreased significantly with ramipril treatment. Endothelial function, as measured by asymmetric dimethylarginine concentrations and vascular responses to ACh, was improved significantly with ramipril treatment. CONCLUSIONS AND IMPLICATIONS Ramipril retards the development of AVS, reduces valvular thioredoxin-interacting protein accumulation and limits endothelial dysfunction in this animal model. These findings provide important insights into the mechanisms of AVS development and an impetus for future human studies of AVS retardation using an angiotensin-converting enzyme inhibitor.

BACKGROUND: Diabetic nephropathy is the leading cause of kidney failure in the developed world. Tranilast has been reported to not only act as an anti-inflammatory and anti-fibrotic compound, but it also exerts anti-oxidative stress effects in diabetic nephropathy. Thioredoxin-interacting protein (Txnip) is the endogenous inhibitor of the anti-oxidant thioredoxin and is highly up-regulated in diabetic nephropathy, leading to oxidative stress and fibrosis. In this study, we aimed to investigate whether tranilast exerts its anti-oxidant properties through the inhibition of Txnip. METHODS: Heterozygous Ren-2 rats were rendered diabetic with streptozotocin. Another group of rats were injected with citrate buffer alone and treated as non-diabetic controls. After 6 weeks of diabetes, diabetic rats were divided into two groups: one group gavaged with tranilast at 200 mg/kg/day and another group with vehicle. RESULTS: Diabetic rats had a significant increase in albuminuria, tubulointerstitial fibrosis, peritubular collagen IV accumulation, reactive oxygen species (ROS) and macrophage infiltration (all P < 0.05). These changes were associated with an increase in Txnip mRNA and protein expression in the tubules and glomeruli of diabetic kidney. Treatment with tranilast for 4 weeks significantly attenuated Txnip up-regulation in diabetic rats and this was associated with a reduction in ROS, fibrosis and macrophage infiltration (all P < 0.05). CONCLUSIONS: This is the first study to demonstrate that tranilast not only has anti-inflammatory and anti-fibrotic effects as previously reported but also attenuates the up-regulation of Txnip and oxidative stress in diabetic nephropathy.

Excessive reactive oxygen species play a key role in the pathogenesis of diabetic nephropathy, but to what extent these result from increased generation, impaired antioxidant systems, or both is incompletely understood. Here, we report the expression, localization, and activity of the antioxidant thioredoxin and its endogenous inhibitor thioredoxin interacting protein (TxnIP) in vivo and in vitro. In normal human and rat kidneys, expression of TxnIP mRNA and protein was most abundant in the glomeruli and distal nephron (distal convoluted tubule and collecting ducts). In contrast, thioredoxin mRNA and protein localized to the renal cortex, particularly within the proximal tubules and to a lesser extent in the distal nephron. Induction of diabetes in rats increased expression of TxnIP but not thioredoxin mRNA. Kidneys from patients with diabetic nephropathy had significantly higher levels of TxnIP than control kidneys, but thioredoxin expression did not differ. In vitro, high glucose increased TxnIP expression in mesangial, NRK (proximal tubule), and MDCK (distal tubule/collecting duct) cells, and decreased the expression of thioredoxin in mesangial and MDCK cells. Knockdown of TxnIP with small interference RNA suggested that TxnIP mediates the glucose-induced impairment of thioredoxin activity. Knockdown of TxnIP also abrogated both glucose-induced 3H-proline incorporation (a marker of collagen production) and oxidative stress. Taken together, these findings suggest that impaired thiol reductive capacity contributes to the generation of reactive oxygen species in diabetes in a site- and cell-specific manner.

Transforming growth factor-beta(1) (TGFbeta(1)) is recognized as both a fibrogenic and inflammatory cytokine and plays a critical role in the kidney pathophysiology. The dysregulation of TGFbeta(1) has been linked with the development of diabetic nephropathy. Connective tissue growth factor (CTGF) is a fibrogenic cytokine and is recognized as a downstream mediator of TGFbeta(1) in kidney fibrosis. TGFbeta(1) is involved in immunomodulation and fibrosis in the kidney. However, CTGF plays a more specific role in the fibrogenic pathways in the kidney proximal tubule cells. Moreover, CTGF facilitates TGFbeta(1) signaling and promotes renal fibrosis. This suggests CTGF could be a potential target for kidney fibrosis. Long-term inhibition and targeting TGFbeta(1) directly is problematic, therefore, a more fruitful direction targeting diabetic nephropathy may involve the development of therapeutic strategies specifically targeting CTGF.

