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Purpose: To investigate whether mesenchymal stem cells (MSCs) could inhibit transforming growth factor beta (TGF-β) signalling pathway by paracrine action. Methods: Bone marrow-derived MSCs were transplanted to streptozotocin-induced diabetic rats via tail vein. MSCconditioned media were used with a model of mesangial cell fibrosis induced by high glucose in vitro. Results: At 8 weeks after MSC treatment, the renal function and the glomerulosclerosis as revealed by periodic acid Schiff stain was dramatically attenuated. The expression of collagen I, collagen IV and α-smooth muscle actin (SMA) in diabetic kidney was decreased, and E-cadherin increased after MSC treatment. The TGF-β signalling pathway was suppressed both in vivo and in vitro. MSCs secreted a significant amount of bone morphogenetic protein 7 (BMP7), in vitro, MSC-conditioned media inhibited TGF-β signalling stimulated by high glucose, and BMP7 neutralizing antibody blocked the inhibitory effect of MSC-conditioned media. Conclusion: MSCs ameliorated glomerular fibrosis in vivo and in vitro by inhibiting TGF-β/Smad signalling pathway via secretion of BMP7.

Diabetic nephropathy is a major cause of morbidity and mortality in diabetic patients. Effective therapies to prevent the development of this disease are required. Berberine (BBR) has several preventive effects on diabetes and its complications. However, the molecular mechanism of BBR on kidney function in diabetes is not well defined. Here, we reported that activation of AMP-activated protein kinase (AMPK) is required for BBR-induced improvement of kidney function in vivo. AMPK phosphorylation and activity, productions of reactive oxygen species (ROS), kidney function including serum blood urea nitrogen (BUN), creatinine clearance (Ccr), and urinary protein excretion, morphology of glomerulus were determined in vitro or in vivo. Exposure of cultured human glomerulus mesangial cells (HGMCs) to BBR time- or dose-dependently activates AMPK by increasing the thr172 phosphorylation and its activities. Inhibition of LKB1 by siRNA or mutant abolished BBR-induced AMPK activation. Incubation of cells with high glucose (HG, 30 mM) markedly induced the oxidative stress of HGMCs, which were abolished by 5-aminoimidazole-4-carboxamide ribonucleoside, AMPK gene overexpression or BBR. Importantly, the effects induced by BBR were bypassed by AMPK siRNA transfection in HG-treated HGMCs. In animal studies, streptozotocin-induced hyperglycemia dramatically promoted glomerulosclerosis and impaired kidney function by increasing serum BUN, urinary protein excretion, and decreasing Ccr, as well as increased oxidative stress. Administration of BBR remarkably improved kidney function in wildtype mice but not in AMPKα2-deficient mice. We conclude that AMPK activation is required for BBR to improve kidney function in diabetic mice.

Nitroalkene derivative of oleic acid (OA-NO), due to its ability to mediate revisable Michael addition, has been demonstrated to have various biological properties and become a therapeutic agent in various diseases. Though its antioxidant properties have been reported in different models of acute kidney injury (AKI), the mechanism by which OA-NO attenuates intracellular oxidative stress is not well investigated. Here, we elucidated the anti-oxidative mechanism of OA-NO in an in vitro model of renal ischemia/reperfusion (I/R) injury. Human tubular epithelial cells were subjected to oxygen and glucose deprivation/re-oxygenation (OGD/R) injury. Pretreatment with OA-NO (1.25 μM, 45 min) attenuated OGD/R triggered reactive oxygen species (ROS) generation and subsequent mitochondrial membrane potential disruption. This action was mediated via up-regulating endogenous antioxidant defense components including superoxide dismutase (SOD1), heme oxygenase 1 (HO-1), and γ-glutamyl cysteine ligase modulatory subunits (GCLM). Moreover, subcellular fractionation analyses demonstrated that OA-NO promoted nuclear translocation of nuclear factor-E2- related factor-2 (Nrf2) and Nrf2 siRNA partially abrogated these protective effects. In addition, OA-NO inhibited NADPH oxidase activation and NADPH oxidase 4 (NOX4), NADPH oxidase 2 (NOX2) and p22 up-regulation after OGD/R injury, which was not relevant to Nrf2. These results contribute to clarify that the mechanism of OA-NO reno-protection involves both inhibition of NADPH oxidase activity and induction of SOD1, Nrf2-dependent HO-1, and GCLM.

Background: Nitroalkene derivatives of oleic acid (OA-NO₂) serve as high-affinity ligand for PPAR-γ, which regulates apoptosis, oxidation and inflammation and plays a central role in ischemia-reperfusion injury. In the present study, we elucidated the protective mechanisms of OA-NO₂ against renal ischemia-reperfusion injury. Methods: HK-2 cells were subjected to oxygen and glucose deprivation followed by re-oxygenation (OGD/R) to mimic renal ischemia-reperfusion injury. Cell apoptosis was analyzed by flow cytometry. Bax mitochondrial translocation, cytochrome c and apoptosis-inducing factor (AIF) cytosolic leakage and Akt/Gsk 3β phosphorylation were evaluated by Western blotting. Bax activation was visualized by immunocytochemistry. GW9662 and siRNA transfection were employed to examine the involvement of PPAR-γ. Results: OGD/R injury promoted mitochondrial translocation and activation of Bax, leakage of cytochrome c and AIF, subsequent caspase-3 activation, and eventually cell apoptosis. Pre-incubation with OA-NO₂ (1.25 µM, 45min) inhibited Bax activation and blocked apoptotic cascade, while the protective effects were negated by GW9662 or PPAR-γ siRNA. Moreover, OA-NO₂ restored Akt and Gsk 3β phosphorylation in a PPAR-γ-dependent way. Conclusion: These findings suggest that OA-NO₂ attenuates OGD/R-induced apoptosis by inhibiting Bax translocation and activation and the subsequent mitochondria-dependent apoptotic cascade in a PPAR-γ dependent manner.