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Hypoxia causes proximal tubular cell damage in diabetes, even though proximal tubular cells have an adaptive system to combat hypoxia involving induction of hypoxia factor-1 (HIF-1) and inhibition of mechanistic target of rapamycin complex 1 (mTORC1). Here, we examined the interference effect of altered glucose and lipid metabolism on the hypoxia responses in proximal tubular cells. In culture, hypoxia alone induced HIF-1 and inhibited mTORC1, preventing death in proximal tubular cells. However, hypoxia with high glucose and palmitate increased mTORC1 activity and promoted apoptosis in proximal tubular cells, which was inhibited by pharmacological and genetic inactivation of mTORC1. Since inhibition of all mTORC1's physiological functions regulated by growth factors including insulin causes various adverse effects, we screened for a microRNA that can inhibit only pro-apoptotic effects of mTORC1 to discover a safe therapeutic target. This screen found microRNA-148b-3p was able to specifically inhibit mTORC1-dependent apoptosis in hypoxic proximal tubular cells exposed to high glucose and palmitate, without affecting insulin-dependent mTORC1 activation. Furthermore, tumor necrosis factor receptor (TNFR) 2 was the target of microRNA-148b-3p and its suppression inhibited apoptosis. Finally, enhanced apoptosis with TNFR2 overexpression was found in hypoxic and mTORC1-activated proximal tubular cells in diabetic rats. Thus, diabetes activated mTORC1 even in hypoxic proximal tubular cells, leading to apoptosis by reducing microRNA-148b-3p expression. Modulating this pathogenic pathway may be a novel therapy for proximal tubular cell damage in diabetes.

MAPK/AP-1-dependent regulation of PAI-1 gene expression by TGF-β in rat mesangial cells. Background Receptor-regulated Smads and/or mitogen-activated protein kinases (MAPKs) are involved in transforming growth factor-β (TGF-β)-nduced expression of various genes, including plasminogen activator inhibitor-1 (PAI-1). Because the sequence of the promoter region in rat PAI-1 gene differs from that in the human gene, we examined the mechanisms of TGF-β-induced rat PAI-1 expression in rat mesangial cells. Methods TGF-β1-induced PAI-1 and c-fos mRNA expressions were determined by Northern blot analysis. Activation of MAPKs and Smad proteins was evaluated by an immunoblot analysis. DNA binding activities of nuclear protein were examined by using an electrophoretic mobility shift assay (EMSA). The activities of PAI-1 promoter were measured by a luciferase reporter assay. Results Extracellular-regulated kinase (ERK) and c-Jun NH-terminal kinase (JNK) phosphorylation, c-fos mRNA expression, and activator protein-1 (AP-1) DNA binding activity stimulated by TGF-β1 were completely suppressed by the ERK kinase (MEK) inhibitors. EMSA and reporter analysis revealed that an AP-1-like sequence located in the proximal region of the rat PAI-1 promoter was the target for TGF-β1, and the disruption of this AP-1-like sequence suppressed basal and TGF-β1-induced promoter activation. TGF-β1 also stimulated nuclear translocation of Smads and binding to palindromic Smad binding element (SBE) located in the rat PAI-1 promoter, without being affected by MEK inhibitor. Point mutation and deletion of palindromic SBE did not affect TGF-β1-induced rat PAI-1 promoter activity. Moreover, interferon-γ (IFN-γ) inhibited TGF-β1-induced PAI-1 expression through selectively suppressing the ERK-AP-1 pathway. Conclusion These results suggest that the essential requirement of MAPK/AP-1 activation for TGF-β1-induced PAI-1 expression is unique to rat mesangial cells.

