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The pathology of progressive renal disease is characterized by glomerular and interstitial inflammation, glomerulosclerosis, and tubulointerstitial fibrosis. This is a consequence of excessive matrix synthesis, reduced matrix degradation, and contraction (reorganization) of extracellular matrix. Fibroblasts, and to a lesser degree, other mesenchymal cells, are known to contribute to renal scar formation through local proliferation, synthesis, and reorganization of matrix proteins. Although much work has focused on the balance between collagen synthesis and degradation, the mechanisms of parenchymal collapse and contraction are becoming increasingly important. Like their counterparts in the skin, the contractile properties of renal fibroblasts are now well recognized. This chapter details an in vitro method for studying the contraction of collagens by homogeneous populations of cultured cells. The method can be altered so that reagents influencing this process may also be studied.

Monoclonal antibodies to proliferation associated antigens have long been used to histologically localize mitogenesis. However, techniques that distinguish cells in the synthetic or S phase have tended to rely on the in vivo incorporation of tritiated thymidine or thymidine analogs such as bromodeoxyuridine. The necessity to pulse with these labels before retrieving tissue means that they cannot be used in humans and are not available retrospectively. Measuring expression of histones serves as a useful adjunct to these techniques. As expression of histone proteins (H2A, H2B, H3, H4) are restricted to the synthetic phase of the cell cycle, hybridization for histone mRNA precisely distinguishes those cells in the S phase. Measuring their expression can easily be applied to the histological localization of proliferation, and can be used both prospectively and with archived tissue specimens. Several histone in situ hybridization probes and nonradioactive detection systems are now available commercially. A generalized protocol for their use in measuring in situ proliferation is provided in this chapter.