Hyperglycemia is a causative factor in the pathogenesis of diabetic nephropathy. Here, we demonstrate the transcriptional profiles of the human proximal tubule cell line (HK-2 cells) exposed to high glucose using cDNA microarray analysis. Thioredoxin-interacting protein (Txnip) was the gene most significantly increased among 10 strongly up-regulated and 15 down-regulated genes. Txnip, heat shock proteins 70 and 90, chemokine (C-C motif) ligand 20, and matrix metalloproteinase-7 were chosen for verification of gene expression. Real-time reverse transcriptase-polymerase chain reaction confirmed the mRNA expression levels of these five genes, consistent with microarray analysis. The increased protein expression of Txnip, CCL20, and MMP7 were also verified by Western blotting and enzyme-linked immunosorbent assay. Increased expression of Txnip and of nitrotyrosine, as a marker of oxidative stress, were confirmed in vivo in diabetic Ren-2 rats. Subsequent studies focused on the dependence of Txnip expression on up-regulation of transforming growth factor (TGF)-beta1 under high-glucose conditions. Overexpression of Txnip and up-regulation of Txnip promoter activity were observed in cells in which the TGF-beta1 gene was silenced in HK-2 cells using short interfering RNA technology. High glucose further increased both Txnip expression and its promoter activity in TGF-beta1 silenced cells compared with wild-type cells exposed to high glucose, suggesting that high glucose induced Txnip through a TGF-beta1-indepen-dent pathway.

AIMS: Smad7 is an inhibitory Smad that regulates transforming growth factor-beta (TGF-beta) signaling. Connective tissue growth factor (CTGF) is recognized as a potent downstream mediator of the fibrogenic effects of TGF-beta1. SMAD binding sites have been identified in both TGF-beta and CTGF promoters. The effect of CTGF on Smad7 expression and its role in the regulation of Smad7 induced by TGF-beta1 in renal tubular cells is unknown. METHODS: Human model of proximal tubular cells (HK-2 cells) was used and confirmed using a diabetic rat model. RT-PCR was performed to measure Smad7, TGF-beta1 and Smad2 and ELISA was performed to measure active TGF-beta1. CTGF or TGF-beta1 was silenced in HK-2 cells using siRNA methodology. RESULTS: TGF-beta1 induced Smad7 in a time-dependent manner, peaking at 30 min (P

Abnormal fibroblast function underlies poor wound healing in patients with diabetes; however, the mechanisms that impair wound healing are poorly defined. Here, we evaluated fibroblasts from individuals who had type 1 diabetes (T1D) for 50 years or more (Medalists, n = 26) and from age-matched controls (n = 7). Compared with those from controls, Medalist fibroblasts demonstrated a reduced migration response to insulin, lower VEGF expression, and less phosphorylated AKT (p-AKT), but not p-ERK, activation. Medalist fibroblasts were also functionally less effective at wound closure in nude mice. Activation of the δ isoform of protein kinase C (PKCδ) was increased in postmortem fibroblasts from Medalists, fibroblasts from living T1D subjects, biopsies of active wounds of living T1D subjects, and granulation tissues from mice with streptozotocin-induced diabetes. Diabetes-induced PKCD mRNA expression was related to a 2-fold increase in the mRNA half-life. Pharmacologic inhibition and siRNA-mediated knockdown of PKCδ or expression of a dominant-negative isoform restored insulin signaling of p-AKT and VEGF expression in vitro and improved wound healing in vivo. Additionally, increasing PKCδ expression in control fibroblasts produced the same abnormalities as those seen in Medalist fibroblasts. Our results indicate that persistent PKCδ elevation in fibroblasts from diabetic patients inhibits insulin signaling and function to impair wound healing and suggest PKCδ inhibition as a potential therapy to improve wound healing in diabetic patients.

