Background: Sustained obstruction of urinary flow invariably leads to inflammation, loss of functional renal structures and progressive deposition of extracellular matrix proteins, culminating in renal fibrosis. Although increased renal tissue inhibitor of matrix metalloproteinase (TIMP-1) expression 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 TIMP-1 transcribing cells. Currently, data concerning plasminogen activator inhibitor type 1 (PAI-1) expression in the ligated kidney are unavailable. Our 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 after unilateral ureteral obstruction (UUO) or sham-surgery (SOR). Leukocyte (OX-1+), macrophage (ED1+) and neutrophil infiltration were analyzed using specific antibodies or nuclear morphology. alpha-Smooth muscle actin (alpha-SMA) immunostaining was measured morphometrically. Mitotic figures and nuclei with an apoptotic morphology were quantified in hematoxylin-eosin (H&E)-stained sections. TIMP-1 mRNA transcribing cells were localized with in situ hybridization (ISH) and identified by subsequent immunostainings for alpha-SMA and macrophages. PAI-1 antigenicity was evaluated immunohistochemically in SOR, contralateral 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 compared with CUKs. Neutrophil accumulation in UUO kidneys became apparent from day 5 and large intraluminal leukocyte clusters (neutrophils and macrophages) were found in the lumen of distended tubules, especially at later stages post-obstruction, when collected urine and tissue samples proved to be sterile upon culture. From day 5 on, the number of apoptotic cells started to predominate the number of mitotic cells in the obstructed kidneys. Interstitial 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 early as 24 hours post-ligation, TIMP-1 mRNA transcribing interstitial cells were detected with ISH, while tubular TIMP-1 expression was sparse. Since at that point in time, no interstitial alpha-SMA expressing cells and only few ED1+ macrophages were present, the bulk of the TIMP-1 mRNA transcription occurred in other interstitial cells. Throughout the study period numerous interstitial TIMP-1 expressing cells were detectable in obstructed kidneys 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 addition, dilated tubules containing intraluminal leukocyte casts were surrounded by a corona of intact neutrophils in H&E-stained sections and ISH showed that similar tubules were encircled by TIMP-1 mRNA expressing cells. PAI-1 immunoreactivity appeared to diminish in the early phase following urinary outlet obstruction, but emerged in damaged tubules from day 5 to 10 on. In later 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 of the fibrotic process and we demonstrated that initially TIMP-1 mRNA is transcribed by very few ED1+ macrophages but mainly by other, presently unidentified, interstitial cells. During later stages of post-ligation, both TIMP-1 (transcribed among others by alpha-SMA+ myofibroblasts, ED1+ macrophages, and possibly neutrophils) and PAI-1 are involved in the progression of tubulointerstitial scarring.