Changes in collagenases and TGF-beta precede structural alterations in a model of chronic renal fibrosis

To study the role of collagenases and transforming growth factor-beta (TGF-beta) in the genesis of interstitial fibrosis, we used the model of bromoethylamine (BEA)-induced papillary necrosis, which is known to lead over a period of 1 to 12 months to interstitial fibrosis and renal insufficiency.

Rats were injected with BEA, and urine and kidney tissue (cortex and medulla) were collected after 1, 2, 3, 7, and 30 days. One kidney was perfused and fixed for morphological studies and immunostained for collagen type I, III, and IV. The other kidney was used to prepare cortex and medulla extracts for gelatinases (by fluorometric and zymographic techniques), tissue inhibitors of metalloproteinase-1 (TIMP-1), and TIMP-2 (by enzyme-linked immunosorbent assay, ELISA) and TGF-beta1 (by ELISA).

Albuminuria and interstitial fibrosis were present in BEA rats by day 7, which continued until day 30. Immunocytochemical staining for collagen types showed that collagen III and IV increased in the interstitium by day 30, but collagen I remained unchanged. Gelatinase activity in the medulla decreased by 57% compared with control by day 2 and remained low until day 30. In the cortex, gelatinase activity remained unchanged between 0 and 7 days after BEA but decreased by 72% by day 30. TIMP-1 and TIMP-2 were decreased by 80% compared with day 0 in both the medulla (by day 1) and cortex (by day 2) and remained low up to day 30. TGF-beta1 immunoreactivity increased progressively until day 2 in the medulla (16-fold higher than control) and day 3 in the cortex (8-fold higher than control) and returned to control level by day 3 in the medulla and by day 30 in the cortex. Two days after BEA injection, the mRNA for TGF-beta1 was increased eightfold in the cortex and 12-fold in the medulla, and it remained high for up to 30 days.

The fibrosis that follows papillary necrosis is associated with both high TGF-beta1 expression and depressed gelatinolytic activity.