Renal dysplasia, the major cause of childhood renal failure, is characterized by defective branching morphogenesis and nephrogenesis. Beta-catenin, a transcription factor and cell adhesion molecule, is markedly increased in the nucleus of kidney cells in human renal dysplasia and contributes to its pathogenesis by altering target genes that are essential for kidney development. Quercetin, a naturally occurring flavonoid, reduces nuclear beta-catenin levels and reduces beta-catenin transcriptional activity. In this study, we utilized wild type and dysplastic mouse kidney organ explants to determine if quercetin reduces beta-catenin activity during kidney development and whether it improves the severity of renal dysplasia. In wild type kidney explants, quercetin treatment resulted in abnormal branching morphogenesis and nephrogenesis in a dose dependent manner. In wild type embryonic kidneys, quercetin reduced nuclear beta-catenin expression and decreased expression of beta-catenin target genes Pax2, Six2, and Gdnf, which are essential for kidney development. Our RD B mouse model of renal dysplasia recapitulates the overexpression of beta-catenin and histopathological changes observed in human renal dysplasia. RD B kidneys treated with quercetin resulted in improvements in the overall histopathology, tissue organization, ureteric branching morphogenesis, and nephrogenesis. Quercetin treatment also resulted in reduced nuclear beta-catenin and reduced Pax2 expression. These improvements were associated with the proper organization of vimentin, NCAM, and E-cadherin, and a 45% increase in the number of developing and maturing nephrons. Further, our results show that in human renal dysplasia, beta-catenin, vimentin, and e-cadherin also have abnormal expression patterns. Taken together, these data demonstrate that quercetin treatment reduces nuclear beta-catenin and this is associated with improved epithelial organization of developing nephrons, resulting in increased developing nephrons and a partial rescue of renal dysplasia.

Kidney disease in children often results from the abnormal development of the kidney, termed renal dysplasia (RD). This abnormal development is often associated with poorly formed nephrons, leading to reduced nephron number and poor kidney function. β‐catenin is a dual function protein that complexes with E‐cadherin to form adherens junctions and regulates gene transcription. In human RD, β‐catenin is overexpressed in the nuclei of metanephric mesenchyme (MM), the precursor cells that will form nephrons. Our mouse models of RD where β‐catenin is overexpressed in the MM, termed βcatMM(over), have dysregulated expression of genes involved in nephron formation, resulting in abnormally developed nephrons with poorly formed adherens junctions. Quercetin is a flavonoid that reduces β‐catenin levels in diseases such as cancer and kidney fibrosis. The objective of our study is to test whether quercetin reduces nuclear β‐catenin levels and improves abnormal nephron formation in RD. Dysplastic kidneys from E13.5 βcatMM(over) embryos were cultured in the presence or absence of 40uM quercetin. Immunofluorescence (IF) for Pax2 and Six2 in untreated βcatMM(over) kidneys demonstrated abnormal kidney patterning, clusters of uninduced MM cells, stalled nephrons, and a near absence of normally developing nephrons. Immunohistochemistry (IHC) revealed β‐catenin overexpression in the nuclei of the MM clusters and in the abnormally forming nephrons. In contrast, quercetin treated βcatMM(over) kidneys demonstrated improved kidney patterning, a distinct nephrogenic zone containing Six2 positive MM cells, and a cortical and medullary area containing Pax2 positive developing and maturing nephrons. A 44% increase in normally developing nephrons was observed in the treated kidneys (untreated: 13.25±1.30, N=7 vs. treated: 19.14±2.01, N=8, p=0.03). IHC demonstrated β‐catenin expression was reduced in the nuclei of the MM and developing nephrons and increased in the cell membrane, and this β‐catenin expression pattern was similar to that observed in wildtype kidneys. Quercetin treated kidneys also showed reduced expression levels of β‐catenin target genes: Pax2 (1.5‐fold, p=0.01), Six2 (1.2‐fold, p=0.46) and Gdnf (1.2‐fold, p=0.28). IHC on untreated βcatMM(over) kidneys demonstrated E‐cadherin localization in the cytoplasm of the stalled nephrons instead of the cell membrane. In contrast, quercetin treated βcatMM(over) kidneys demonstrated E‐cadherin expression exclusively in the cell membrane. Analysis of human fetal dysplastic tissue also demonstrated cytoplasmic E‐cadherin localization in abnormally developing nephrons, which was not observed in normally developing nephrons in normal human fetal kidneys. E13.5 wildtype kidneys treated with 40uM, 80uM and 160uM quercetin showed no change in β‐catenin protein levels by Western blot. However, IHC and IF showed that increasing quercetin dose resulted in β‐catenin and E‐cadherin expression being limited to the cell membrane. In conclusion, our data supports that quercetin is effective in improvi...

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...