Molecular Studies Of Corin Expression In The Apical Membrane Of Polarized Renal Epithelial Cells

Objective:Corin is a type II transmembrane serine protease mainly expressed in cardiomyocytes,where corin converts pro-atrial natriuretic peptide(pro-ANP)to active atrial natriuretic peptide(ANP)to regulate salt-water balance and blood pressure.In addition to the heart,corin expression has been found in human,mouse and rat kidneys.In animal models of proteinuric disease and patients with chronic kidney disease,renal and urinary corin levels were significantly decreased compared with normal controls.These results suggest that renal corin may play an important role in kidney function and that impaired renal corin expression and/or function may contribute to kidney diseases.The renal tubular epithelial cells are a single layer of polarized cells on the surface of the renal tubular lumen.Corin protein expression was detected on the luminal side of the renal tubules,where sodium and water reabsorption occur.Studies of corin expression in renal tubular epithelial cells should help to provide new insights into the potential role of corin in kidney function and disease.In this study,we examined corin expression in human kidney tissues to understand the cellular and subcellular distribution of corin protein in renal tubules.We also used Madin-Darby Canine Kidney(MDCK)cells as a model to study the cell membrane distribution of corin in polarized renal epithelial cells.Methods:(1)Immunofluorescent staining and immuno-electron microscopy(immuno-EM)were performed to study corin subcellular distribution in human renal proximal tubular epithelial cells.(2)Immunofluorescent staining was used to examine the cell membrane targeting of corin,neprilysin,hepsin and matriptase in polarized MDCK cells in culture and to study corin membrane targeting in non-polarized human embryonic kidney 293(HEK-293)cells and mouse HL-1 cardiomyocytes.(3)Cell transfection,immunoprecipitation and Western blotting were performed to study the expression,activation and ectodomain shedding of corin in MDCK cells.(4)Glycosidase digestion and Western blotting analysis were used to compare N-glycosylation in corin proteins expressed in polarized MDCK cells and non-polarized HEK-293 cells.(5)Plasmids expressing corin deletion mutants were constructed by QuikChange Lightning Site-Directed Mutagenesis Kit using wild-type(WT)human corin expressing plasmid as a template.(6)Immunofluorescent staining was used to examine the effect of corin extracellular domain deletions on corin cell membrane targeting in polarized MDCK cells.Results:(1)In human renal proximal tubular epithelial cells,neprilysin was detected in the brush border region,whereas corin was detected under the brush border in immunofluorescent staining experiments.Consistent with these results,immuno-EM confirmed corin expression in the apical membrane of human proximal tubular epithelial cells.(2)In cultured MDCK cells,we found that corin and neprilysin were located at the apical membrane.In contrast,hepsin and matriptase were expressed on both apical and basolateral membranes in MDCK cells.In HEK-293 cells and HL-1 cardiomyocytes,corin was detected on both apical and basolateral membranes.(3)By immunoprecipitation and Western analysis,we found that corin underwent zymogen activation and extodomain shedding in transfected MDCK cells.These post-translational processes of corin were similar to those reported in HEK-293 cells.(4)On Western blots,corin fragments shed into the conditioned medium migrated slower than those fragments from HEK-293 cells.Glycosidase digestion experiments indicated that the apparent molecular mass differences between corin fragments from MDCK and HEK-293 cells were due to different amounts of N-glycans on these protein fragments.(5)In MDCK cells,we found that corin WT and mutants with truncated N-terminal cytoplasmic tails were expressed similarly on the apical membrane,suggesting that the cytoplasmic tail of corin is an apical sorting signal in the polarized renal epithelial cells.(6)We also tested a series of corin mutants that lacked different extracellular domains.Corin mutants lacking LDLR6-8 or SR domain were found to be expressed on both the apical and basolateral membranes of MDCK cells.Conversely,the corin mutant that contained only SR and the protease domains in the extracellular region retained the characteristic of the apical targeting.(7)We further tested corin mutants that lacked N-glycosylation sites in the SR domain.We found that the corin mutant without N-glycosylation sites in the SR domain were still targeted to the apical membrane in MDCK cells,indicating that N-glycosylation in the SR domain is not a corin apical sorting signal in the polarized renal epithelial cells.(8)By constructing and testing additional corin mutants with truncated sequences in the SR domain,we found that an intact SR domain is essential for the apical membrane targeting of corin in MDCK cells.Conclusions:(1)In human kidneys,corin was expressed predominantly on the apical membrane of renal epithelial cells.(2)In polarized MDCK cells,corin was specifically targeted to the apical membrane.In contrast,corin was present on both apical and basolateral membranes in non-polarized HEK-293 and HL-1 cells.(3)In MDCK cells,corin underwent zymogen activation and ectodomain shedding,which were similar to those reported in HEK-293 and HL-1 cells.Corin protein expressed in MDCK cells,however,contained more abundant N-glycans than corin proteins from HEK-293 cells.(4)The cytoplasmic tail and N-glycosylation in the SR domain are not corin apical sorting signals.(5)LDLR8 and SR domains are required for corin apical membrane sorting in MDCK cells.It is possible that the presence of the LDLR8 is a requirement for the integrity of the SR domain,which serves as an apical membrane signal in polarized renal epithelial cells.In summary,our studies show that corin is specifically expressed on the apical membrane of the polarized renal epithelial cells.Such a cell membrane expression pattern differs from the corin expression in non-polarized HEK-293 cells and cardiomyocytes.Our results also uncovered the structural elements in the corin extracellular region that are critical for the apical membrane targeting of corin in polarized renal epithelial cells.These findings provide important new insights into the potential corin function in kidney biology and diseases.