15-Deoxy-Δ 12,14 -prostaglandin J 2 inhibits IL-1β–induced cyclooxygenase-2 expression in mesangial cells . Background Cyclooxygenase-2 (COX-2), a key enzyme in the synthesis of prostaglandins, is induced in mesangial cells in response to proinflammatory cytokines. Recently, 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ), one of the natural ligands of peroxisome proliferator-activated receptor γ (PPARγ), has been reported to have an anti-inflammatory effect. Therefore, we examined the effect of 15d-PGJ 2 on COX-2 expression in cultured rat mesangial cells. Methods Mesangial cells were incubated with 15d-PGJ 2 for 30 minutes and then exposed to interleukin-1β (IL-1β). The expression of COX-2 mRNA and proteins was determined by Northern blot and immunoblot analyses, respectively. Accumulation of prostaglandin E 2 (PGE 2 ) was measured by an enzyme-linked immunosorbent assay (ELISA). Activities of mitogen-activated protein kinases (MAPKs) were evaluated by an immunoblot analysis. DNA binding activities of activator protein-1 (AP-1) or nuclear factor-κB (NF-κB) were examined by an electrophoretic mobility shift assay (EMSA). The activities of PPAR responsive elements (PPRE) and COX-2 promoter were measured by a luciferase reporter assay. Results 15D-PGJ 2 significantly suppressed IL-1β–induced COX-2 expression and PGE 2 production, but thiazolidinediones, synthetic PPARγ ligands, did not affect COX-2 expression. Moreover, the cells transfected with a PPRE luciferase reporter did not respond to 15d-PGJ 2 . IL-1β rapidly activated extracellular signal-regulated kinase (ERK) and c-Jun NH 2 -terminal kinase (JNK), which were involved in the up-regulation of COX-2 induction, but 15d-PGJ 2 inhibited the activation of these kinases. 15d-PGJ 2 inhibited the IL-1β–induced increase in binding activities of nuclear proteins to consensus AP-1 site and AP-1–like site of COX-2 promoter but not of NF-κB. IL-1β was unable to activate the COX-2 promoter when the AP-1–like site was mutated. Conclusions These data suggest that 15d-PGJ 2 inhibits IL-1β–induced COX-2 expression, independent of PPARγ activation, by suppression of ERK and JNK pathways and AP-1 activation in mesangial cells. Thus, 15d-PGJ 2 may play an important role in the negative feedback mechanism of COX-2 expression in renal inflammation and may be useful as an anti-inflammatory agent.

Integrin β1 and dipeptidyl peptidase (DPP)-4 play roles in endothelial cell biology. Vascular endothelial growth factor (VEGF)-A inhibits endothelial-to-mesenchymal transition (EndMT) through VEGF-R2, but through VEGF-R1 promotes EndMT by reducing the bioavailability of VEGF-A. Here we tested whether DPP-4-integrin β1 interactions have a role in EndMT in the renal fibrosis of diabetic nephropathy. In streptozotocin-induced fibrotic kidneys in diabetic CD-1 mice, levels of endothelial DPP-4, integrin β1, and phospho-integrin β1 were all higher and associated with plasma cystatin C elevation. The DPP-4 inhibitor linagliptin ameliorated kidney fibrosis, reduced plasma cystatin C levels, and suppressed endothelial levels of DPP-4, integrin β1, and phospho-integrin β1. In cultured endothelial cells, DPP-4 and integrin β1 physically interacted. Suppression of DPP-4 by siRNA was associated with suppression of integrin β1 and vice versa. Knockdown of either integrin β1 or DPP-4 resulted in the silencing of TGF-β2-induced TGF-β receptor heterodimer formation, smad3 phosphorylation, and EndMT. DPP-4 negatively regulated endothelial viability signaling by VEGF-R2 suppression and VEGF-R1 induction in endothelial cells. Thus, DPP-4 and integrin β1 interactions regulate key endothelial cell signal transduction in both physiological and pathological conditions including EndMT. Hence, inhibiting DPP-4 may be a therapeutic target for treating kidney fibrosis in diabetes.

Dinucleotide repeat polymorphism of matrix metalloproteinase-9 gene is associated with diabetic nephropathy.BackgroundAlthough genetic susceptibility has been proposed as an important factor for the development and progression of diabetic nephropathy, the definitive gene has not been identified. To identify the genetic marker for diabetic nephropathy, we examined the association between the (A-C)n dinucleotide repeat polymorphism upstream of the matrix metalloproteinase-9 (MMP-9) gene and diabetic nephropathy in a group of Japanese patients with type 2 diabetes.MethodsPatients were divided into three groups based on their urinary albumin excretion rate (AER) and the stage of diabetic retinopathy as follows: uncomplicated group (U), normal albuminuria (AER

