TIMP-1 gene expression and PAI-1 antigen after unilateral ureteral obstruction in the adult male rat

Background. Sustained obstruction of urinary flow invariably leads to infla mmation. loss of functional renal structures and progressive deposition of extracellular matrix proteins, culminating in renal fibrosis. Although incr eased renal tissue inhibitor of matrix metalloproteinase (TIMP-1) expressio n is one of the: early events following experimental hydronephrosis. little is known about its cellular source. Both the recruited macrophage and the resident/recruited (myo)fibroblast have been postulated to be candidate TIM P-1 transcribing cells. Currently, data concerning plasminogen activator in hibitor type 1 (PAI-I) expression in the ligated kidney are unavailable. Ou r study concentrated on the localization of TIMP-1 expressing cells and PAI 1 immunoreactive cells in the obstructed rat kidney. Methods. Rats were sacrificed 1, 5, 10, 15. 20 and 26 days alter unilateral ureteral obstruction (UUO) or sham-surgery (SOR). Leukocyte (OX-1(+)), mac rophage (ED1(+)) and neutrophil infiltration were analyzed using specific a ntibodies or nuclear morphology. alpha-Smooth muscle actin (alpha-SMA) immu nostaining was measured morphometrically. Mitotic figures and nuclei with a n apoptotic morphology were quantified in hematoxylin-eosin (H&E)-stained s ections. TIMP-1 mRNA transcribing cells were localized with in situ hybridi zation (ISH) and identified by subsequent immunostainings for a-SMA and mac rophages. PAI-1 antigenicity was evaluated immunohistochemically in SOR, co ntralateral unobstructed kidneys (CUK), and UUO kidneys. Results. The number of leukocytes and macrophages in the ligated rat kidney increased progressively in time, starting from day 5 post-surgery when com pared with CUKs. Neutrophil accumulation in UUO kidneys became apparent fro m day 5 and large intraluminal leukocyte clusters (neutrophils and macropha ges) were found in the lumen of distended tubules. especially at later stag es post-obstruction, when collected urine and tissue samples proved to be s terile upon culture. From day 5 on, the number of apoptotic cells started t o predominate the number of mitotic cells in the obstructed kidneys. Inters titial alpha-SMA immunoreactivity in the ligated kidney expanded from day 5 on and was most pronounced in the inner stripe of the outer medulla. As ea rly as 24 hours post-ligation, TIMP-1 mRNA transcribing interstitial cells were detected with ISH, while tubular TIMP-1 expression was sparse. Since a t that point in time, no interstitial alpha-SMA expressing cells and only f ew ED1(+) macrophages were present, the bulk of the TIMP-1 mRNA transcripti on occurred in other interstitial cells. Throughout the study period numero us interstitial TIMF-1 expressing cells were detectable in obstructed kidne ys and from day 5 after ligation on, we could identify alpha-SMA(+) and to a lesser degree ED1(+) macrophages as TIMP-1 transcribing cells. In additio n, dilated tubules containing intraluminal leukocyte casts were surrounded by a corona of intact neutrophils in H&E-stained sections and ISH showed th at similar tubules were encircled by TIMP-1 mRNB expressing cells. PAI-I im munoreactivity appeared to diminish in the early phase following urinary ou tlet obstruction, but emerged in damaged tubules from day 5 to 10 on. In la ter stages post-ligation, PAI-1(+) cells and PAI-1 immunoreactive material were found embedded in the extracellular matrix. Conclusions. Our results confirm that TIMP-1 is active in the early phase o f the fibrotic process and we demonstrated that initially TIMP-1 mRNA is tr anscribed by very few ED1(+) macrophages but mainly by other, presently uni dentified, interstitial cells. During later stages of post ligation, both T IMP-1 (transcribed among others by alpha-SMA(+) myofibroblasts, ED1(+) macr ophages, and possibly neutrophils) and PAI-1 are involved in the progressio n of tubulointerstitial scarring.