Quercetin improves abnormal nephron formation in a mouse model of renal dysplasia
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abstract
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 improving nephron formation in RD by reducing nuclear β‐catenin and redistributing β‐catenin and E‐cadherin to the cell membrane which improves epithelial integrity during nephron formation.Support or Funding InformationNSERC, CIHR, KFOC
