Laboratory Investigation

Kidney International (1991) 40, 470–476; doi:10.1038/ki.1991.234

Demonstration of PDGF B-chain mRNA in glomeruli in mesangial proliferative nephritis by in situ hybridization

Ashio Yoshimura1, Kathy Gordon1, Charles E Alpers1, Jürgen Floege1, Pam Pritzl1, Russell Ross1, William G Couser1, Daniel F Bowen-Pope1 and Richard J Johnson1

1Division of Nephrology, Departments of Medicine and Pathology, University of Washington Medical Center, Seattle, Washington, USA

Correspondence: Dr Ashio Yoshimura, Division of Nephrology, RM-11, University of Washington Medical Center, Seattle, Washington 98195, USA.

Received 21 January 1991; Revised 15 April 1991; Accepted 16 April 1991.

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Abstract

Demonstration of PDGF B-chain mRNA in glomeruli in mesangial proliferative nephritis by in situ hybridization. We used the technique of in situ hybridization to determine if cells expressing PDGF B-chain mRNA can be detected in a model of mesangial proliferative nephritis in the rat induced with antibody directed against the Thy 1 antigen present on the mesangial cell membrane. The method involved hybridization with a digoxigenin-labeled cRNA probe for the murine PDGF B-chain followed by detection with an anti-digoxigenin-alkaline phosphatase conjugate and subsequent colorimetric reaction. In normal rats (N = 4), the majority of glomeruli (74%) were negative for PDGF B-chain mRNA, whereas 65% of glomeruli from rats with mesangial proliferative nephritis (N = 4) had segmental or diffuse staining for PDGF B-chain mRNA in a mesangial pattern. The difference, as measured using a semiquantitative scale, was significant (mean scores 0.4 plusminus 0.2 vs. 1.9 plusminus 0.2; scale 0 to 3+ ; P < 0.001). The increase in PDGF B-chain mRNA positive cells localized to areas of hypercellularity and was associated with a significant increase in cells positive for PDGF B-chain by immunostaining with a specific monoclonal antibody (0.8 plusminus 0.1 vs. 1.7 plusminus 0.4, scale 0 to 3+ , normal vs. diseased rats, P < 0.005). Complement depletion, which prevents the mesangial cell proliferation, also prevented the increase in cells expressing PDGF B-chain mRNA and protein. Thus, this method of in situ hybridization can successfully detect cells expressing PDGF mRNA in active glomerulonephritis, and may be useful for detecting cells expressing genes for other growth factors and cytokines in both human and experimental models of glomerular injury.

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