MicroRNA-5010-5p ameliorates high-glucose induced inflammation in renal tubular epithelial cells by modulating the expression of PPP2R2D.

INTRODUCTION: We previously reported the significant upregulation of eight circulating exosomal microRNAs (miRNAs) in patients with diabetic kidney disease (DKD). However, their specific roles and molecular mechanisms in the kidney remain unknown. Among the eight miRNAs, we evaluated the effects of miR-5010-5p on renal tubular epithelial cells under diabetic conditions in this study. RESEARCH DESIGN AND METHODS: We transfected the renal tubular epithelial cell line, HK-2, with an miR-5010-5p mimic using recombinant plasmids. The target gene of hsa-miR-5010-5p was identified using a dual-luciferase assay. Cell viability was assessed via the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Moreover, mRNA and protein expression levels were determined via real-time PCR and western blotting, respectively. RESULTS: High glucose levels did not significantly affect the intracellular expression of miR-5010-5p in HK-2 cells. Transfection of the miR-5010-5p mimic caused no change in cell viability. However, miR-5010-5p-transfected HK-2 cells exhibited significantly decreased expression levels of inflammatory cytokines, such as the monocyte chemoattractant protein-1, interleukin-1ß, and tumor necrosis factor-ɑ, under high-glucose conditions. These changes were accompanied by the restored expression of phosphorylated AMP-activated protein kinase (AMPK) and decreased phosphorylation of nuclear factor-kappa B. Dual-luciferase assay revealed that miR-5010-5p targeted the gene, protein phosphatase 2 regulatory subunit B delta (PPP2R2D), a subunit of protein phosphatase 2A, which modulates AMPK phosphorylation. CONCLUSIONS: Our findings suggest that increased miR-5010-5p expression reduces high glucose-induced inflammatory responses in renal tubular epithelial cells via the regulation of the target gene, PPP2R2D, which modulates AMPK phosphorylation. Therefore, miR-5010-5p may be a promising therapeutic target for DKD.

Attenuation of diabetic kidney injury in DPP4-deficient rats; role of GLP-1 on the suppression of AGE formation by inducing glyoxalase 1.

Dipeptidyl peptidase 4 (DPP4) inactivates incretin hormone glucagon-like peptide-1. DPP4 inhibitors may exert beneficial effects on diabetic nephropathy (DN) independently of glycemic control; however, the mechanisms underlying are not fully understood. Here, we investigated the mechanisms of the beneficial effects of DPP4 inhibition on DN using DPP4-deficient (DPP4-def) rats and rat mesangial cells.Blood glucose and HbA1c significantly increased by streptozotocin (STZ) and no differences were between WT-STZ and DPP4-def-STZ. The albumin level in urine decreased significantly and the albumin/creatinine ratio decreased slightly in DPP4-def-STZ. The glomerular volume in DPP4-def-STZ significantly decreased compared with that of WT-STZ. Advanced glycation end products formation, receptor for AGE (RAGE) protein expression, and its downstream inflammatory cytokines and fibrotic factors in kidney tissue, were significantly suppressed in the DPP4-def-STZ compared to the WT-STZ with increasing glyoxalase-1 (GLO-1) expression responsible for the detoxification of methylglyoxal (MGO). In vitro, exendin-4 suppressed MGO-induced AGEs production by enhancing the expression of GLO-1 and nuclear factor-erythroid 2 p45 subunit-related factor 2, resulting in decreasing pro-inflammatory cytokine levels. This effect was abolished by GLO-1 siRNA.Our data suggest that endogenously increased GLP-1 in DPP4-deficient rats contributes to the attenuation of DN partially by regulating AGEs formation via upregulation of GLO-1 expression.

Lysophosphatidic acid receptor 1 inhibitor, AM095, attenuates diabetic nephropathy in mice by downregulation of TLR4/NF-κB signaling and NADPH oxidase.

Diabetic nephropathy (DN) is one of the major long-term complications of diabetes. Lysophosphatidic acid (LPA) signaling has been implicated in renal fibrosis. In our previous study, we found that the LPA receptor 1/3 (LPAR1/3) antagonist, ki16425, protected against DN in diabetic db/db mice. Here, we investigated the effects of a specific pharmacological inhibitor of LPA receptor 1 (LPA1), AM095, on DN in streptozotocin (STZ)-induced diabetic mice to exclude a possible contribution of LPAR3 inhibition. AM095 treatment significantly reduced albuminuria and the albumin to creatinine ratio and significantly decreased the glomerular volume and tuft area in the treated group compared with the STZ-vehicle group. In the kidney of STZ-induced diabetic mice, the expression of LPAR1 mRNA and protein was positively correlated with oxidative stress. AM095 treatment inhibited LPA-induced reactive oxygen species production and NADPH oxidase expression as well as LPA-induced toll like receptor 4 (TLR4) expression in mesangial cells and in the kidney of STZ-induced diabetic mice. In addition, AM095 treatment suppressed LPA-induced pro-inflammatory cytokines and fibrotic factors expression through downregulation of phosphorylated NFκBp65 and c-Jun N-terminal kinases (JNK) in vitro and in the kidney of STZ-induced diabetic mice. Pharmacological or siRNA inhibition of TLR4 and NADPH oxidase mimicked the effects of AM095 in vitro. In conclusion, AM095 is effective in preventing the pathogenesis of DN by inhibiting TLR4/NF-κB and the NADPH oxidase system, consequently inhibiting the inflammatory signaling cascade in renal tissue of diabetic mice, suggesting that LPAR1 antagonism might provide a potential therapeutic target for DN.