Matrix accumulation in the renal tubulointerstitium is predictive of a progressive decline in renal function. Transforming growth factor-β1 (TGF-β1) and, more recently, connective tissue growth factor (CTGF) are recognized to play key roles in mediating the fibrogenic response, independently of the primary renal insult. Further definition of the independent and interrelated effects of CTGF and TGF-β1 is critical for the development of effective antifibrotic strategies. CTGF (20 ng/ml) induced fibronectin and collagen IV secretion in primary cultures of human proximal tubule cells (PTC) and cortical fibroblasts (CF) compared with control values ( P < 0.005 in all cases). This effect was inhibited by neutralizing antibodies to either TGF-β or to the TGF-β type II receptor (TβRII). TGF-β1 induced a greater increase in fibronectin and collagen IV secretion in both PTC ( P < 0.01) and CF ( P < 0.01) compared with that observed with CTGF alone. The combination of TGF-β1 and CTGF was additive in their effects on both PTC and CF fibronectin and collagen IV secretion. TGF-β1 (2 ng/ml) stimulated CTGF mRNA expression within 30 min, which was sustained for up to 24 h, with a consequent increase in CTGF protein ( P < 0.05), whereas CTGF had no effect on TGF-β1 mRNA or protein expression. TGF-β1 (2 ng/ml) induced phosphorylated (p)Smad-2 within 15 min, which was sustained for up to 24 h. CTGF had a delayed effect on increasing pSmad-2 expression, which was evident at 24 h. In conclusion, this study has demonstrated the key dependence of the fibrogenic actions of CTGF on TGF-β. It has further uniquely demonstrated that CTGF requires TGF-β, signaling through the TβRII in both PTCs and CFs, to exert its fibrogenic response in this in vitro model.

Background/Aims: Approximately 30% of individuals with diabetes mellitus are susceptible to diabetic nephropathy, whereas ischemic injury uniformly induces renal impairment. As matrix accumulation correlates with progressive renal disease we assessed parameters associated with matrix turnover in response to high glucose ± hypoxia in human cortical fibroblasts (CF). Methods: CF were grown to confluence and exposed to media containing 5 or 25 mmol/l D-glucose for 72 h with or without a superimposed hypoxic insult. Results: High glucose increased cellular protein content (p < 0.05). Combined high glucose and hypoxia induced a further increase in cellular protein content (p < 0.005), suggestive of a synergistic hypertrophic effect. MMP secretion corresponded inversely with changes in TIMP expression. In cell cultures derived from 2/3 of patients, high glucose increased MMP-9 (p < 0.0005) and MMP-2 (p < 0.005) while TIMP-1 was reduced (p = 0.05). In the remaining cell cultures derived from 1/3 of patients, MMP-2 was reduced (p < 0.0001) while TIMP-1 and TIMP-2 were both increased (p < 0.05). In contrast, hypoxia induced uniform reductions in MMP-9 and MMP-2 in both normal and high glucose conditions. High glucose increased the expression of PAI-1 mRNA (p < 0.05) in all patients independent of changes in the MMP-TIMP axis. Conclusions: In summary, variability was observed in the MMP-TIMP axis following exposure to high glucose. In contrast, high glucose uniformly induces PAI-1 expression. Hypoxic insults uniformly reduce matrix breakdown independent of the prevailing glucose conditions.

