OBJECTIVEAlthough Smad3 has been considered as a downstream mediator of transforming growth factor-β (TGF-β) signaling in diabetes complications, the role of Smad7 in diabetes remains largely unclear. The current study tests the hypothesis that Smad7 may play a protective role and has therapeutic potential for diabetic kidney disease.RESEARCH DESIGN AND METHODSProtective role of Smad7 in diabetic kidney disease was examined in streptozotocin-induced diabetic mice that have Smad7 gene knockout (KO) and in diabetic rats given Smad7 gene transfer using an ultrasound-microbubble-mediated technique.RESULTSWe found that mice deficient for Smad7 developed more severe diabetic kidney injury than wild-type mice as evidenced by a significant increase in microalbuminuria, renal fibrosis (collagen I, IV, and fibronectin), and renal inflammation (interleukin-1β [IL-1β], tumor necrosis factor-α [TNF-α], monocyte chemoattractant protein-1 [MCP-1], intracellular adhesion molecule-1 [ICAM-1], and macrophages). Further studies revealed that enhanced renal fibrosis and inflammation in Smad7 KO mice with diabetes were associated with increased activation of both TGF-β/Smad2/3 and nuclear factor-κB (NF-κB) signaling pathways. To develop a therapeutic potential for diabetic kidney disease, Smad7 gene was transferred into the kidney in diabetic rats by an ultrasound-microbubble-mediated technique. Although overexpression of renal Smad7 had no effect on levels of blood glucose, it significantly attenuated the development of microalbuminuria, TGF-β/Smad3-mediated renal fibrosis such as collagen I and IV and fibronectin accumulation and NF-κB/p65-driven renal inflammation including IL-1β, TNF-α, MCP-1, and ICAM-1 expression and macrophage infiltration in diabetic rats.CONCLUSIONSSmad7 plays a protective role in diabetic renal injury. Overexpression of Smad7 may represent a novel therapy for the diabetic kidney complication.

Loss of miR-29 is associated with cardiac fibrosis. This study examined the role and therapeutic potential of miR-29 in mouse model of hypertension induced by angiotensin II (AngII). By using microRNA microarray, in situ hybridization, and real-time polymerase chain reaction, we found that AngII-induced cardiac fibrosis in the hypertensive heart and in cultured cardiac fibroblasts were associated with downregulation of miR-29a-c via a Smad3-dependent mechanism. In vitro knockdown of miR-29b enhanced but overexpression of miR-29b inhibited AngII-induced fibrosis, revealing a protective role of miR-29b in cardiac fibrosis in response to AngII. This was further demonstrated in vivo by the ability of overexpressing miR-29b in the mouse heart to prevent AngII-mediated cardiac fibrosis and cardiac dysfunction. Importantly, we also found that restored miR-29b in the established hypertensive heart was capable of blocking progressive cardiac fibrosis and improving cardiac dysfunction, demonstrating a therapeutic potential of miR-29b for chronic heart disease. Further studies revealed that targeting the transforming growth factor (TGF)-β1 coding sequence region, thereby inhibiting TGF-β/Smad3 signaling, could be a new mechanism by which miR-29b inhibited AngII-induced cardiac fibrosis. In conclusion, miR-29b plays a protective role in AngII-mediated cardiac remodeling and may be a therapeutic agent for cardiac fibrosis by targeting the TGF-β/Smad3 pathway.

The pattern of glomerulonephritis (GN) developing in response to a planted antigen (sheep anti-mouse GBM globulin) was compared in two strains of mice which demonstrated either a predominant Th1 (C57BL/6) or Th2 (BALB/c) response to this antigen. GN was induced with a subnephritogenic i.v. dose of sheep anti-mouse GBM globulin in mice presensitized to sheep globulin. Sensitized C57BL/6 mice showed pronounced cutaneous delayed-type hypersensitivity (DTH) following the challenge with sheep globulin, low titers of circulating anti-sheep globulin antibody and high interferon gamma (IFN-gamma) and low interleukin 4 (IL-4) production by splenic T cells, consistent with a predominant Th1 pattern of immune response. Sensitized BALB/c mice did not develop DTH following cutaneous challenge with sheep globulin, had higher circulating anti-sheep globulin antibody titers, and showed high IL-4 and low IFN gamma production by splenic T cells compared with C57BL/6 mice, consistent with a predominant Th2 response. In C57BL/6 mice, GN developing in response to sheep globulin exhibited a severe crescentic pattern with prominent glomerular T cell and macrophage influx and fibrin deposition. In vivo depletion with a monoclonal anti-CD4 antibody demonstrated that this injury was T helper cell dependent. Treatment with monoclonal anti-mouse IFN gamma antibody significantly reduced glomerular injury and crescent formation and attenuated the cutaneous DTH response. GN induced by the same protocol in BALB/c mice exhibited pronounced glomerular IgG and complement deposition. Crescent formation, fibrin deposition, and glomerular T cell and macrophage infiltration were significantly less than observed in C57BL/6 mice, and injury was not T cell dependent in the effector phase. These data suggest that the pattern of glomerular injury induced by a planted antigen can be determined by the balance of T helper cell subset activation. A Th1 response induces a severe crescentic pattern of GN, which like cutaneous DTH, is T helper cell and IFN gamma dependent.

The mechanism by which TGF-β regulates renal inflammation and fibrosis is largely unclear; however, it is well accepted that its biological effects are mediated through Smad2 and Smad3 phosphorylation. Following activation, these Smads form heteromeric complex with Smad4 and translocate into the nucleus to bind and regulate the expression of target genes. Here we studied the roles of Smad4 to regulate TGF-β signaling in a mouse model of unilateral ureteral obstruction using conditional Smad4 knockout mice and in isolated Smad4 mutant macrophages and fibroblasts. Disruption of Smad4 significantly enhanced renal inflammation as evidenced by a greater CD45(+) leukocyte and F4/80(+) macrophage infiltration and upregulation of IL-1β, TNF-α, MCP-1, and ICAM-1 in the obstructed kidney and in IL-1β-stimulated macrophages. In contrast, deletion of Smad4 inhibited renal fibrosis and TGF-β1-induced collagen I expression by fibroblasts. Further studies showed that the loss of Smad4 repressed Smad7 transcription, leading to a loss of functional protein. This, in turn, inhibited IκBα expression but enhanced NF-κB activation, thereby promoting renal inflammation. Interestingly, deletion of Smad4 influenced Smad3-mediated promoter activities and the binding of Smad3 to the COL1A2 promoter, but not Smad3 phosphorylation and nuclear translocation, thereby inhibiting the fibrotic response. Thus, Smad4 may be a key regulator for the diverse roles of TGF-β1 in inflammation and fibrogenesis by interacting with Smad7 and Smad3 to influence their transcriptional activities in renal inflammation and fibrosis.