No abstract

We recently showed that Toll-like receptor (TLR) TLR4 was overexpressed in the human diabetic kidney, which could promote tubular inflammation. Here we explored whether the TLR4 antagonist, CRX-526, has therapeutic potential to attenuate renal injuries and slow the progression of advanced diabetic nephropathy in wild-type and endothelial nitric oxide synthase (eNOS) knockout mice. In the latter, the endogenous TLR4 ligand, high-mobility group box 1, was upregulated more than in wild-type animals. Four weeks after streptozotocin induction of diabetes, mice were injected with either CRX-526 or vehicle for 8 weeks. CRX-526 significantly reduced albuminuria and blood urea nitrogen without altering blood glucose and systolic blood pressure in diabetic mice. Glomerular hypertrophy, glomerulosclerosis, and tubulointerstitial injury were attenuated by CRX-526, which was associated with decreased chemokine (C-C motif) ligand (CCL)-2, osteopontin, CCL-5 overexpression, subsequent macrophage infiltration, and collagen deposition. These effects were associated with inhibition of TGF-β overexpression and NF-κB activation. In vitro, CRX-526 inhibited high glucose-induced osteopontin upregulation and NF-κB nuclear translocation in cultured human proximal tubular epithelial cells. Thus, we provided evidence that inhibition of TLR4 with the synthetic antagonist CRX-526 conferred renoprotective effects in eNOS knockout diabetic mice with advanced diabetic nephropathy.

Kallistatin is a serine protease inhibitor with anti-inflammatory, anti-angiogenic, and anti-oxidative properties. Since oxidative stress plays a critical role in the pathogenesis of diabetic nephropathy, we studied the effect and mechanisms of action of kallistatin superinduction. Using ultrasound-microbubble-mediated gene transfer, kallistatin overexpression was induced in kidney tubules. In db/db mice, kallistatin overexpression reduced serum creatinine and BUN levels, ameliorated glomerulosclerosis and tubulointerstitial injury, and attenuated renal fibrosis by inhibiting TGF-β signaling. Additionally, downstream PAI-1 and collagens I and IV expression were reduced and kallistatin partially suppressed renal inflammation by inhibiting NF-κB signaling and decreasing tissue kallikrein activity. Kallistatin lowered blood pressure and attenuated oxidative stress as evidenced by suppressed levels of NADPH oxidase 4, and oxidative markers (nitrotyrosine, 8-hydroxydeoxyguanosine, and malondialdehyde) in diabetic renal tissue. Kallistatin also inhibited RAGE expression in the diabetic kidney and AGE-stimulated cultured proximal tubular cells. Reduced AGE-induced reactive oxygen species generation reflected an anti-oxidative mechanism via the AGE-RAGE-reactive oxygen species axis. These results indicate a renoprotective role of kallistatin against diabetic nephropathy by multiple mechanisms including suppression of oxidative stress, anti-fibrotic and anti-inflammatory actions, and blood pressure lowering.