Background/Aims: Several recent studies have suggested that angiotensin-converting enzyme (ACE) inhibitors ameliorate chronic cyclosporin A (CyA) tubulo-interstitial disease by mechanisms independent of their antihypertensive effects. The aim of the present study was to determine whether ACE inhibition exerts a direct beneficial effect on the tubulo-interstitium in an in vitro model of chronic CyA nephropathy. Methods: Primary cultures of human proximal tubular cells (PTC) and renal cortical fibroblasts (CF) were exposed for 24 h to CyA in the presence or absence of enalaprilat. Parameters of tubulo-interstitial nephrotoxicity were then measured including collagen synthesis (proline incorporation), tubular viability and function (thymidine incorporation, lactate dehydrogenase release, and apical sodium-hydrogen exchange), and secretion of insulin-like growth factor I, transforming growth factor beta 1 (TGFβ1), and platelet-derived growth factor. Results: CyA promoted CF collagen synthesis, PTC cytotoxicity (suppressed viability, growth and sodium transport), and tubulo-interstitial fibrogenic cytokine release (CF secretion of insulin-like growth factor I and PTC secretion of TGFβ1 and platelet-derived growth factor). Enalaprilat completely reversed the stimulatory effects of CyA on CF collagen synthesis (CyA + enalaprilat 6.40 ± 0.50% vs. CyA alone 8.33 ± 0.56% vs. control 6.57 ± 0.62% vs. enalaprilat alone 5.55 ± 0.93%, p < 0.05) and PTC secretion of TGFβ1 (0.71 ± 0.11, 1.13 ± 0.09, 0.89 ± 0.07, and 0.67 ± 0.09 ng/mg protein/day, respectively, p < 0.05). However, the other manifestations of CyA toxicity were not significantly reversed by concomitant enalaprilat administration. Conclusions: ACE inhibition directly prevents CyA-induced interstitial fibrosis, but not proximal tubule cytotoxicity, independently of haemodynamic and systemic renin-angiotensin system effects. Renoprotection may be partially afforded by directly preventing the tubular secretion of TGFβ1.

Rationale: Loss of insulin action in the endothelium can cause endothelial dysfunction and atherosclerosis. Hyperglycemia and elevated fatty acids induced by diabetes mellitus can activate protein kinase C-β isoforms and selectively inhibit insulin signaling via phosphatidylinositol 3-kinase/Akt pathway to inhibit the activation of endothelial nitric oxide synthase and metabolic actions. Objective: To demonstrate that overexpressing protein kinase C-β2 isoform in endothelial cells can cause selective insulin resistance and exacerbate atherosclerosis in the aorta. Methods and Results: Protein kinase C-β2 isoform was overexpressed in endothelial cells using a promoter of vascular endothelial cell cadherin. These mice were cross-bred with apoE −/− mice [Tg (Prkcb) apoE −/− ]. On a Western diet, Tg(Prkcb) apoE −/− and apoE −/− mice did not differ in systemic insulin sensitivity, glucose tolerance, plasma lipid, or blood pressure. Insulin action in endothelial cells and femoral artery from Tg(Prkcb) apoE −/− mice was impaired by ≈40% with respect to Akt/endothelial nitric oxide synthase activation, and leukocyte-endothelial cell binding increased in cultured lung endothelial cells from Tg(Prkcb) apoE −/− mice compared with that from apoE −/− mice. Basal and angiotensin-stimulated big endothelin-1 levels were elevated in Tg(Prkcb) apoE −/− mice compared with apoE −/− mice. The severity of atherosclerosis in the aorta from Tg(Prkcb) apoE −/− mice increased by ≈70% as measured by en face fat staining and plaque content of the number of smooth muscle cells, macrophages, and extracellular matrix. Conclusions: Specific protein kinase C-β2 activation in the endothelial cells caused dysfunction and accelerated atherosclerosis because of loss of insulin-stimulated Akt/endothelial nitric oxide synthase activation and angiotensin-induced increases in endothelin-1 expression.

