Abstract-Previous studies have reported that uric acid stimulates vascular smooth muscle cell (VSMC) proliferation in vitro. We hypothesized that uric acid may also have direct proinflammatory effects on VSMCs. Crystal-and endotoxin-free uric acid was found to increase VSMC monocyte chemoattractant protein-1 (MCP-1) expression in a time-and dose-dependent manner, peaking at 24 hours. Increased mRNA and protein expression occurred as early as 3 hours after uric acid incubation and was partially dependent on posttranscriptional modification of MCP-1 mRNA. In addition, uric acid activated the transcription factors nuclear factor-B and activator protein-1, as well as the MAPK signaling molecules ERK p44/42 and p38, and increased cyclooxygenase-2 (COX-2) mRNA expression. Inhibition of p38 (with SB 203580), ERK 44/42 (with UO126 or PD 98059), or COX-2 (with NS398) each significantly suppressed uric acid-induced MCP-1 expression at 24 hours, implicating these pathways in the response to uric acid. The ability of both N-acetyl-cysteine and diphenyleneionium (antioxidants) to inhibit uric acid-induced MCP-1 production suggested involvement of intracellular redox pathways. Uric acid regulates critical proinflammatory pathways in VSMCs, suggesting it may have a role in the vascular changes associated with hypertension and vascular disease.

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TGF-␤ has been shown to play a critical role in anti-inflammation; however, the signaling mechanisms of TGF-␤ in anti-inflammatory response remains largely unclear. This study reported that mice that overexpress latent TGF-␤1 on skin are protected against renal inflammation in a model of obstructive kidney disease and investigated the signaling mechanism of TGF-␤1 in inhibition of renal inflammation in vivo and in vitro. Seven days after urinary obstruction, wild-type mice developed severe renal inflammation, including massive T cell and macrophage infiltration and marked upregulation of IL-1␤, TNF-␣, and intercellular adhesion molecule-1 (all P < 0.001). Surprising, renal inflammation was prevented in transgenic mice. This was associated with an increase in latent TGF-␤1 in circulation (a 10-fold increase) and renal tissues (a 2.5-fold increase). Further studies showed that inhibition of renal inflammation in TGF-␤1 transgenic mice was also associated with a marked upregulation of renal Smad7 and IB␣ and a suppression of NF-B activation in the diseased kidney (all P < 0.01). These in vivo findings suggested the importance of TGF-␤-NF-B cross-talk signaling pathway in regulating renal inflammation. This was tested in vitro in a doxycycline-regulated Smad7-expressing renal tubular cell line. Overexpression of Smad7 was able to upregulate IB␣ directly in a time-and dose-dependent manner, thereby inhibiting NF-B activation and NF-B-driven inflammatory response. In conclusion, latent TGF-␤ may have protective roles in renal inflammation. Smad7-mediated inhibition of NF-B activation via the induction of IkB␣ may be the central mechanism by which latent TGF-␤ prevents renal inflammation.

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.

ABSTRACT. It has been shown that transforming growth factor–β (TGF-β) is a potent mediator in renal fibrosis and that Smad proteins are critical intracellular mediators in TGF-β signaling. It is here reported that TGF-β mediates renal fibrogenesis in tubular epithelial cells (TEC) in association with the activation of Smad2 and that overexpression of Smad7 blocks this fibrotic process. Using a normal rat kidney tubular epithelial cell line (NRK52E), it was determined that TGF-β1 induces Smad2 phosphorylation and nuclear localization in both a dose- and time-dependent manner. The activation of Smad2 was evident at 5 min (20%), peaked at 15 to 30 min (85%), and declined to baseline levels by 2 h (5 to 10%). This was associated with de novo expression of collagens I, III, and IV and the transformation of TEC into a “myofibroblast” phenotype with de novo expression of α-smooth muscle actin (α-SMA) and with the loss of E-cadherin (>50%). To investigate a negative regulatory role of Smad7 in renal fibrosis, the Smad 7 gene was stably transfected and its expression was tightly controlled by doxycycline into NRK52E cells. Overexpression of Smad7 induced by doxycycline results in marked inhibition of TGF-β–induced Smad2 activation (90%↓) with the prevention of collagen synthesis and myofibroblast transformation. Thus, Smad2 activation occurs in the fibrogenic response of TEC to TGF-β, and this process is blocked by overexpression of Smad7. This indicates that Smad signaling is a key pathway of TGF-β–mediated renal fibrosis and suggests that treatments targeting the inactivation of Smad2 by overexpression of Smad7 may provide a new therapeutic strategy for renal fibrosis.