The role of WT1 in nephron endowment and glomeruloscerosis (GS) / Chronic Kidney Disease (CKD) /

Abstract

The DDS/+ mouse is a model for glomerulosclerosis in Denys Drash Syndrome (DDS). The mutation results in a 20% reduction in nephron number that triggers a compensatory cascade of events leading to glomerulosclerosis. However, the mechanisms controlling the rate of maturation of nephrons and determining final nephron number within a kidney are not fully known. Stereological examinations on newborn kidneys were performed for glomerular count. Microarray analysis of foetal kidneys was carried out to reveal differentially expressed genes. 1136 differentially expressed genes were identified by microarray, of which the top 5 genes encoded mitochondrial tRNAs. Ingenuity Pathway Analysis indicated the involvement of renal developmental and tRNA splicing in the underlying mechanisms. Cross-database analyses revealed at least 216 genes that contain intragenic WT1 target sequence therefor represent potential direct targets of WT1 that are mis expressed due to the mutation. Given the expression changes of apoptosis related genes, TUNEL staining to identify apoptosis was carried out to reveal insignificant differences between genotypes. Given abnormal expression of mitochondrial tRNAs, immunofluorescence and western blotting were carried out and confirmed the presence of WT1 protein in mitochondrial isolates. Furthermore, metabolic profiling revealed abnormalities in the oxidative phosphorylation system in WT1 mutant cells compared with wild type. This study identified a list of genes that are associated with WT1 induced nephron underdosing, many of which have not previously been linked with nephrogenesis. The data suggests a role for apoptosis in nephron underdosing, however, more strikingly, the differentially expressed genes point to more complex molecular interactions involving mitochondria and WT1. To date, there are no reports of WT1 involvement in any aspect of mitochondrial biology. Molecular analyses verified WT1 protein is found in mitochondria indicating the potential for direct involvement of WT1 in mitochondria and, therefore, a potential for mitochondrial involvement in nephron development and endowment. These data suggest a previously unrecognised component of nephrogenesis that should be considered in future investigations to further uncover the various pathways involved in final nephron endowment.