To correlate changes between VEGF expression with systemic and retinal oxidative stress and inflammation in rodent models of obesity induced insulin resistance and diabetes.Retinal VEGF mRNA and protein levels were assessed by RT-PCR and VEGF ELISA, respectively. Urinary 8-hydroxydeoxyguanosine (8-OHdG), blood levels of C-reactive protein (CRP), malondialdehyde (MDA), and CD11b/c positive cell ratio were used as systemic inflammatory markers. Retinal expression of Nox2, Nox4, and p47phox mRNA levels were measured as oxidative stress markers. TNF-α, inter-cellular adhesion molecule-1 (ICAM-1), IL1β, and activation of nuclear factor κB (NF-κB) were used as retinal inflammatory markers.Retinal VEGF mRNA and protein expression increased in Zucker diabetic fatty (ZDF(fa/fa)) rats and streptozotosin (STZ) induced diabetic Sprague-Dawley rats, after two months of disease, but not in Zucker fatty (ZF) rats. Systemic markers of oxidative stress and inflammation were elevated in insulin resistant and diabetic rats. Some oxidative stress and inflammatory markers (TNF-α, IL-6, ICAM-1, and IL1-β) were upregulated in the retina of ZDF(fa/fa) and STZ diabetic rats after 4 months of disease. In contrast, activation of NF-κB in the retina was observed in high fat fed nondiabetic and diabetic cis-NF-κB(EGFP) mice, ZF, ZDF(fa/fa), and STZ-induced diabetic rats.Only persistent hyperglycemia and diabetes increased retinal VEGF expression. Some markers of inflammation and oxidative stress were elevated in the retina and systemic circulation of obese and insulin resistant rodents with and without diabetes. Induction of VEGF and its associated retinal pathologies by diabetes requires chronic hyperglycemia and factors in addition to inflammation and oxidative stress.

This study characterizes the effect of glucose-induced activation of protein kinase Cδ (PKCδ) and Src homology-2 domain-containing phosphatase-1 (SHP-1) expression on vascular endothelial growth factor (VEGF) actions in glomerular podocytes in cultures and in glomeruli of diabetic rodents. Elevation of glucose levels induced PKCδ and p38 mitogen-activated protein kinase (p38 MAPK) to increase SHP-1 expression, increased podocyte apoptosis, and inhibited VEGF activation in podocytes and glomerular endothelial cells. The adverse effects of high glucose levels can be negated by molecular inhibitors of PKCδ, p38MAPK, and SHP-1 and only partially reduced by antioxidants and nuclear factor-κB (NF-κB) inhibitor. Increased PKCδ activation and SHP-1 expression correlated with loss of VEGF signaling and podocyte numbers in the glomeruli of diabetic rats and mice. In contrast, diabetic PKCδ-knockout (Prkcd−/−) mice did not exhibit activation of p38 MAPK and SHP-1 or inhibition of VEGF signaling in renal glomeruli. Functionally, diabetic Prkcd−/− mice had decreased expressions of TGFβ, VEGF, and extracellular matrix and less albuminuria than diabetic Prkcd+/+ mice. Hyperglycemia and diabetes can cause glomerular podocyte apoptosis and endothelial dysfunction partly due to increased PKCδ/p38 MAPK activation and the expression of SHP-1 to cause VEGF resistance, independent of NF-κB activation.—Mima, A., Kitada, M., Geraldes, P., Li, Q., Matsumoto, M., Mizutani, K., Qi, W., Li, C., Leitges, M., Rask-Madsen, C., King, G. L. Glomerular VEGF resistance induced by PKCδ/ SHP-1 activation and contribution to diabetic nephropathy.

BackgroundDiabetic cardiomyopathy (DCM) is an increasingly recognized cause of chronic heart failure amongst diabetic patients. Both increased reactive oxygen species (ROS) generation and impaired ROS scavenging have been implicated in the pathogenesis of hyperglycemia-induced left ventricular dysfunction, cardiac fibrosis, apoptosis and hypertrophy. We hypothesized that 3',4'-dihydroxyflavonol (DiOHF), a small highly lipid soluble synthetic flavonol, may prevent DCM by scavenging ROS, thus preventing ROS-induced cardiac damage.Methodology/principal findingsSix week old homozygous Ren-2 rats were randomized to receive either streptozotocin or citrate buffer, then further randomized to receive either DiOHF (1 mg/kg/day) by oral gavage or vehicle for six weeks. Cardiac function was assessed via echocardiography and left ventricular cardiac catheterization before the animals were sacrificed and hearts removed for histological and molecular analyses. Diabetic Ren-2 rats showed evidence of diastolic dysfunction with prolonged deceleration time, reduced E/A ratio, and increased slope of end-diastolic pressure volume relationship (EDPVR) in association with marked interstitial fibrosis and oxidative stress (all PConclusionsInhibition of ROS production and action by DiOHF improved diastolic function and reduced myocyte hypertrophy as well as collagen deposition. These findings suggest the potential clinical utility of antioxidative compounds such as flavonols in the prevention of diabetes-associated cardiac dysfunction.

