ARHGDIA: a novel gene implicated in nephrotic syndrome.

BACKGROUND: Congenital nephrotic syndrome arises from a defect in the glomerular filtration barrier that permits the unrestricted passage of protein across the barrier, resulting in proteinuria, hypoalbuminaemia, and severe oedema. While most cases are due to mutations in one of five genes, in up to 15% of cases, a genetic cause is not identified. We investigated two sisters with a presumed recessive form of congenital nephrotic syndrome. METHODS AND RESULTS: Whole exome sequencing identified five genes with diallelic mutations that were shared by the sisters, and Sanger sequencing revealed that ARHGDIA that encodes Rho GDP (guanosine diphosphate) dissociation inhibitor α (RhoGDIα, OMIM 601925) was the most likely candidate. Mice with targeted inactivation of ARHGDIA are known to develop severe proteinuria and nephrotic syndrome, therefore this gene was pursued in functional studies. The sisters harbour a homozygous in-frame deletion that is predicted to remove a highly conserved aspartic acid residue within the interface where the protein, RhoGDIα, interacts with the Rho family of small GTPases (c.553_555del(p.Asp185del)). Rho-GTPases are critical regulators of the actin cytoskeleton and when bound to RhoGDIα, they are sequestered in an inactive, cytosolic pool. In the mouse kidney, RhoGDIα was highly expressed in podocytes, a critical cell within the glomerular filtration barrier. When transfected in HEK293T cells, the mutant RhoGDIα was unable to bind to the Rho-GTPases, RhoA, Rac1, and Cdc42, unlike the wild-type construct. When RhoGDIα was knocked down in podocytes, RhoA, Rac1, and Cdc42 were hyperactivated and podocyte motility was impaired. The proband's fibroblasts demonstrated mislocalisation of RhoGDIα to the nucleus, hyperactivation of the three Rho-GTPases, and impaired cell motility, suggesting that the in-frame deletion leads to a loss of function. CONCLUSIONS: Mutations in ARHGDIA need to be considered in the aetiology of heritable forms of nephrotic syndrome.

Morphogenesis during mouse embryonic kidney explant culture.

BACKGROUND: Renal organogenesis is routinely studied using cultured murine embryonic kidneys, but the application of this model has not yet been subjected to rigorous standards. METHODS: We measured ex vivo growth and morphogenesis of day 13 murine kidneys and evaluated the importance of culture conditions and biological variables. RESULTS: Kidney size was measured in two dimensions as planar surface area and was shown to correlate highly with volume (R2 = 0.60, P < 0.005). The final surface area of kidneys was directly dependent on the initial starting size (R2 = 0.61, P < 0.05), suggesting that the final surface area is not a valid outcome measurement unless starting size is equal among treatments. Relative growth rate, defined as (final surface area - initial surface area)/initial surface area, was a good measure of growth and independent of size and anatomical position (P> 0.05). Significant differences in size and growth rates were observed among litters (P < 0.05), implying that kidneys from a given litter must be randomized to avoid confounding results. Planar surface area of each explant increased in proportion to ureteric bud branching (R2 = 0.6854, P < 0.05). In a comparison of a variety of base media and supplements, kidney explants were observed to grow best in Dulbecco's modified Eagle's medium (DMEM)/F12 with 5% fetal bovine serum and to sustain growth for up to 96 hours, despite decreased proliferation and increased apoptosis at this time point. CONCLUSIONS: These results represent an important step in establishing standardized procedures for the use of cultured embryonic kidneys and will improve our ability to apply the model to better understand kidney morphogenesis.