Xiaohe Cai

Urokinase Receptor Deficiency Accelerates Renal Fibrosis in Obstructive Nephropathy

The urokinase cellular receptor (uPAR) recognizes the N-terminal growth factor domain of urokinase-type plasminogen activator (uPA) and is expressed by several cell types. The present study was designed to test the hypothesis that uPAR regulates the renal fibrogenic response to chronic injury. Groups of uPAR wild-type (+/+) and deficient (-/-) mice were investigated between 3 and 14 d after unilateral ureteral obstruction (UUO) or sham surgery. Not detected in normal kidneys, uPAR mRNA was expressed in response to UUO in the +/+ mice. By in situ hybridization, uPAR mRNA transcripts were detected in renal tubules and interstitial cells of the obstructed uPAR+/+ kidneys. The severity of renal fibrosis, based on the measurement of total collagen (13.5 +/- 1.5 versus 9.8 +/- 1.0 microg/mg kidney on day 14; -/- versus +/+) and interstitial area stained by Masson trichrome (22 +/- 4% versus 14 +/- 3% on day 14; -/- versus +/+) was significantly greater in the uPAR-/- mice. In the absence of uPAR, renal uPA activity was significantly decreased compared with the wild-type animals after UUO (62 +/- 20 versus 135 +/- 13 units at day 3 UUO; 74 +/- 17 versus 141 +/- 16 at day 7 UUO; 98 +/- 20 versus 165 +/- 10 at day 14 UUO; -/- versus +/+). In contrast, renal expression of several genes that regulate plasmin activity were similar in both genotypes, including uPA, tPA, PAI-1, protease nexin-1, and alpha2-antiplasmin. Worse renal fibrosis in the uPAR-/- mice appears to be TGF-beta-independent, as TGF-beta activity was actually reduced by 65% in the -/- mice despite similar renal TGF-beta1 mRNA levels. Significantly lower levels of the major 2.3-kb transcript and the 69-kd active protein of hepatocyte growth factor (HGF), a known anti-fibrotic growth factor, in the uPAR-/- mice suggests a potential link between HGF and the renoprotective effects of uPAR. These data suggest that renal uPAR attenuates the fibrogenic response to renal injury, an outcome that is mediated in part by urokinase-dependent but plasminogen-independent functions.

Cell Biology – Immunology – PathologyMultifunctionality of PAI-1 in fibrogenesis: Evidence from obstructive nephropathy in PAI-1–overexpressing mice

BACKGROUND Plasminogen activator inhibitor-1 (PAI-1) has been implicated in the pathogenesis of chronic kidney disease based on its up-regulated expression and on the beneficial effects of PAI-1 inhibition or depletion in experimental models. PAI-1 is a multifunctional protein and the mechanisms that account for its profibrotic effects have not been fully elucidated. METHODS The present study was designed to investigate PAI-1-dependent fibrogenic pathways by comparing the unilateral ureteral obstruction model (UUO) (days 3, 7, and 14) in PAI-1-overexpressing mice (PAI-1 tg) to wild-type mice, both on a C57BL6 background. RESULTS Following UUO, total kidney PAI-1 mRNA and/or protein levels were significantly higher in the PAI-1 tg mice (N= 6 to 8/group) and fibrosis severity was significantly worse (days 3, 7, and 14), measured both as Sirius red-positive interstitial area (e.g., 10 +/- 3.2% vs. 4.5 +/- 1.0%) (day 14) and total kidney collagen (e.g., 11.1 +/- 1.7 vs. 6.2 +/- 1.3 microg/mg) (day 14). By day 14, the expression of two normal tubular proteins, E-cadherin and Ksp-cadherin, were significantly lower in the PAI-1 tg mice (3.2 +/- 0.5% vs. 11.7 +/- 5.9% and 2.6 +/- 1.6) vs. 6.2 +/- 0.8%, respectively), implying more extensive tubular damage. At least four fibrogenic pathways were differentially expressed in the PAI-1 tg mice. First, interstitial macrophage recruitment was more intense (P < 0.05 days 3 and 14). Second, interstitial myofibroblast density was greater (P < 0.05 days 3 and 7) despite similar numbers of proliferating tubulointerstitial cells. Third, transforming growth factor-beta1 (TGF-beta1) and collagen I mRNA were significantly higher. Finally, urokinase activity was significantly lower (P < 0.05 days 7 and 14) despite similar mRNA levels. Gene microarray studies documented that that the deletion of this single profibrotic gene had far-reaching consequences on renal cellular responses to chronic injury. CONCLUSION These data provide further evidence that PAI-1 is directly involved in interstitial fibrosis and tubular damage via two primary overlapping mechanisms: early effects on interstitial cell recruitment and late effects associated with decreased urokinase activity.