Krüppel-like factor 6 (KLF6) is a DNA-binding protein containing a triple zinc-fingered motif and plays a key role in the regulation of cell proliferation, differentiation, and development. More recently it has been implicated in hepatic fibrosis via its binding to the transforming growth factor (TGF)-β control element. In the kidney, epithelial-mesenchymal transition (EMT) is a major contributor to the pathogenesis of renal fibrosis, with TGF-β1 being a key mediator of EMT. The present study aimed to determine the role of KLF6 and TGF-β1 in EMT in proximal tubule cells. To determine the relevance in clinical disease, KLF6 was measured in kidneys of streptozotocin-induced diabetic Ren-2 rats and in cells exposed to high (30 mM) glucose. TGF-β1 was confirmed to induce EMT by morphological change, loss of E-cadherin, and gain in vimentin expression. KLF6 mRNA expression was concomitantly measured. To determine the role of KLF6 in EMT, the above markers of EMT were determined in KLF6-silenced (small interfering RNA) and KLF6-overexpressing proximal tubule cells. KLF6 overexpression significantly promoted a phenotype consistent with EMT. High glucose induced KLF6 in proximal tubule cells ( P < 0.05). This increase in KLF6 in response to high glucose was TGF-β1 mediated. In an in vivo model of diabetic nephropathy KLF6 increased at week 8 ( P < 0.05). KLF6 plays a permissive role in TGF-β1-induced EMT in proximal tubule cells. Its upregulation in in vivo models of diabetic nephropathy suggests it as a potential therapeutic target.

Hyperglycaemia is a causative factor in the pathogenesis of diabetic nephropathy, known to induce chemokines in the kidney. Macrophage inflammatory protein-3 alpha (MIP-3 alpha) is a CC chemokine that has been reported to attract memory T lymphocytes. Our previous microarray study showed significant increased level of MIP-3 alpha in high glucose-induced transcriptional profile in renal proximal tubule cells. Transforming growth factor-beta1 (TGF-beta1) is a key regulator in inflammation and fibrosis in diabetes mellitus setting.This study aimed to determine the role of TGF beta 1 in high glucose-induced MIP-3 alpha expression. An in vitro model of human proximal tubular cells (HK-2 cells) and an in vivo model of the transgenic (mRen-2)27 diabetic rat, well characterized as a model of human diabetic nephropathy, were used. Small interfering RNA technology was used to silence TGF-beta1 gene in HK-2 cells and subsequent experiments were performed to measure mRNA and protein levels of MIP-3 alpha using real time reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was used to measure the protein level of MIP-3 alpha and CD3 a marker of T lymphocytes in the in vivo model.MIP-3 alpha mRNA and protein expression was increased in HK-2 cells by high glucose and TGF-beta1. MIP-3 alpha was up-regulated in the dilated tubules of diabetic rats compared with non-diabetic control animals and CD3 was found to be present around the dilated tubules expressing MIP-3 alpha. This up-regulation was attenuated in the presence of an angiotensin-converting enzyme (ACE) inhibitor. MIP-3 alpha expression significantly decreased in cells in which the TGF-beta1 gene was silenced using small interfering RNA. Furthermore, exposure to high glucose did not induce MIP-3 alpha expression in TGF-beta1 gene silenced cells compared with wild-type cells.In summary, we have uniquely demonstrated that high glucose increases MIP-3 alpha through a TGF beta 1 dependent pathway, suggesting the centrality of TGF-beta1 in both the inflammatory and previously demonstrated fibrotic responses in diabetic nephropathy.