As renal lipotoxicity can lead to chronic kidney disease (CKD), we examined the role of peroxisome proliferator-activated receptor (PPAR)-α, a positive regulator of renal lipolysis. Feeding mice a high-fat diet induced glomerular injury, and treating them with fenofibrate, a PPARα agonist, increased the expression of lipolytic enzymes and reduced lipid accumulation and oxidative stress in glomeruli, while inhibiting the development of albuminuria and glomerular fibrosis. In mice given an overload of free fatty acid-bound albumin to induce tubulointerstitial injury, fenofibrate attenuated the development of oxidative stress, macrophage infiltration, and fibrosis, and enhanced lipolysis in the renal interstitium. Fenofibrate inhibited palmitate-induced expression of profibrotic plasminogen activator inhibitor-1 (PAI-1) in cultured mesangial cells, and the expression of both monocyte chemoattractant protein-1 and PAI-1 in proximal tubular cells along with the overexpression of lipolytic enzymes. Thus, fenofibrate can attenuate lipotoxicity-induced glomerular and tubulointerstitial injuries, with enhancement of renal lipolysis. Whether amelioration of renal lipotoxicity by PPARα agonists will turn out to be a useful strategy against CKD will require direct testing.

Alteration of mesangial response to ANP and angiotensin II by glucose. To test the hypothesis that the function of glomerular mesangial cells is impaired in diabetes, we examined the responsiveness of mesangial cells cultured under high concentrations of glucose to atrial natriuretic peptide (ANP 1 ) and angiotensin II (Ang II). The ANP-induced accumulation of cGMP was enhanced in mesangial cells cultured under high glucose conditions, possibly due to the activation of particulate guanylate cyclase. Ang II action in mesangial cells was evaluated by measuring the ability of Ang II to inhibit ANP-induced cGMP accumulation through both activating phosphodiesterase (initial phase) and inhibiting guanylate cyclase (maintenance phase). The inhibition of both ANP-induced cellular cGMP accumulation and particulate guanylate cyclase activity by Ang II was significantly reduced in mesangial cells cultured under high concentrations of glucose. Moreover, in the cells exposed to high concentrations of glucose, both basal and Ang II-stimulated levels of inositol 1,4,5-trisphosphate (IP 3 ) were significantly reduced. These results indicate that, in high glucose conditions, the actions of ANP and Ang II are modulated differently, resulting in the impairment of contractile responsiveness of mesangial cells.

Dual mechanism of angiotensin II inhibits ANP-induced mesangial cGMP accumulation. To evaluate an interaction between vasoconstrictive (Ang II) 1 and vasodilating (Ang) peptides, we examined the effect of Ang II on ANP-induced accumulation of cGMP in cultured glomerular mesangial cells. ANP rapidly increased intracellular cGMP levels, with a peak stimulation at one minute in the absence of IBMX and at ten minutes in the presence of IBMX. The ANP-induced cGMP accumulation was significantly inhibited when the cells were treated with Ang II simultaneously with ANP for one minute in the absence of IBMX. This inhibitory effect of Ang II was completely abolished by IBMX and significantly reduced in calcium-free media or by W7, but not affected by H7. Similar inhibitory effect was observed when cells were treated with A23187 but not with TPA for one minute. In the presence of IBMX, Ang II inhibited ANP-induced cGMP accumulation when cells were treated with Ang II for 15 minutes prior to the stimulation by ANP. This inhibition by Ang II was blocked by H7. ANP-induced increase in particulate guanylate cyclase activity was significantly reduced in the cells treated with Ang II or TPA. This reduction of enzyme activity was also prevented by H7. These results indicate that Ang II inhibits ANP-induced cGMP accumulation in cultured glomerular mesangial cells through at least two mechanisms; one is the activation of calcium-dependent, calmodulin-stimulated cyclic nucleotide phosphodiesterase in the initial phase, and the other is the inhibition of guanylate cyclase resulting from protein kinase C activation in the maintenance phase.