Plasmin(ogen) Promotes Renal Interstitial Fibrosis by Promoting Epithelial-to-Mesenchymal Transition: Role of Plasmin-Activated Signals

Plasminogen (Plg) activator inhibitor-1 (PAI-1) is an important fibrosis-promoting molecule. Whether this effect can be attributed to PAI-1s activity as an inhibitor of plasmin generation is debated. This study was designed to investigate the role of Plg in renal fibrosis using in vivo and in vitro approaches. Plg-deficient (Plg-/-) and wild-type (Plg+/+) C57BL/6 mice were subjected to unilateral ureteral obstruction or sham surgery (n = 8/group; sham, days 3, 7, 14, and 21). Plg deficiency was confirmed by the absence of Plg mRNA, protein, and plasmin activity. After 21 d of unilateral ureteral obstruction, total kidney collagen was significantly reduced by 35% in the Plg-/- mice. Epithelial-to-mesenchymal transition (EMT), as typified by tubular loss of E-cadherin and acquisition of alpha-smooth muscle actin, was also significantly reduced in Plg-/- mice, 76% and 50%, respectively. Attenuation of EMT and fibrosis severity in the Plg-/- mice was associated with significantly lower levels of phosphorylated extracellular signal-regulated kinase (ERK) and active TGF-beta. In vitro, addition of plasmin (20 microg/ml) to cultures of murine tubular epithelial cells initiated ERK phosphorylation within minutes, followed by phenotypic transition to fibroblast-specific protein-1+, alpha-smooth muscle actin+, fibronectin-producing fibroblast-like cells. Both plasmin-induced ERK activation and EMT were significantly blocked in vitro by the protease-activated receptor-1 (PAR-1) silencing RNA; by pepducin, a specific anti-PAR-1 signaling peptide; and by the ERK kinase inhibitor UO126. Plasmin-induced ERK phosphorylation was enhanced in PAR-1-overexpressing tubular cells. These findings support important profibrotic roles for plasmin that include PAR-1-dependent ERK signaling and EMT induction.

Urokinase Receptor Modulates Cellular and Angiogenic Responses in Obstructive Nephropathy

Interstitial cells have been implicated in the pathogenesis of renal fibrosis. Given that the urokinase receptor (uPAR) is known to play a role in cell adhesion, migration, and angiogenesis, the present study was designed to evaluate the role of uPAR in the regulation of the phenotypic composition of interstitial cells (macrophages, myofibroblasts, capillaries) in response to chronic renal injury. Groups of uPAR wild-type (+/+) and knockout (-/-) mice were investigated between 3 and 14 d after unilateral ureteral obstruction (UUO) or sham surgery (n = 8 mice per group). The density of F4/80+ interstitial macrophages (Mphi) was significantly lower in the -/- mice (3.3 +/- 0.4 versus 6.9 +/- 1.7% area at day 3 UUO; 10.8 +/- 1.6 versus 15.7 +/- 1.0% at day 14 UUO; -/- versus +/+). In contrast, in the -/- mice there were significantly more alpha smooth muscle actin (alphaSMA)-positive cells (12.9 +/- 3.2 versus 7.8 +/- 1.5% area at day 3 UUO; 21.0 +/- 4.7 versus 9.7 +/- 1.9% at day 14 UUO) and CD34-positive endothelial cells (8.4 +/- 1.9 versus 4.0 +/- 1.1% area at day 14 UUO). These differences were associated with significantly more interstitial fibrosis in the -/- mice based on Sirius red staining (4.6 +/- 0.9 versus 2.3 +/- 0.9% area at 14 d UUO). Absence of the uPAR scavenger receptor was associated with significantly greater accumulation of plasminogen activator inhibitor-1 protein (PAI-1) (20.5 +/- 3.5 versus 9.1 +/- 2.9% area, day 14 UUO) and vitronectin protein (2.4 +/- 1.1 versus 0.9 +/- 0.4% area, day 14 UUO). By immunostaining alphaSMA+ cells, CD34+ cells, vitronectin and PAI-1 co-localized to the same tubulointerstitial area. The number of apoptotic cells increased in response to UUO but was significantly higher in the -/- mice (2.0 +/- 0.2 versus 1.2 +/- 0.2 per 100 tubulointerstitial cells, day 14 UUO) while the number of proliferating cells was significantly lower in the uPAR-/- mice. These data suggest that uPAR deficiency suppresses renal Mphi recruitment, but the absence of this scavenger receptor actually accentuates the fibrogenic response, likely due in part to the delayed clearance of angiogenic/profibrotic molecules such as PAI-1 and decreased receptor-associated uPA activity.