Activation of protein kinase C (PKC) isoforms has been implicated as a central mediator in the pathogenesis of diabetic nephropathy. Although high glucose levels stimulate catalytic activity of PKC, the effects of high glucose levels on the expression of genes encoding PKC isoforms are unknown. We sought to determine whether in addition to activation, diabetes may lead to increased transcription of two PKC isoforms that have been implicated in the pathogenesis of diabetic nephropathy, PKC-alpha and PKC-beta.Recent advances in molecular biological techniques now permit quantitative analysis of mRNA from archival, formalin-fixed, paraffin-embedded tissue sections. RNA was extracted from scraped 6 microm sections of biopsy tissue, and PRKC-alpha and PRKC-beta (also known as PRKCA and PRKCB) mRNA measured using real-time PCR. Expression of genes encoding PKC isoforms was examined in renal biopsies (n=25) with classical histological features of diabetic nephropathy and compared with that in normal control tissue (n=6). Peptide localisation of PKC-alpha, PKC-beta and the activated forms phosphorylated PKC-alpha and -beta was also performed on matched paraffin-embedded sections of renal biopsies using immunohistochemistry. The effects of high glucose on PRKC-beta expression and peptide production in cultured human proximal tubular epithelial cells were assessed.Quantitative real-time PCR demonstrated a 9.9-fold increase in PRKC-beta mRNA in kidney biopsies of diabetic patients relative to control (p0.001). No increase in PRKC-alpha expression was seen. In addition, a correlation between renal PRKC-beta mRNA and HbA(1c) was observed in diabetic patients (r=0.63, p0.05). There was co-localisation of PKC-beta and phospho-PKC-beta predominantly to proximal tubules. A 60% increase in PRKC-beta mRNA and peptide in cultured human proximal tubular epithelial cells exposed to high glucose (p0.05) was seen in vitro.PKC-beta is upregulated at the gene expression level in human diabetic nephropathy. PRKC-beta mRNA correlates closely with serum HbA(1c), possibly partially explaining the relationship between glycaemic control and progression of diabetic nephropathy. Archival human tissue provides a valuable resource for molecular analyses.

Transforming growth factor-β1 (TGF-β1) functions as an important immunomodulatory cytokine in human kidney. Evidence suggests that connective tissue growth factor (CTGF) is an important downstream mediator of the profibrotic effects of TGF-β1. However, the role of CTGF in TGF-β1-induced chemokine production remains unknown. This study was undertaken to determine whether CTGF is involved in mediating TGF-β1-induced chemokine production in renal proximal tubular (HK-2) cells. Interleukin-8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) were measured. TGF-β1 induced an increase in IL-8 and MCP-1 (both P < 0.05) compared with control levels. CTGF was effectively silenced using small interference RNA (siRNA) in HK-2 cells. RT-PCR and real-time PCR confirmed a 94% reduction in CTGF mRNA. In the CTGF-silenced cells, TGF-β1-stimulated IL-8 and MCP-1 secretion was not altered compared with control cells. Similarly, basal secretion of IL-8 and MCP-1 was not changed in CTGF-silenced cells. The direct effect of CTGF (20, 200, and 400 ng/ml) on IL-8 and MCP-1 was assessed at 24-, 48-, and 72-h time points and no stimulation was observed. Our studies further demonstrate that in the CTGF gene-silenced cells, CTGF partially mediates TGF-β1-induced fibronectin and collagen IV secretion. These data suggest that TGF-β1 induced IL-8 and MCP-1 via CTGF-independent pathway. TGF-β mediates both fibrosis and chemokine production in the proximal tubule of the kidney. However, CTGF plays a more specific role as a downstream mediator of TGF-β1-induced fibrosis.

Background/Aims: Tubulointerstitial pathology with the accumulation of extracellular matrix are pathological hallmarks of diabetic nephropathy that are directly related to declining renal function. Tranilast (N-[3,4-dimethoxycinnamoyl]anthranilic acid), an inhibitor of transforming growth factor-β (TGF-β), used to treat hypertrophic scars has recently been shown in pilot studies to exert a beneficial effect in advanced diabetic nephropathy in humans. However, its effects on diabetic renal pathology are unknown. Methods: Studies were conducted using a transgenic model, the diabetic (mRen-2)27 rat, which develops many of the structural and functional characteristics of human diabetic nephropathy when diabetes is induced with streptozotocin (STZ). An experimental design was chosen to mimic, in part, the clinical context with drug therapy (tranilast 400 mg/kg/ day) initiated in established disease (8 weeks after STZ) and in the presence of persistent hyperglycaemia and hypertension. Results: At 16 weeks, diabetes was associated with progressive albuminuria, tubulointerstitial fibrosis and tubular atrophy. Without affecting blood pressure or blood glucose, tranilast treatment was associated with a 83% reduction in tubulointerstitial fibrosis (p < 0.001), a 58% reduction in tubular atrophy (p < 0.01) and near normalization of albuminuria (p < 0.05) in diabetic Ren-2 rats. In vitro studies in primary cultures of human renal cortical fibroblasts demonstrated a reduction in TGF-β-induced hydroxyproline incorporation and fibronectin synthesis with tranilast 100 µM. Conclusion: Tranilast, when administered during the course of experimental diabetic nephropathy, attenuates tubulointerstitial pathology and albuminuria. These findings are consistent with the antagonist effects of tranilast on TGF-β actions in the diabetic kidney.

