Trpsl Haploinsufficiency Promotes Renal Fibrosis By Increasing Arkadia Expression

[Perpose]Mutations in TRPS1cause tricho-rhino-pharyngeal syndrome (TRPS). Trps1is essential for nephron development, acting downstream of Bmp7.1Because Bmp7counteracts epithelial-to-mesenchymal transition (EMT) and reverses chronic renal injury, we examined the function of Trps1in renal fibrosis. Methods:Trps1HT mice, Immunohistochemistry, Immunocytochemistry, Real-time PCR, Western blot, primary cell culture。[Results]Trps1is localized to the nuclei of proximal tubular epithelial cells in adult micePreviously we demonstrated that Trps1was expressed in ureteric buds, renal vesicles and cap mesenchyme cells in embryonic kidneys. To examine the precise localization of Trps1protein in kidneys of adult mice, we performed immunohistochemistry with antibodies against Trps1. Positive staining was seen in the nuclei of tubular epithelial cells in the cortex region. To further characterize the precise localization, antibodies directed against villin, a specific marker for the brush border of the proximal tubules, was used for immunohistochemistry. The villin-positive area was restricted to the cortex regions where Trps1was stained. Thus, double immunostaining confirmed that Trps1was localized to the proximal tubular epithelial cells.Heterozygous loss of Trps1promotes renal fibrosis after UUOLigation of a ureter causes renal interstitial fibrosis characterized by tubular atrophy and deposition of interstitial matrices in the obstructed kidney. Hematoxylin and eosin staining showed representative histological findings on day7and14after UUO or sham-operation (sham), in WT) and HT mice. The obstructed kidneys showed tubular dilation, atrophy, a widened interstitial space, and a greater number of interstitial cells in both WT and HT mice. We found that the degree of these changes was greater in HT mice than in WT mice. Interstitial collagen deposition as determined by immunohistochemistry with an anti-type Ⅰ collagen antibody was more pronounced in HT obstructed kidneys than in WT obstructed kidneys. These data suggest that heterozygous loss of Trps1enhances renal fibrosis after UUO.Heterozygous loss of Trps1promotes TGF-β/Smad signaling after UUOAs TGF-P signaling is proposed to be the major contributor to renal fibrosis, we tested whether the promotion of renal fibrosis in HT mice was associated with TGF-β signaling. Consistent with a previous finding, RT-PCR and ELISA assays showed that TGF-β signaling was markedly upregulated at both the protein and mRNA levels after UUO in both WT and HT mice compared with sham-operated mice. However, these levels were not different between WT and HT kidneys that received UUO (data not shown). Next, we examined the phosphorylation of Smad3, a key mediator of TGF-β in fibrosis, in UUO kidneys. Immunohistochemistry showed that nuclear phosphorylated Smad3(pSmad3) staining was markedly increased in the UUO kidneys compared with sham-operated kidneys in both WT and HT mice at day7and day14, and the extent of the positive staining was greater in HT than in WT kidneys. A quantitative analysis showed that the amount of the phosphorylated Smad3in HT kidneys was approximately two-fold higher than in WT kidneys after UUO.The extent of renal fibrosis is associated with Trps1and Smad7protein levelsNext, we checked the expression levels of Trps1in the normal and obstructed kidneys from WT and HT mice to examine the relationship between Trps1and renal fibrosis. As shown in Fig.4A and B, the amount of Trps1protein was lower on day7and day14after UUO in a time-dependent manner in both WT and HT kidneys and was reduced by approximately half in HT kidneys compared with WT kidneys.Emerging evidence indicates that Smad7is a major inhibitor of TGF-β signaling and negatively regulates its intensity and duration. Given the fact that overactivation of Smad2/3in fibrotic kidneys is associated with a reduction in Smad7, we examined whether the expression of Smad7was altered in UUO kidneys. Western blotting showed that Smad7protein levels declined in a time-dependent manner in the UUO kidneys from both genotypes. Interestingly, Smad7was two-fold lower in the UUO kidneys from HT mice than in UUO kidneys from WT mice. However, virtually no difference was seen in Smad7mRNA expression in either WT or HT kidneys after UUO (data not shown). Western blots also showed that heterozygous loss of Trps1significantly increased the amount of-SMA and phosphor-Smad3in the obstructed kidneys at day7and day14after UUO. From these results, it is conceivable that the amount of Trps1protein is negatively correlated with the extent of fibrosis in obstructed kidneys and that a reduction in Trps1promotes renal fibrosis through a reduction in Smad7protein levels.Heterozygous loss of Trps1increases TGF-β-mediated EMTTo confirm whether lower levels of Trps1can influence the TGF-β/Smad3signaling pathway, we compared the expression levels of molecules that are involved in EMT using cultured PTECs from WT and HT mice. Cells from both genotypes appeared morphologically similar. At12h and24h after treatment with5ng/ml TGF-β1to induce EMT, HT cells showed a greater degree of staining for α-SMA than WT cells, indicating more HT cells transdifferentiated into myofibroblasts. The positive Trps1staining disappeared by24h after TGF-β1treatment in both genotypes. Consistent with the immunostaining, immunoblots revealed that the increase in α-SMA after TGF-β1treatment was more pronounced in HT cells than in WT cells. In contrast, E-cadherin decreased more quickly in HT cells than in WT cells. These results suggest that Trps1inhibits TGF-β-induced EMT of cultured renal epithelial cells.TGF-β1/Smad3signaling is upregulated by reductions in Smad7in HT cells than WT cells during EMT.To address the molecular mechanism underlying the inhibition of EMT by Trps1, we examined whether a reduction in Trps1affected TGF-/Smad3signaling in PTECs. Smad3phosphorylation peaked at6h and then decreased gradually in WT cells, whereas it continued to increase until it peaked12h after TGF-1treatment in HT cells. On the other hand, the amount of Smad7protein decreased in a time-dependent manner after TGF-1treatment in both WT and HT cells, and we found that the amount of Smad7in HT cells was approximately half of that in WT cells. Heterozygous loss of Trps1decreases Smad7levels by increasing Arkadia expressionBecause Trps1is a transcriptional repressor that binds the GATA sites of target genes and Smad7mRNA levels were similar between WT and HT cells, it is unlikely that Trps1regulates Smad7gene expression directly. Therefore, we examined the expression of Smurf1, Smurf2, and Arkadia, three E3ubiquitin ligases, that degrade Smad7and regulate TGF-β signaling in EMT of renal tubular epithelial cells. The amount of Arkadia increased in a time-dependent manner in WT cells, whereas virtually no difference was seen in the levels of Smurf1and Smurf2(data not shown). More interestingly, heterozygous loss of Trps1increased Arkadia levels four-fold compared to those seen in WT cells, and this difference was further augmented in a time-dependent manner after TGF-(31treatment. Taken together, these results indicate TGF-(31/Smad3signaling is promoted by reduced expression of Trps1, which results in lower Smad7protein levels through up-regulation of Arkadia expression.[Conclusion] Trps1haploinsufficiency promotes renal fibrosis by increasing Arkadia expression.