Shroom3 regulates epithelial differentiation during tubular repair after ischemia reperfusion injury

다국어 초록 (Multilingual Abstract)

Shroom3 is an actin‐binding protein that modulates actomyosin dynamics to alter epithelial cell morphology. In adult kidneys, Shroom3 is apically localized in tubular epithelial cells of cortical nephron segments, such as S1 of proximal tubules, distal tubules, and collecting ducts. Genomic variants in Shroom3 are strongly associated with poor renal function and poor outcomes in kidney transplant recipients. After a kidney insult such as ischemia reperfusion injury from renal or cardiac surgeries, the damaged renal epithelium undergoes cell morphology changes to regenerate normal renal epithelial structure. This repair process includes actomyosin remodelling, allowing damaged epithelial cells to undergo de‐differentiation into a mesenchymal cell type, proliferate and re‐differentiate to tubular epithelial cells. In this study, our objective is to determine whether Shroom3 plays a role in renal epithelial repair after an ischemic kidney insult. We performed bilateral renal ischemia/reperfusion (I/R) on 3‐month‐old Shroom3 heterozygous mutant mice (Shrm3+/‐)and wild type (WT) mice. Compared to WT at 10 days post‐I/R, most of the epithelial cells in cortical tubules of Shrm3+/‐ kidneys had F‐actin and phospho‐Myosin Light Chain‐2 (pMLC‐2) sporadically distributed in the cytoplasm rather than being apically localized. In these cortical tubular cells, immunohistochemistry (IHC) demonstrated that mesenchymal marker Vimentin was highly expressed while epithelial markers N‐cadherin and E‐cadherin were significantly lower than in WT. These findings suggest that Shroom3 regulates tubular repair in an actin dependent manner that facilitates proper epithelial re‐differentiation. The inability to complete tubular epithelial re‐differentiation would result in a worsened kidney histopathology. Analysis of Shrm3+/‐ kidneys compared to WT at 10 days post‐I/R showed higher levels of Kidney Injury Molecule‐1 in the apical surface of cortical tubular cells as shown by IHC, a 2.5‐fold increase in cell proliferation shown by KI‐67 (Shrm3+/‐=16.5±0.8 vs. WT=6.6±1.8), a 4.4‐fold increase in apoptosis shown by Caspase‐3 (Shrm3+/‐ =1.4±0.09 vs. WT=0.32±0.04), a 2.4‐fold increase in inflammation levels shown by F4/80 (Shrm3+/‐ =64.5±2.2 vs. WT=26.4±2.5), and a 4.3‐fold increase in fibrosis levels shown by Picrosirius red (Shrm3+/‐ =10.3±0.73 vs. WT=2.4±0.15). These histopathological changes translated to severe impairments in kidney function: Shrm3+/‐ mice had a 4.0‐fold increase in serum creatinine levels at 24 hours post‐I/R and did not return to baseline levels until after 7 days, while WT mice had a 2.0‐fold increase after 24 hours and returned to baseline by 48 hours. Shrm3+/‐ mice also had higher mortality rates over a 10‐day period (Shrm3+/‐ =34.6% vs. WT=14.3%). Taken together, our findings demonstrate that Shroom3 plays key roles in renal epithelium repair after ischemia reperfusion injury likely by regulating actin cytoskeleton remodeling during the repair phase and thus modulating epithelial re‐differentiation. Our results could help explain why patients with genetic anomalies in Shroom3 show worse renal outcomes after an ischemic kidney insult.