Differential inhibition of mesangial MAP kinase cascade by cyclic nucleotides. The agents which increase intracellular cyclic AMP (cAMP) or cyclic GMP (cGMP) have been found to counteract the effects of the vasoconstrictive agents such as endothelin-1 (ET-1). To clarify the mechanism of this interaction, we evaluated the activities of mitogen-activated protein kinase (MAPK) cascade, one of the important signal transduction system of ET-1. Beraprost sodium, an analogue of PGI 2 , and adrenomedullin, a cAMP-raising agent, inhibited ET-1-induced activation of MAPK. Dibutyryl cAMP (Bt 2 -cAMP) and 8-bromo-cGMP (8-Br-cGMP), cell permeable analogues of cAMP and cGMP, were also able to inhibit the activation of MAPK and MAPK kinase (MAPKK) by ET-1 without interfering basal activities. In contrast, phorbol 12,13-dibutylate (PDBu)-induced activation of MAPK and MAPKK was inhibited by Bt 2 -cAMP but not by 8-Br-cGMP. Interestingly, atrial natriuretic peptide (ANP) partially inhibited PDBu-induced activation of MAPK and MAPKK. These results indicate that cAMP and cGMP inhibit ET-1-induced activation of MAPK in cultured mesangial cells at different steps; the former might inhibit at a step downstream of PKC and the latter prior to PKC. The data also suggest that ANP might have cGMP-independent effect on MAPK.

TGF-β1 stimulates glucose uptake by enhancing GLUT1 expression in mesangial cells. Background An increase in the expression of transforming growth factor-β1 (TGF-β1) has been proposed to play an important role in the excessive production of extracellular matrix (ECM) proteins seen in diabetes. Because the linkage between glucose metabolism and ECM protein production was found in mesangial cells overexpressed with the brain-type glucose transporter (GLUT1), we hypothesized that TGF-β1 could affect glucose metabolism. Methods To prove this hypothesis, we examined the effect of TGF-β1 on glucose uptake, the first step of glucose metabolism, in mesangial cells. 2-Deoxy-D-glucose (2DOG) uptake and the expression of GLUT1 were measured in mesangial cells exposed to various concentrations of TGF-β1. The kinetic constants were determined using 2DOG and 3-O-methyl-D-glucose (3OMG). The effect of anti–TGF-β neutralizing antibody on 2DOG uptake and GLUT1 mRNA was also examined in mesangial cells cultured under high-glucose (22.2mm) conditions for 72hours. Results TGF-β1 stimulated 2DOG uptake in mesangial cells by approximately 2.5-fold in a dose- (1.25ng/ml maximum) and time-dependent manner, with a peak stimulation at nine hours. The increase in 2DOG uptake by TGF-β1 was completely abolished by the addition of 1 μg/ml cycloheximide, and kinetic analysis of 2DOG or 3OMG uptake revealed an increase in V max by TGF-β1. Furthermore, TGF-β1 enhanced the expression of GLUT1 mRNA from one hour, followed by an enhancement of the expression of GLUT1 protein at nine hours. Finally, 2DOG uptake was significantly enhanced in cells cultured under high-glucose (22.2mm) conditions as compared with that in cells under normal glucose (5.6mm) conditions, and this increase in 2DOG uptake in cells under high-glucose conditions was inhibited by the addition of anti–TGF-β neutralizing antibody. Conclusions TGF-β1 stimulates glucose uptake by enhancing the expression of GLUT1 in mesangial cells, which leads to the acceleration of intracellular metabolic abnormalities in diabetes.

Role of mitogen-activated protein kinases as downstream effectors of transforming growth factor-β in mesangial cells. Transforming growth factor-β (TGF-β) is a multifunctional cytokine that regulates cell proliferation, differentiation, and production of extracellular matrix proteins in various types of cells including mesangial cells. Although TGF-β has been also known as an important player in the pathogenesis of various fibrotic diseases including glomerulosclerosis, signal-transduction cascades of TGF-β have remained to be clarified. However, emerging evidence indicates that TGF-β can activate various signal transduction cascades such as Smad proteins and mitogen-activated protein kinases (MAPKs) in many types of cells. Here, we examine the role of MAPKs in TGF-β-induced gene expression of extracellular matrix proteins in mesangial cells. TGF-β increases extracellular signal-regulated kinase (ERK) activity, one of the MAPKs, and the expression of fibronectin mRNA and protein in rat mesangial cells. Furthermore, PD98059, a specific inhibitor of MAPK/ERK kinase (MEK), can inhibit this TGF-β-induced fibronectin expression. These data suggest that MAPKs play an important role in TGF-β-mediated extracellular matrix production in mesangial cells.