Determination of collagen content, concentration, and sub-types in kidney tissue.

Fibrosis and sclerosis are widely recognized as hallmarks of progressive renal disease and are caused by the excessive accumulation of connective tissue, mostly collagen. The detection of collagen content, concentration (collagen content/dry weight tissue), and sub-types from kidney tissues is therefore an important part of determining the extent of renal fibrosis in ageing and diseased states. This chapter describes a colorimetric-based hydroxyproline assay used to estimate total collagen content and concentration. Based on the method of Bergman and Loxley (8), this spectrophotometric technique estimates total collagen by measuring the hydroxyproline content of tissue. The assay relies on the fact that the collagen triple helix is one of the few proteins that contain the amino acid hydroxyproline. The second part of this chapter describes the use of sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) to isolate, detect and quantify changes in the soluble and insoluble interstitial collagen sub-types. This technique complements the hydroxyproline assay by providing a means of identifying which interstitial collagens are altered in renal disease.