Mannose Receptor 2 Attenuates Renal Fibrosis

Mannose receptor 2 (Mrc2) expresses an extracellular fibronectin type II domain that binds to and internalizes collagen, suggesting that it may play a role in modulating renal fibrosis. Here, we found that Mrc2 levels were very low in normal kidneys but subsets of interstitial myofibroblasts and macrophages upregulated Mrc2 after unilateral ureteral obstruction (UUO). Renal fibrosis and renal parenchymal damage were significantly worse in Mrc2-deficient mice. Similarly, Mrc2-deficient Col4α3(-/-) mice with hereditary nephritis had significantly higher levels of total kidney collagen, serum BUN, and urinary protein than Mrc2-sufficient Col4α3(-/-) mice. The more severe phenotype seemed to be the result of reduced collagen turnover, because procollagen III (α1) mRNA levels and fractional collagen synthesis in the wild-type and Mrc2-deficient kidneys were similar after UUO. Although Mrc2 associates with the urokinase receptor, differences in renal urokinase activity did not account for the increased fibrosis in the Mrc2-deficient mice. Treating wild-type mice with a cathepsin inhibitor, which blocks proteases implicated in Mrc2-mediated collagen degradation, worsened UUO-induced renal fibrosis. Cathepsin mRNA profiles were similar in Mrc2-positive fibroblasts and macrophages, and Mrc2 genotype did not alter relative cathepsin mRNA levels. Taken together, these data establish an important fibrosis-attenuating role for Mrc2-expressing renal interstitial cells and suggest the involvement of a lysosomal collagen turnover pathway.

Plasminogen Activator Inhibitor-1 Deficiency Has Renal Benefits but Some Adverse Systemic Consequences in Diabetic Mice

Background:Elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1) are observed in patients with obesity, hypertension and diabetes, and several observations suggest that PAI-1 mediates diabetic vascular complications. Although increased intrarenal expression of PAI-1 is also a feature of diabetic nephropathy, evidence that PAI-1 plays a primary pathogenetic role in the renal pathology is lacking. Methods: This study was designed to investigate the renal effects of genetic PAI-1 deficiency in db/db mice with obesity, hyperinsulinemia and hyperglycemia. For comparison the effects of PAI-1 deficiency were also examined in a cohort of mice with insulin-deficient streptozotocin (STZ)-induced diabetes. The findings are reported for 4 study groups at 8 months of age: PAI-1+/+ controls, PAI-1+/+ diabetics, PAI-1–/– controls and PAI-1–/– diabetics. Results: PAI-1 deficiency had an unexpected negative impact on the db/db mice. Overall 33% of the diabetic mice died prematurely, and 63% of the db/db PAI-1–/– males had an obese body habitus but were runts. The final analyses were limited to the female db/db mice. Several nephropathy parameters were improved in the db/db PAI-1–/– group compared to the db/db PAI-1+/+ group including: albumin-to-creatinine ratios (57 ± 45 vs. 145 ± 71 µg/mg ×10), change in glomerular extracellular matrix (ECM) area (decrease of 10% compared to controls vs. an increase of 31%) and increased total kidney collagen (47% increased vs. 96% in the PAI-1+/+ diabetics). The serum glucose levels were 15–25% lower in the PAI-1–/– nondiabetic control groups and remained lower in the db/dbPAI-1–/– mice. The STZ study was performed in males. None of the mice developed a runted phenotype or died prematurely. After diabetes of 6 months’ duration changes in glomerular ECM area (–15 vs. +64%) and total kidney collagen (+8 vs. +40%) were lower in the PAI-1–/– mice compared to the PAI-1+/+ mice. The serum cholesterol levels were significantly lower in the PAI-1–/– mice, both controls (47 ± 3 vs. 53 ± 10 mg/dl) and diabetics (48 ± 3 vs. 74 ± 9 mg/dl). Conclusion: These data suggest a direct role for PAI-1 in renal matrix expansion and metabolic control in diabetes, but they also highlight important adverse outcomes that include male runting and premature death in mice with diabetes due to an inactive leptin receptor.

Exogenous Bone Morphogenetic Protein-7 Fails to Attenuate Renal Fibrosis in Rats with Overload Proteinuria

