Identification, cellular distribution and potential function of MDC9 in rat kidney
Description
Little information is available concerning the role of MDCs in the kidney or their role in renal pathophysiology. Using degenerate primers for the metalloprotease and disintegrin domains of the MDC family, cDNA templates from tubules, glomeruli and glomerular epithelial cells yielded a single, 195 bp product which upon sequence analysis corresponded to the disintegrin domain of mouse MDC9. Northern analysis on the tubules, glomeruli and GECs, revealed a 3.9 kb transcript identical to mouse MDC9. Western analysis of ConcanavalinA enriched glomerular extracts revealed 116 kDa and 76 kDa proteins, which upon reduction changed to the corresponding 124 kDa and 84 kDa forms. Histochemical studies revealed a basolateral distribution of MDC9 in tubules and glomerular epithelial cells, which colocalized with the betal integrin chain Expression of MDC9-GFP fusion protein in renal epithelial cells revealed a punctate basolateral distribution. Transient overexpression of MDC9 disintegrin domain led to detachment of cultured renal epithelial cells and MDC9 disintegrin domain complexed with alpha3, alpha5 and alpha6 integrins. Overexpression of MDC9-GFP facilitated fusion of renal epithelial cells compared to control GFP expressing cells. Enrichment of rat cortical lysates, using anti-MDC9 IgGs, immunoprecipitated a 116 kDa renal gelatinase. However recombinant MDC9 metalloprotease domain lacked gelatinase activity To explore the role of MDC9 in renal pathophysiology, its expression and localization was examined in rats with experimental (puromycin aminonucleoside/PAN) nephrotic syndrome. MDC9 mRNA levels (RT-PCR) and protein levels (Western) were not significantly altered, in glomeruli and tubules, prepared from rats sacrificed at ONSET of proteinuria (urinary protein/24 hours: PAN 86 + 1.9; control = 18 + 0.8) and PEAK of proteinuria (urinary protein/24 hours: PAN 1470 + 7.6; control = 26 + 1). However immunoperoxidase chemistry revealed a gradual shift in localization of MDC9 with progression of proteinuria: from exclusively basolateral in controls; to basolateral and luminal at the onset of proteinuria; to primarily luminal at the peak of proteinuria. Similar changes in localization were also observed for the EGF receptor. These observations and the ability of MDC9 to cleave HB-EGF, a mitogenic EGF receptor ligand, suggest a role of MDC9 in renal epithelial repair and/or regeneration