Daryl M. Okamura

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.

Investigating mechanisms of chronic kidney disease in mouse models

Animal models of chronic kidney disease (CKD) are important experimental tools that are used to investigate novel mechanistic pathways and to validate potential new therapeutic interventions prior to pre-clinical testing in humans. Over the past several years, mouse CKD models have been extensively used for these purposes. Despite significant limitations, the model of unilateral ureteral obstruction (UUO) has essentially become the high-throughput in vivo model, as it recapitulates the fundamental pathogenetic mechanisms that typify all forms of CKD in a relatively short time span. In addition, several alternative mouse models are available that can be used to validate new mechanistic paradigms and/or novel therapies. Here, we review several models—both genetic and experimentally induced—that provide investigators with an opportunity to include renal functional study end-points together with quantitative measures of fibrosis severity, something that is not possible with the UUO model.

Galectin-3 preserves renal tubules and modulates extracellular matrix remodeling in progressive fibrosis

Renal tubular cell apoptosis is a critical detrimental event that leads to chronic kidney injury in association with renal fibrosis. The present study was designed to investigate the role of galectin-3 (Gal-3), an important regulator of multiple apoptotic pathways, in chronic kidney disease induced by unilateral ureteral obstruction (UUO). After UUO, Gal-3 expression significantly increased compared with basal levels reaching a peak increase of 95-fold by day 7. Upregulated Gal-3 is predominantly tubular at early time points after UUO but shifts to interstitial cells as the injury progresses. On day 14, there was a significant increase in TdT-mediated dUTP nick end labeling-positive cells (129%) and cytochrome c release (29%), and a decrease in BrdU-positive cells (62%) in Gal-3-deficient compared with wild-type mice. The degree of renal damage was more extensive in Gal-3-deficient mice at days 14 and 21, 35 and 21% increase in total collagen, respectively. Despite more severe fibrosis, myofibroblasts were significantly decreased by 58% on day 14 in the Gal-3-deficient compared with wild-type mice. There was also a corresponding 80% decrease in extracellular matrix synthesis in Gal-3-deficient compared with wild-type mice. Endo180 is a recently recognized receptor for intracellular collagen degradation that is expressed by interstitial cells during renal fibrogenesis. Endo180 expression was significantly decreased by greater than 50% in Gal-3-deficient compared with wild-type mice. Taken together, these results suggested that Gal-3 not only protects renal tubules from chronic injury by limiting apoptosis but that it may lead to enhanced matrix remodeling and fibrosis attenuation.

Tipping the redox balance of oxidative stress in fibrogenic pathways in chronic kidney disease

Patients with moderate to advanced chronic kidney disease or end-stage renal disease have a greatly increased cardiovascular risk that cannot be explained entirely by traditional cardiovascular risk factors. An increase in oxidative stress and inflammation have been proposed as nontraditional cardiovascular risk factors in this patient population. Oxidative stress reflects the redox balance between oxidant generation and antioxidant mechanisms. The generation of reactive oxygen species is not simply a random process that oxidizes nearby macromolecules, but, in many instances, the oxidants target particular amino acid residues or lipid moieties. Oxidant mechanisms are now recognized to be intimately involved in cell signaling and to be vital components of the immune response. This is equally true for antioxidant mechanisms as well. In the progression of chronic kidney disease, the redox balance is not in equilibrium and is tipped toward oxidation, resulting in the dysregulation of cellular process and subsequent tissue injury. In this review we discuss the major oxidant and antioxidant pathways and the biomarkers to assess redox status. We also review the data linking the pathogenesis of oxidative stress, inflammation, and the progressive loss of kidney function in chronic kidney disease.

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.

The balance of powers: Redox regulation of fibrogenic pathways in kidney injury.

