Functional And Mechanistic Studies Of Corin In Patients With Chronic Kidney Disease And Pregnancy-Induced Hypertension

Part Ⅰ. Reduced Urinary and Renal Corin Levels in Patients withChronic Kidney DiseaseObjective:Human corin is a type II transmembrane serine protease discovered in the heart.Corin regulates blood pressure and cardiac function by activating natriuretic peptides.Corin mRNA and protein were detected in other tissues such as kidney, bone andpregnant uterus. Recent studies reported that renal corin expression was reduced in ratmodels of kidney disease, suggesting that corin may act locally in regulating renalfunction. Studies in cardiomyocytes have shown that corin is shed from the cellsurface and that soluble corin is present in human blood. We hypothesize that a similarshedding mechanism may occur to corin in the kidney and that corin protein may bepresent in human urine. To test this hypothesis, we measured corin in plasma andurine samples from normal individuals and patients with chronic kidney disease (CKD).We also examined corin protein expression in kidney tissues from normal controls andpatients with CKD. Our study may help to understand the role of corin in renalfunction and kidney disease.Methods:Blood, urine and kidney biopsy samples were obtained from normal individualsand patients with CKD. Blood samples were collected into tubes containing EDTAas an anticoagulant. Plasma samples were prepared by centrifugation. Urine sampleswere collected and centrifuged to remove cell debris. Plasma and urinary corin levelswere measured by ELISA. Corin protein expression in kidney tissues from normalcontrols and patients with CKD was examined by immunohistochemistry followed by computer-assisted quantitative analysis.Results:1. We detected soluble corin in human urine samples, indicating that corin is shedfrom the cell surface in the kidney. In normal males (n=101), urinary corin levels were227107pg/mL, which was significantly higher than that in normal females161108pg/mL (n=104)(P=0.00003). In both males and females, urinary corin levels appearedto be similar in different age groups.2. In patients with CKD, urinary corin levels in males (n=103) were significantlylower than that of normal males (156124vs.227107pg/mL, P=0.0002). Infemale patients with CKD (n=111), urinary corin levels also appeared to be lower thanthat of normal females (144100vs.161108pg/mL). The difference, however,was not statistically significant (P=0.181).3. In multiple linear-regression analysis, chronic glomerulonephritis (CGN)(P<0.001), nephrotic syndrome (NS)(P<0.001), systemic lupus erythematosus (SLE)(P=0.004), and glomerular filtration rate (GFR)(P<0.001) were found to beindependent predictors for urine corin levels. Diastolic blood pressure (DBP)(P=0.037) and gender (P=0.011) were also found to be an independent predictor.4. In both male and female patients with CKD, urinary corin levels correlatedwith the severity of the disease. Patients with stage5CKD had the lowest levels ofurinary corin (both P values <0.01).5. In both male and female groups, patients with CKD and hypertension hadsignificantly lower urinary corin levels than those of normal controls and patients withCKD but without hypertension (both P values <0.01).6. By immunohistochemistry, corin protein staining was identified on the surfaceof epithelial cells in renal tubules. The staining was significantly weaker in samplesfrom patients with CKD than that of normal controls.Conclusions:1. We detected soluble corin in human urine, indicating that corin is shed fromthe renal cell surface and that urinary corin levels may reflect corin expression in thekidney.2. Compared with that in normal controls, corin levels in urine samples and kidney tissues were significantly reduced in patients with CKD.3. In patients with CKD, urinary corin levels were progressively lower in patientswith more advanced disease. Our results suggest that corin deficiency may contributeto the pathogenesis of CKD and that urinary corin may be used as a biomarker in thediagnosis and monitoring of CKD. Part Ⅱ. Identification and Functional Studies of a Novel CorinMutation in Patients with Pregnancy-Induced HypertensionObjective:Corin is a cardiac protease that regulates blood pressure and cardiac function.Unexpectedly, corin mRNA and protein were detected in the pregnant uterus. Inmouse models, corin deficiency caused pregnancy-induced hypertension and proteinuria,which are characteristics of preeclampsia in patients. The results suggested that corinmay play a role in the uterus to prevent hypertension during pregnancy and that corindeficiency may represent an underlying mechanism in pregnancy-induced hypertensionin patients. To test this hypothesis, we sequenced the corin gene in preeclampticpatients and examined the effect of naturally occurring gene mutation on corin proteinstructure and function. These studies should help us to understand the role of corin inpregnancy-induced hypertension.Methods:Blood samples were obtained from65patients with preeclampsia and124normalcontrols. Genomic DNAs were isolated. Polymerase chain reaction (PCR) was usedto amplify the sequence of all22exons of the corin gene. PCR products were isolatedand sequenced. Plasmids expressing corin mutants were constructed by site-directedmutagenesis. Recombinant wild-type (WT) and mutant corin proteins were expressedin transfected human embryonic kidney (HEK)293cells. Corin expression level andzymogen activation were examined by Western blotting. The activity of recombinantWT and mutant corin was determined in a cell-based pro-atrial natriuretic peptide(pro-ANP) processing assay. Results:1. We sequenced corin gene exons in65patients with preeclampsia andidentified a patient who had a corin allele containing an A to G change at the nucleotideposition1507in exon11. The mutation is predicted to cause a Ser to Gly substitutionat amino acid472in Frizzled2domain of corin. No other corin gene mutations werefound in this patient. The S472G mutation was not found in the other64patients and124normal controls.2. In transfected HEK293cells, S472G mutation did not alter corin proteinexpression, as indicated by Western blotting. In cell-based pro-ANP processing assay,corin S472G mutant had~10%activity compared to that of WT corin. Westernanalysis indicated that the zymogen activation of S472G mutant was impaired.3. Additional cellular and biochemical studies showed that S472G mutation alsoaltered corin shedding from the cell surface. In the conditioned medium fromtransfected HEK293cells, an alternatively cleaved corin fragment was detected in cellsexpressing corin S472G mutant.Conclusions:1. We sequenced the corin gene in patients with preeclampsia and identified anovel corin mutation that caused S472G changes in corin Frizzled domain2.2. In transfected HEK293cells, S472G mutation did not alter corin proteinexpression but reduced corin activity by~90%in pro-ANP processing assay.3. Biochemical studies indicated that S472G mutation prevented corin zymogeneactivation and altered corin shedding on the cell surface.4. Our results indicate that the mutation may reduce corin activity in vivo,contributing to the pathogenesis of preeclampsia in the patient. Our results illustratethe physiological importance of the corin-mediated pathway in maintaining normalblood pressure during pregnancy.5. Our results also indicate the important of corin Frizzled domain2in regulatingcorin protein conformation and zymogen activation.