Expression of Embryonic Fibronectin Isoform EIIIA Parallels α-Smooth Muscle Actin in Maturing and Diseased Kidney

Abstract

In this study we examined if an association exists between expression of an alternatively spliced "embryonic" fibronectin isoform EIIIA (Fn-EIIIA) and alpha-smooth muscle actin (alpha-SMA) in the maturing and adult rat kidney and in two unrelated models of glomerular disease, passive accelerated anti-glomerular basement membrane (GBM) nephritis and Habu venom (HV)-induced proliferative glomerulonephritis, using immunohistochemistry and in situ hybridization. Fn-EIIIA and alpha-SMA proteins were abundantly expressed in mesangium and in periglomerular and peritubular interstitium of 20-day embryonic and 7-day (D-7) postnatal kidneys in regions of tubule and glomerular development. Staining was markedly reduced in these structures in maturing juvenile (D-14) kidney and was largely lost in adult kidney. Expression of Fn-EIIIA and alpha-SMA was reinitiated in the mesangium and the periglomerular and peritubular interstitium in both models and was also observed in glomerular crescents in anti-GBM nephritis. Increased expression of Fn-EIIIA mRNA by in situ hybridization corresponded to the localization of protein staining. Dual labeling experiments verified co-localization of Fn-EIIIA and alpha-SMA, showing a strong correlation of staining between location and staining intensity during kidney development, maturation, and disease. Expression of EIIIA mRNA corresponded to protein expression in developing and diseased kidneys and was lost in adult kidney. These studies show a recapitulation of the co-expression of Fn-EIIIA and alpha-SMA in anti-GBM disease and suggest a functional link for these two proteins.

Origin of Interstitial Fibroblasts in an Accelerated Model of Angiotensin II-Induced Renal Fibrosis

“…39,40 Kidneys of rats receiving HV alone showed a course of Fn-EIIIA expression similar to that previously described 37,39 peaking at 7 days and diminishing at 14 days (Figure 2a). Fn-EIIIA expression in kidneys of rats administered Ang II alone was increased in the glomerular mesangium at all time points examined.…”

“…The rats were unilaterally nephrectomized to increase the incidence of glomerular lesions after HV as previously described. [37][38][39][40] Twenty-four hours later the rats were divided into four groups and subjected to the following protocol:…”

“…Renal expression of extracellular matrix was assessed by indirect or direct immunofluorescence or immunoperoxidase microscopy as described previously 37,39,40 using a mouse monoclonal antibody specific for the alternatively spliced EIIIA domain of fibronectin (Fn-EIIIA, clone IST-9, Serotec; Harlan Bioproducts for Science, Indianapolis, IN) and rabbit polyclonal antibodies to collagen type I and type IV (Chemicon Int., Temecula, CA). These antibodies are highly specific to their respective types with less than 0.1% cross reactivity with other collagens.…”

“…Dual-label immunofluorescence was used to assess the expression of the individual marker proteins in peritubular myofibroblasts and tubular epithelial cells relative to the tubular basement membrane (TBM) using laminin as an indicator of tubular boundaries and using fluorescence techniques similar to those previously described. 40,46 To detect myofibroblasts relative to the TBM, a Cy3-labeled mouse anti-␣-SMA antibody (clone 1A4, Sigma) was used at a 1:100 dilution then the sections were incubated with rabbit anti-laminin IgG, 20 g/ml (Lab Vision Corporation, Fremont, CA), followed by incubation with FITC-labeled mouse anti-rabbit IgG to detect TBM in green. To examine epithelial cell location relative to the TBM and interstitium, a monoclonal antibody to Na ϩ ,K ϩ -ATPase (clone ␣5; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) was used followed by staining for laminin as above.…”

“…Kidneys of rats given HV alone showed typical progression of mesangioproliferative glomerular characterized by microaneurysms in early lesions followed by micronodules as previously described. [37][38][39][40] The lesions begin to resolve and decrease in size by 14 days after HV (Figure 1a). Glomerular lesions were observed with an incidence of 10.2 Ϯ 1.7%, 11.8 Ϯ 3.1%, and 5.8 Ϯ 0.7% at 48 hours and 7 and 14 days after HV, respectively.…”