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Aims/hypothesis: The accumulation of extracellular matrix (ECM) is a characteristic of diabetic nephropathy, and is partially caused by profibrotic proteins TGF-β and connective tissue growth factor (CTGF). We aimed to identify microRNAs (miRNAs) targeting CTGF on podocytes in diabetic nephropathy., Methods: We investigated miRNAs targeting CTGF on podocytes with miRNA array analysis and identified a candidate miRNA, miR-26a. Using overexpression and silencing of miR-26a in cultured podocytes, we examined changes of ECM and its host genes. We further investigated glomerular miR-26a expression in humans and in mouse models of diabetic nephropathy., Results: miR-26a, which was downregulated by TGF-β1, was expressed in glomerular cells including podocytes and in tubules by in situ hybridisation. Glomerular miR-26a expression was downregulated by 70% in streptozotocin-induced diabetic mice. Transfection of miR-26a mimics in cultured human podocytes decreased the CTGF protein level by 50%, and directly inhibited CTGF expression in podocytes, as demonstrated by a reporter assay with the 3'-untranslated region of the CTGF gene. This effect was abolished by a mutant plasmid. miR-26a mimics also inhibited TGF-β1-induced collagen expression, SMAD-binding activity and expression of its host genes CTDSP2 and CTDSPL. Knockdown of CTDSP2 and CTDSPL increased collagen expression in TGF-β-stimulated podocytes, suggesting that host genes also regulate TGF-β/SMAD signalling. Finally, we observed a positive correlation between microdissected glomerular miR-26a expression levels and estimated GFR in patients with diabetic nephropathy., Conclusions/interpretation: The downregulation of miR-26a is involved in the progression of diabetic nephropathy both in humans and in mice through enhanced TGF-β/CTGF signalling.

Aims/hypothesis: Hyperlipidaemia is an independent risk factor for the progression of diabetic nephropathy, but its molecular mechanism remains elusive. We investigated in mice how diabetes and hyperlipidaemia cause renal lesions separately and in combination, and the involvement of Toll-like receptor 4 (TLR4) in the process., Methods: Diabetes was induced in wild-type (WT) and Tlr4 knockout (KO) mice by intraperitoneal injection of streptozotocin (STZ). At 2 weeks after STZ injection, normal diet was substituted with a high-fat diet (HFD). Functional and histological analyses were carried out 6 weeks later., Results: Compared with treatment with STZ or HFD alone, treatment of WT mice with both STZ and HFD markedly aggravated nephropathy, as indicated by an increase in albuminuria, mesangial expansion, infiltration of macrophages and upregulation of pro-inflammatory and extracellular-matrix-associated gene expression in glomeruli. In Tlr4 KO mice, the addition of an HFD to STZ had almost no effects on the variables measured. Production of protein S100 calcium binding protein A8 (calgranulin A; S100A8), a potent ligand for TLR4, was observed in abundance in macrophages infiltrating STZ-HFD WT glomeruli and in glomeruli of diabetic nephropathy patients. High-glucose and fatty acid treatment synergistically upregulated S100a8 gene expression in macrophages from WT mice, but not from KO mice. As putative downstream targets of TLR4, phosphorylation of interferon regulatory factor 3 (IRF3) was enhanced in kidneys of WT mice co-treated with STZ and HFD., Conclusions/interpretation: Activation of S100A8/TLR4 signalling was elucidated in an animal model of diabetic glomerular injury accompanied with hyperlipidaemia, which may provide novel therapeutic targets in progressive diabetic nephropathy.