Lipid deposits can injury the kidney of diabetic patients and models. Sterol regulatory element binding protein-1 (SREBP-1) is transcription factor regulating the synthesis of fatty acid and triglyceride. At present whether the expression of SREBP-1 makes some effects on the lipid accumulation in diabetic kidney is not still clear completely. The purpose of our in vivo and in vitro study is to investigate the relationship between the expression of SREBP-1 and lipid abnormal metabolism in the type 1 diabetic rats and explore to inhibit SREBP-1 gene expression by RNA interfere in human renal proximal tubular epithelial cells line (HKC cells). The animal experiment showed that triglyceride and SREBP-1 were up-regulated in proximal tubule of diabetic rats' kidney, which may result in increase of transforming growth factor-beta1 (TGF-beta1) and accumulation of extracellular matrix (ECM). The further HKC cells experiment confirmed SREBP-1 increasing resulted into lipid droplet formation. The expression of fatty acid synthase (FAS) in HKC cells transfected with specific plasmid for SREBP-1 gene was significantly more than that of the cells transfected with the control plasmid pcDNA3.1 and that of the untransfected cells. Simultaneously, up-regulation of TGF-beta1 and fibronectin, an ECM glycoprotein, was evident in HKC cells transfected by specific SREBP-1 plasmid. Furthermore, we found that high glucose was a positive factor on the expression of SREBP-1 at protein and mRNA levels in HKC cells. High glucose makes effects on SREBP-1 in time-dependent manner, and the greatest effect was at 48 h. In addition, two effective eukaryotic expression plasmid vectors of shRNA aimed at SREBP-1 were designed and constructed successfully. Compared with the negative control plasmid group, the levels of the expression of SREBP-1 were inhibited by 24.11 and 36.15%, respectively, at mRNA level, 20.80 and 37.59%, respectively, at precursor segment of protein level, and 38.12 and 52.24%, respectively, at mature segment of protein level at 48 h after transfection. In vivo and in vitro study suggested that high glucose caused increasing SREBP-1 mRNA and protein in renal proximal tubule epithelial cells of type 1 diabetic rats. Increasing SREBP-1 plays an important role in the pathogenesis of renal lipid accumulation by up-regulation of FAS and ECM accumulation by inducing TGF-beta1 expression. The application of vector-mediated RNAi could markedly inhibit the expression of SREBP-1 in HKC cells, which is a promising tool for future research into the mechanisms of renal lipid accumulation in vivo.

The objective of this study was to investigate the role of miR-148a-3p in lupus nephritis (LN) based on data from previous studies and a microRNA assay. We evaluated the miR-148a-3p expression level in LN renal tissues and blood serum to determine its clinicopathological significance and effect on glomerular cell proliferation. Then, we collected renal glomeruli from LN mice and determined the miR-148a-3p, proliferating cell nuclear antigen (PCNA), and PCNA/Thy1 expression. We performed functional analyses of miR-148a-3p in vitro and in vivo. We also investigated the target gene of miR-148a-3p in LN. The results showed that miR-148a-3p expression levels were significantly higher not only in glomeruli but also in the blood serum during LN and increased in the glomeruli of LN mice and that at the same time there was positive correlation between miR-148a-3p and PCNA expression of glomruli. Overexpression of miR-148a-3p accelerated cell proliferation and PCNA expression, while a miR-148a-3p inhibitor inhibited cell proliferation via the Akt/cyclin D1 pathway. Furthermore, miR-148a-3p overexpression reduced the phosphatase and tensin homology deleted on chromosome ten (PTEN) expression level, while miR-148a-3p silencing increased its expression in high-mobility group box 1 (HMGB1)-induced mouse mesangial cells (MMCs). Luciferase assays demonstrated that miR-148a-3p could directly bind to the PTEN 3'-UTR. PTEN overexpression inhibited MMC proliferation considerably, resembling the results observed during miR-148a-3p inhibition. Reducing miR-148a-3p expression upregulated PTEN in the glomeruli and improved renal function in LN mice. Thus miR-148a-3p may promote proliferation and contribute to LN progression by targeting PTEN.

Previous studies have shown that high glucose stimulates renal SREBP-1 gene expression and increases renal tubular cells lipid metabolism, however, the mechanisms remain elusive. In the present study we demonstrated that PI3K/Akt pathway was activated in human renal proximal tubular cell line (HKC) exposed to high glucose accompanied with up-regulation of SREBP-1, TGF-β1, lipid droplets deposits and extracellular matrix production. Inhibition of PI3K/Akt pathway by chemical LY294002 or specific short hairpin RNA (shRNA) vector prevented SREBP-1 and TGF-β1 up-regulation, as well as ameliorated HKC cells lipogenesis and extracellular matrix accumulation. These findings indicate that PI3K/Akt pathway potentially mediates high glucose-induced lipogenesis and extracellular matrix accumulation in HKC cells.

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Extracellular matrix accumulation and fibrosis are the features of diabetic nephropathy. PI3K (phosphatidylinositol 3-kinase)/Akt (protein kinase B) signal pathway and its inhibitor PTEN (phosphatase and tensin homolog deleted on chromosome 10) are revealed to modulate renal fibrosis. However, the exact mechanism is still not well known. In the present study we found that compared with normal mice, diabetic mice showed decreased PTEN, increased phospho-Akt (Ser 473), phospho-Akt (Thr 308), CTGF (connective tissue growth factor), α-SMA (α-smooth muscle actin), and matricellular protein in kidney. Knocking down of PTEN caused an increase in phospho-Akt (Ser 473), phospho-Akt (Thr 308), CTGF, secreted fibronectin, and secreted Col 3 in HKC cells (human renal tubular epithelial cells). Again, in vitro experiment revealed 1.89, 2.18, 1.92, 3.06, 2.06-fold increases of phospho-Akt (Ser 473), phospho-Akt (Thr 308), CTGF, secreted fibronectin, and secreted Col 3 in high glucose-stimulated HKC cells in comparison with normal control cells. Furthermore, knocking down of CTGF reversed increased secreted fibronectin and Col 3 in high glucose-treated HKC cells. Moreover, transfection of PTEN expression vector prevented high glucose-caused these changes in HKC cells. Especially, CTGF expression, secretion of fibronectin and Col 3 were, respectively, decreased by 38.81, 53.85, and 39.12%. The treatment of LY294002 inhibited phospho-Akt (Ser 473) and phospho-Akt (Thr 308) expression followed by decreased CTGF, secretory fibronectin and secretory Col 3 in high glucose-treated HKC cells. In the end our study suggests that PTEN regulates renal extracellular matrix production via activated Akt and increased CTGF in diabetes mellitus.