Oxidative stress plays a central role in the pathogenesis of diverse chronic inflammatory disorders including diabetic complications, cardiovascular disease, aging, and chronic kidney disease (CKD). Patients with moderate to advanced CKD have markedly increased levels of oxidative stress and inflammation that likely contribute to the unacceptable high rates of morbidity and mortality in this patient population. Oxidative stress is defined as an imbalance of the generation of reactive oxygen species (ROS) in excess of the capacity of cells/tissues to detoxify or scavenge them. Such a state of oxidative stress may alter the structure/function of cellular macromolecules and tissues that eventually leads to organ dysfunction. The harmful effects of ROS have been largely attributed to its indiscriminate, stochastic effects on the oxidation of protein, lipids, or DNA but in many instances the oxidants target particular amino acid residues or lipid moieties. Oxidant mechanisms are intimately involved in cell signaling and are linked to several key redox-sensitive signaling pathways in fibrogenesis. Dysregulation of antioxidant mechanisms and overproduction of ROS not only promotes a fibrotic milieu but leads to mitochondrial dysfunction and further exacerbates kidney injury. Our studies support the hypothesis that unique reactive intermediates generated in localized microenvironments of vulnerable tissues such as the kidney activate fibrogenic pathways and promote end-organ damage. The ability to quantify these changes and assess response to therapies will be pivotal in understanding disease mechanisms and monitoring efficacy of therapy.

Cysteamine modulates oxidative stress and blocks myofibroblast activity in CKD.

Therapy to slow the relentless expansion of interstitial extracellular matrix that leads to renal functional decline in patients with CKD is currently lacking. Because chronic kidney injury increases tissue oxidative stress, we evaluated the antifibrotic efficacy of cysteamine bitartrate, an antioxidant therapy for patients with nephropathic cystinosis, in a mouse model of unilateral ureteral obstruction. Fresh cysteamine (600 mg/kg) was added to drinking water daily beginning on the day of surgery, and outcomes were assessed on days 7, 14, and 21 after surgery. Plasma cysteamine levels showed diurnal variation, with peak levels similar to those observed in patients with cystinosis. In cysteamine-treated mice, fibrosis severity decreased significantly at 14 and 21 days after unilateral ureteral obstruction, and renal oxidized protein levels decreased at each time point, suggesting reduced oxidative stress. Consistent with these results, treatment of cultured macrophages with cysteamine reduced cellular generation of reactive oxygen species. Furthermore, treatment with cysteamine reduced α-smooth muscle actin-positive interstitial myofibroblast proliferation and mRNA levels of extracellular matrix proteins in mice and attenuated myofibroblast differentiation and proliferation in vitro, but did not augment TGF-β signaling. In a study of renal ischemia reperfusion, cysteamine therapy initiated 10 days after injury and continued for 14 days decreased renal fibrosis by 40%. Taken together, these data suggest previously unrecognized antifibrotic actions of cysteamine via TGF-β-independent mechanisms that include oxidative stress reduction and attenuation of the myofibroblast response to kidney injury and support further investigation into the potential benefit of cysteamine therapy in the treatment of CKD.

Significant Reductions in Mortality in Hospitalized Patients with Systemic Lupus Erythematosus in Washington State from 2003 to 2011

Background Systemic lupus erythematosus (SLE or lupus) is an autoimmune multisystem disease. While a complete understanding of lupus’ origins, mechanisms, and progression is not yet available, a number of studies have demonstrated correlations between disease prevalence and severity, gender, and race. There have been few population based studies in the United States Objectives To assess temporal changes in demographics and hospital mortality of patients with lupus in Washington State from 2003 to 2011 Study Design This study used data from the Healthcare Cost and Utilization Project (HCUP), a patient information database, and data from the Washington State census to study a group of patients in the state. Lupus hospitalizations were defined as any hospitalization with an ICD-9-CM diagnosis code for systemic lupus erythematosus. Regression analysis was used to assess the effect of calendar time on demographics and hospital outcomes. Results There were a total of 18,905 patients in this study with a diagnostic code for lupus. The mean age of the group was 51.5 years (95% CI: 50.6-52.3) in 2003 and 51.3 years (95% CI: 50.6-52.0) in 2011. The population was 88.6% female. Blacks were 2.8 times more likely to have a lupus hospitalization than whites when compared to the Washington population. While hospital mortality decreased during this eight year period (3.12% in 2003 to 1.28% in 2011, p=0.001) hospital length of stay remained statistically unchanged at an average of 4.9 days during that eight year period. We found a significant decrease in annual hospital mortality over the study period [odds ratio(OR): 0.92 per year, 95% CI 0.88-0.96, P<0.001]. Hospital mortality was higher in males (2.6% male death to 1.8% female death) Conclusions In this large group of hospitalized lupus patients in Washington, hospital length of stay remained relatively stable over time but hospital mortality decreased by over 50% over the eight year study period.