Background: Bone morphogenetic protein-7 (BMP-7) plays a critical role in renal development, accelerates recovery from acute renal injury, and more recently it has been shown to delay progressive renal disease. The present study was designed to investigate the effect of BMP-7 on interstitial fibrosis in the rat protein-overloaded model. Methods: Renal disease was induced in 26 rats by daily intraperitoneal injections of bovine serum albumin (BSA); controls (n = 28) were injected with saline. Half of the rats in each group were treated with human recombinant BMP-7 (300 µg/kg i.p. 3 times weekly) and half with placebo. Animals were killed after 3 or 6 weeks. Results: Compared to the saline control groups, the BSA groups had evidence of chronic renal disease: significantly increased urinary protein excretion rates; total kidney collagen content, and increased fibronectin and collagen III interstitial areas. By 6 weeks the BSA + BMP-7 group compared to the BSA + placebo group had a nonsignificant decrease in blood urea nitrogen (40 ± 13 vs. 46 ± 11 mg/dl), total kidney collagen (10.8 ± 2.1 vs. 12.2 ± 3.5 µg/kidney), fibronectin interstitial area (23 ± 4 vs. 25 ± 8%) and collagen III interstitial area (22 ± 6 vs. 28 ± 7%). Despite these results, renal gene expression profiles actually predicted worse fibrosis in the BSA + BMP-7 group with significantly higher total kidney mRNA levels for α1(III) procollagen (2.8 ± 0.5 vs. 1.6 ± 0.6, p < 0.05) and fibronectin at 6 weeks (1.9 ± 0.3 vs. 1.2 ± 0.5, p < 0.05). Renal BMP-7 mRNA levels at 6 weeks were significantly increased in the BSA + placebo group compared to the saline + placebo group with no difference between the BSA + BMP-7 and the BSA + placebo groups. Both cortical and medullary tubules expressed BMP-7 protein but BMP-7 was only detected in the tubular lumina and urine of proteinuric animals. Conclusions: In rats with protein-overload proteinuria, renal tubules continue to express BMP-7 but some of the endogenous protein is secreted into the urinary space. Administration of exogenous recombinant BMP-7 had no effect on proteinuria but was associated with a nonsignificant trend towards less interstitial fibrosis at 6 weeks despite significantly higher kidney extracellular matrix gene mRNA levels. These findings suggest that BMP-7 treatment may have anti-fibrotic effects through enhancement of matrix turnover, although overall these effects are modest in proteinuric states in the absence of significant tubular epithelial cell apoptosis and epithelial-mesenchymal transition.

Mitogenic Signaling of Urokinase Receptor–Deficient Kidney Fibroblasts: Actions of an Alternative Urokinase Receptor and LDL Receptor–Related Protein

The urokinase receptor (uPAR) attenuates myofibroblast recruitment and fibrosis in the kidney. This study examined the role of uPAR and its co-receptor LDL receptor-related protein (LRP) in the regulation of kidney fibroblast proliferation and extracellular signal-regulated kinase (ERK) signaling. Compared with uPAR+/+ cells, uPAR-/- kidney fibroblasts were hyperproliferative. UPAR-/- fibroblast proliferation was 60% inhibited by an ERK kinase inhibitor. LRP protein was reduced and extracellular accumulation of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) proteins were greater in uPAR-/- cultures. Addition of functional uPA protein or LRP antisense RNA significantly increased ERK signaling and cell mitosis in both genotypes. Enhanced uPAR-/- fibroblast proliferation was reversed by a recombinant nonfunctional uPA peptide. The density of cell-bound fluor-uPA was similar between uPAR-/- and uPAR+/+ fibroblasts (78 +/- 6 versus 92 +/- 16 units). These data suggest that uPAR-deficient kidney fibroblasts express lower levels of its scavenger co-receptor LRP, resulting in greater extracellular accumulation of uPA and PAI-1. Enhanced proliferation of uPAR-/- fibroblasts seems to be mediated by uPA-dependent ERK signaling via an alternative urokinase receptor.

Vitronectin accumulates in the interstitium but minimally impacts fibrogenesis in experimental chronic kidney disease

Vitronectin (Vtn) is a glycoprotein found in normal serum and pathological extracellular matrix. Given its known interactions with plasminogen activator inhibitor-1 (PAI-1) and Vtn cellular receptors, especially αvβ3 integrin and the urokinase receptor (uPAR), this study was designed to investigate its role in renal fibrogenesis in the mouse model of unilateral ureteral obstruction (UUO). Kidney Vtn mRNA levels were increased ×1.8-5.1 and Vtn protein levels ×1.9-3 on days 7, 14, and 21 after UUO compared with sham kidney levels. Groups of age-matched C57BL/6 wild-type (Vtn+/+) and Vtn-/- mice (n = 10-11/group) were killed 7, 14, or 21 days after UUO. Absence of Vtn resulted in the following significant differences, but only on day 14: fewer αSMA+ interstitial myofibroblasts (×0.53), lower procollagen III mRNA levels (×0.41), lower PAI-1 protein (×0.23), higher uPA activity (×1.1), and lower αv protein (×0.32). The number of CD68+ macrophages did not differ between the genotypes. Despite these transient differences on day 14, the absence of Vtn had no effect on fibrosis severity based on both picrosirius red-positive interstitial area and total kidney collagen measured by the hydroxyproline assay. These findings suggest that despite significant interstitial Vtn deposition in the UUO model of chronic kidney disease, its fibrogenic role is either nonessential or redundant. These data are remarkable given Vtns strong affinity for the potent fibrogenic molecule PAI-1.