Serine proteases, inhibitors and receptors in renal fibrosis

Abstract

Chronic kidney disease (CKD) is estimated to affect one in eight adults. Their kidney function progressively deteriorates as inflammatory and fibrotic processes damage nephrons. New therapies to prevent renal functional decline must build on basic research studies that identify critical cellular and molecular mediators. Plasminogen activator inhibitor-1 (PAI-1), a potent fibrosis-promoting glycoprotein, is one promising candidate. Absent from normal kidneys, PAI-1 is frequently expressed in injured kidneys. Studies in genetically engineered mice have demonstrated its potency as a pro-fibrotic molecule. Somewhat surprising, its ability to inhibit serine protease activity does not appear to be its primary pro-fibrotic effect in CKD. Both tissue-type plasminogen activator and plasminogen deficiency significantly reduced renal fibrosis severity after ureteral obstruction, while genetic urokinase (uPA) deficiency had no effect. PAI-1 expression is associated with enhanced recruitment of key cellular effectors of renal fibrosis - interstitial macrophages and myofibroblasts. The ability of PAI-1 to promote cell migration involves interactions with the low-density lipoprotein receptor-associate protein-1 and also complex interactions with uPA bound to its receptor (uPAR) and several leukocyte and matrix integrins that associate with uPAR as co-receptors. uPAR is expressed by several cell types in damaged kidneys, and studies in uPAR-deficient mice have shown that its serves a protective role. uPAR mediates additional anti-fibrotic effects - it interacts with specific co-receptors to degrade PAI-1 and extracellular collagens, and soluble uPAR has leukocyte chemoattractant properties. Molecular pathways activated by serine proteases and their inhibitor, PAI-1, are promising targets for future anti-fibrotic therapeutic agents.

Figure 1

Schematic overview of the major pathways involved in the pathogenesis of chronic kidney disease (CKD). The initial phase is characterized by an influx of macrophages and (myo)fibroblasts) into the interstitium. Macrophage recruitment is facilitated by increased permeability of the interstitial capillaries and by release of chemoattractant molecules from activated tubular epithelial cells. Locally macrophages produce a variety of biologically active products that can propagate injury and stimulate synthesis of extracellular matrix proteins by myofibroblasts. These proteins are the precursors of the scar tissue that begins to form within the interstitium. The interstitial myofibroblasts that actively secrete extracellular matrix appear to have several possiblel origins – resident kidney fibroblasts, circulating fibrocytes, vascular pericytes/fibroblasts and damaged tubular epithelial cells that transdifferentiate into mesenchymal cells. In additional to enhanced synthesis rates, endogenous matrix turnover pathways or often inhibited to accelerate fibrosis. The photomicrographs illustrate the consequences of these pathological events which convert normal kidney architecture (left) to regions of interstitial inflammation and fibrosis and tubular loss (right). The normal kidney, characterized by intact glomeruli (G) and tubules (T) that sit back-to-back with minimal intervening interstitial space are transformed into a kidney characterized by a prominent and abnormal interstitial region consisting of extracellular matrix proteins and inflammatory cells. These changes have detrimental consequences on the number of functional intact nephrons, as illustrated by the shrunken glomerulus and atrophic tubules (right).

Figure 2

PAI-1 biological effects that may enhance renal fibrosis. PAI-1 expression is markedly increased in damaged kidneys, as illustrated for the mouse unilateral ureteral obstruction (UUO) model. PAI-1 is a glycoprotein that normally has a short half-life due to intracellular degradation after binding to the tertiary complex of uPA + uPAR + LRP-1. High affinity binding to vitronectin competes with uPAR binding and prolongs PAI-1 survival, as may occur when vitronectin accumulates in damaged renal interstitial regions. PAI-1 regulates the activity of two distinct molecular pathways, each of which may potentially be involved with kidney fibrosis: the protease activity-dependent pathway (leading to inhibition of tPA, uPA and plasmin activity) and a cellular pathway that involves PAI-1 interactions with uPAR and its co-receptors that is largely protease-activity-independent. Although PAI-1 blocks tPA and uPA activation, which has downstream effects on plasmin generation from plasminogen, these effects do not appear to enhance renal fibrosis based on the data that are currently available from experimental CKD models. In fact, the number of interstitial myofibroblasts and fibrosis severity are significantly reduced in tPA- and plasminogen-deficient mice compared to wild-type mice. In contrast, the ability of PAI-1 to alter interactions between uPAR and its co-receptors (such as several intregin family members) leads to enhanced recruitment of fibrosis-promoting cells, including macrophages and myofibroblasts. The PAI-1 northern blot was reproduced from Oda et al, PAI-1 deficiency attenuates the fibrogenic response to ureteral obstruction, Kidney Int 60:587-596, 2001 with copyright permission (28).

Figure 3

PAI-1 and serine proteases: effects of genetically altered expression levels on renal fibrosis severity (sirius red+ interstitial collagen), F4/80+ interstitial macrophages and SMA+ interstitial myofibroblasts in the mouse unilateral ureteral obstruction (UUO) model. The data are expressed as the percent interstitial area stained relative to levels in wild-type mice 14 days after UUO (except for the tPA−/− study and the uPA−/− study for which day 7 data for total kidney collagen levels and macrophage area are respectively shown, and indicated by the *). Data are taken from the following publications: (10, 28, 29, 37, 44, 47). The photomicrographs A. B and D are reproduced from Oda et al, PAI-1 deficiency attenuates the fibrogenic response to ureteral obstruction, Kidney Int 60:587-596, 2001 with copyright permission (28). ND – not done.