Reghuvaran Anand, Kumar Ashwani, Lin Qisheng, Rajeevan Nallakandi, Sun Zeguo, Shi Hongmei, Barsotti Gabriel, Tanvir E M, Pell John, Perincheri Sudhir, Wei Chengguo, Planoutene Marina, Eichmann Anne, Mas Valeria, Zhang Weijia, Das Bhaskar, Cantley Lloyd, Xu Leyuan, He Cijiang John, Menon Madhav C
Section of Nephrology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA.
Department of Nephrology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, PRC.
bioRxiv. 2024 Nov 23:2024.11.22.624409. doi: 10.1101/2024.11.22.624409.
Common intronic enhancer SNPs in Shroom3 associate with CKD in GWAS, although there is paucity of detailed mechanism. Previously, we reported a role for Shroom3 in mediating crosstalk between TGFβ1- & Wnt/Ctnnb1 pathways promoting renal fibrosis (TIF). However, beneficial roles for Shroom3 in proteinuria have also been reported suggesting pleiotropic effects. Here we focused on identifying the specific profibrotic Shroom3 motif. Given known therapeutic roles for Rho-kinase inhibitors in experimental CKD, and the established interaction between Shroom3 and Rock via its ASD2 domain, we hypothesized that Shroom3-mediated ROCK activation played a crucial role in its profibrotic function in high expressors. To test this hypothesis, we developed transgenic mice and cell lines that inducibly overexpressed wild-type- (WT-Sh3) or ASD2-domain deletion- Shroom3 (ASD2Δ-Sh3). Prior scRNAseq data showed that during TIF, Shroom3 and Rock co-expression occurred in injured tubular cells and fibroblasts, highlighting cell-types where this mechanism could be involved. Using HEK293T cells, we first confirmed absent ROCK binding and inhibited TGFβ1-signaling with ASD2Δ-Sh3-overexpression vs WT-Sh3. In mIMCD cells, ASD2Δ-Sh3 overexpression, reduced Rock activation (phospho-MYPT1), pro-fibrotic and pro-inflammatory transcripts vs WT-Sh3. Fibroblast proliferation (3T3) was also reduced with ASD2Δ-Sh3. , we studied ureteric obstruction (UUO) and Aristolochic nephropathy (AAN) as TIF models. In AAN, inducible global-, or Pan-tubular specific-, WTSh3-overexpression showed increased azotemia, and TIF vs ASD2Δ-Sh3 mice. WT-Sh3 mice consistently showed significant enrichment of Rho-GTPase, TGFβ1- and Wnt/CtnnB1- signaling in kidney transcriptome, paralleling Shroom3-coexpressed genes in tubulo-interstitial transcriptomes from human CKD. In UUO, again WT-Sh3 mice recapitulated increased fibrosis vs ASD2Δ-Sh3. Importantly, ASD2Δ-Sh3 did not develop albuminuria vs WT-Sh3, while mutating a disparate Fyn-binding Shroom3 motif induced albuminuria in mice, suggesting motif-specific roles for Shroom3 in the kidney. Hence, our data show a critical role for the Rock-binding, ASD2-domain in mediating TIF in milieu of Shroom3 excess, with relevance to human CKD.
Shroom3基因常见的内含子增强子单核苷酸多态性（SNP）在全基因组关联研究（GWAS）中与慢性肾脏病（CKD）相关，尽管其详细机制尚不清楚。此前，我们报道了Shroom3在介导转化生长因子β1（TGFβ1）与Wnt/β-连环蛋白1（Wnt/Ctnnb1）信号通路之间的串扰以促进肾纤维化（TIF）中发挥作用。然而，也有报道称Shroom3在蛋白尿中具有有益作用，提示其具有多效性。在此，我们专注于鉴定Shroom3的特定促纤维化基序。鉴于Rho激酶抑制剂在实验性CKD中已知的治疗作用，以及Shroom3与Rho相关卷曲螺旋蛋白激酶（Rock）通过其ASD2结构域建立的相互作用，我们推测Shroom3介导的ROCK激活在其高表达者的促纤维化功能中起关键作用。为了验证这一假设，我们构建了可诱导过表达野生型（WT-Sh3）或ASD2结构域缺失的Shroom3（ASD2Δ-Sh3）的转基因小鼠和细胞系。先前的单细胞RNA测序（scRNAseq）数据显示，在TIF过程中，Shroom3和Rock在受损的肾小管细胞和成纤维细胞中共表达，突出了该机制可能涉及的细胞类型。利用人胚肾293T细胞（HEK293T），我们首先证实ASD2Δ-Sh3过表达与WT-Sh3相比，不存在ROCK结合且抑制了TGFβ1信号传导。在小鼠肾小管间质细胞（mIMCD）中，ASD2Δ-Sh3过表达与WT-Sh3相比，降低了Rock激活（磷酸化肌球蛋白磷酸酶靶向亚基1，phospho-MYPT1）、促纤维化和促炎转录本。ASD2Δ-Sh3也降低了成纤维细胞（3T3）的增殖。我们研究了输尿管梗阻（UUO）和马兜铃酸肾病（AAN）作为TIF模型。在AAN中，可诱导的全身性或肾小管泛特异性WTSh3过表达与ASD2Δ-Sh3小鼠相比，显示出氮质血症增加和TIF。WT-Sh3小鼠的肾脏转录组中始终显示Rho鸟苷三磷酸酶（Rho-GTPase）、TGFβ1和Wnt/Ctnnb1信号显著富集，与人CKD肾小管间质转录组中与Shroom3共表达的基因相似。在UUO中，与ASD2Δ-Sh3相比，WT-Sh3小鼠再次出现纤维化增加。重要的是，与WT-Sh3相比，ASD2Δ-Sh3未出现蛋白尿，而突变一个不同的Fyn结合Shroom3基序可诱导小鼠出现蛋白尿，提示Shroom3在肾脏中具有基序特异性作用。因此，我们的数据表明，在Shroom3过量的情况下，与Rock结合的ASD2结构域在介导TIF中起关键作用，这与人类CKD相关。
