Detection of mRNA for Alpha-3 Chain of Type IV Collagen in the Glomerular Epithelium, and the Effect of Perfused Elastase on Its Expression

Abstract

Background: The type IV collagen is a complex of trimetric molecule composed of six genetically distinct polypeptide chains; α1–6(IV). Since α3(IV) distribute specifically in the glomerular basement membrane (GBM) of glomerular capillary, we tried to develop the detection methods for the transcripts of α3(IV) in glomerular epithelial cells (GEC) which produce most of the components for GBM. Then, using these molecular techniques, the influence of elastase, one of the proteases released from activated polymorphonuclear leukocytes at the site of inflammation, on GEC was determined as manifested by expressional alteration of α3(IV) mRNA. Methods: DIG-labeled oligo-DNA probe designating non-collagenous region of α3(IV) was used for in situ hybridization. Semiquantitative measurement of α3(IV) in the renal cortex was performed by PCR reactions, each reaction being normalized by that for GAPDH. Then, the femoral artery of each of 18 Sprague-Dawley rats was catheterized and the left kidney was perfused with saline alone (0.5 ml) or saline containing 100 µg/ml elastase. After collection of urine for 24 h, the left kidney was harvested for analysis of mRNA (4 for in situ hybridization and 5 kidneys for PCR analysis). Results: Antisense cDNA probe and PCR reaction well identified α3(IV) mRNA in the cytoplasm of GEC and in the renal cortex, respectively. Urinary protein excreted by rats with elastase perfusion was 47.2 ± 3.8 mg/24 h but this was only 13.9 ± 1.1 mg/24 h in control rats (mean ± SEM, p < 0.05). In situ hybridization demonstrated that expression of α3(IV) mRNA in GEC was focally or diffusely reduced in the glomeruli of rats with elastase perfusion, whereas the transcripts were well stained in GEC of controls. PCR analysis showed about 25% decrease in transcripts of α3(IV) in the renal cortex of rats with elastase perfusion compared to those of control rats. Conclusions: α3(IV) mRNA was identified specifically in the GEC in the glomeruli. Co-incidence of proteinuria and reduced α3(IV) expression by elastase suggests adverse effects of elastase on GEC and close association between proteinuria and GEC injury.