Aims/hypothesis. We investigated the effects of constant and intermittently increased glucose concentrations on human proximal tubule cells and cortical fibroblasts in primary culture. Methods. Cells were grown to confluence and then exposed for 4 days to 6.1 mmol/l d-glucose (normal), 25 mmol/l d-glucose (high), or 6.1 mmol/l alternating with 25 mmol/l d-glucose on a daily basis. Results. In proximal tubular cells, exposure to high glucose caused an 11 % increase in thymidine uptake (p < 0.05), a 230 % increase in secretion of transforming growth factor beta 1 (TGF-β1; p < 0.05) and a 393 % increase in platelet derived growth factor. Intermittent exposure to high glucose caused thymidine uptake to further increase by 42 % (p < 0.01) and TGF-β1 secretion by 352 % (p < 0.01) but no additional increase in platelet-derived growth factor secretion was observed. Cellular protein content increased by 27 % (p < 0.05) and collagen synthesis by 29 % (p < 0.05), changes that were not observed in cells constantly exposed to high glucose. In cortical fibroblasts constant exposure to high glucose caused a 35 % increase in thymidine uptake (p < 0.01). Intermittently high glucose increased thymidine incorporation a further 58 % (p < 0.001), collagen synthesis by 65 % (p < 0.01) and insulin-like growth factor binding protein 3 secretion by 216 % (p < 0.01). Conclusion/interpretation. In cultured human tubulointerstitial cells, increased glucose concentrations change cell growth, collagen synthesis and cytokine secretion. These effects are enhanced following intermittent exposure to high glucose, indicating that short lived excursions in glycaemic control have important pathological effects on the human tubulointerstitium. [Diabetologia (1999) 42: 1113–1119]

Tranilast (N-[3,4-dimethoxycinnamoyl]anthranilic acid) is a synthetic compound that we have recently reported to inhibit transforming growth factor-beta1 (TGF-beta1)-induced tubulointerstitial fibrosis in the kidney. Connective tissue growth factor (CTGF) is recognized as a potent downstream mediator of TGF-beta1. Both proximal tubule cells (PTCs) and cortical fibroblasts (CFs) are considered to be responsible for the production of tubulointerstitial extracellular matrix (ECM). These studies were undertaken to assess the profibrotic effects of CTGF in an in vitro model of the human PTCs and CFs, and to determine whether tranilast is effective in limiting the in vitro matrix responses induced by CTGF. Primary cultures of PTCs and CFs were exposed to CTGF (20 ng/ml)+/-tranilast (100 microM). Cell hypertrophy and the secretion of the ECM proteins fibronectin and collagen IV were determined. The effects of tranilast on TGF-beta1-induced CTGF mRNA expression and on phosphorylation of Smad2 were determined. CTGF significantly induced cell hypertrophy, increased fibronectin, and collagen IV secretion in PTCs and CFs. In all cases, the CTGF-induced increase in ECM protein was inhibited in the presence of tranilast. Tranilast reduced CTGF mRNA and phosphorylation of Smad2, which were induced by TGF-beta1 in PTCs and CFs. These results suggest that tranilast is a potential effective antifibrotic compound in the kidney, exerting its effects via inhibition of TGF-beta1-induced CTGF expression and downstream activation of the Smad2 pathway in both PTCs and CFs.