Gender-Specific Renoprotective Pathways in αMUPA Transgenic Mice Subjected to Acute Kidney Injury.

Acute kidney injury (AKI) is a serious health concern with high morbidity and high mortality worldwide. Recently, sexual dimorphism has become increasingly recognized as a factor influencing the severity of the disease. This study explores the gender-specific renoprotective pathways in αMUPA transgenic mice subjected to AKI. αMUPA transgenic male and female mice were subjected to ischemia-reperfusion (I/R)-AKI in the presence or absence of orchiectomy, oophorectomy, and L-NAME administration. Blood samples and kidneys were harvested 48 h following AKI for the biomarkers of kidney function, renal injury, inflammatory response and intracellular pathway sensing of or responding to AKI. Our findings show differing responses to AKI, where female αMUPA mice were remarkably protected against AKI as compared with males, as was evident by the lower SCr and BUN, normal renal histologically and attenuated expression of NGAL and KIM-1. Moreover, αMUPA females did not show a significant change in the renal inflammatory and fibrotic markers following AKI as compared with wild-type (WT) mice and αMUPA males. Interestingly, oophorectomized females eliminated the observed resistance to renal injury, highlighting the central protective role of estrogen. Correspondingly, orchiectomy in αMUPA males mitigated their sensitivity to renal damage, thereby emphasizing the devastating effects of testosterone. Additionally, treatment with L-NAME proved to have significant deleterious impacts on the renal protective mediators, thereby underscoring the involvement of eNOS. In conclusion, gender-specific differences in the response to AKI in αMUPA mice include multifaceted and keen interactions between the sex hormones and key biochemical mediators (such as estrogen, testosterone and eNOS). These novel findings shed light on the renoprotective pathways and mechanisms, which may pave the way for development of therapeutic interventions.

The Protective Pathways Activated in Kidneys of αMUPA Transgenic Mice Following Ischemia\Reperfusion-Induced Acute Kidney Injury.

Despite the high prevalence of acute kidney injury (AKI), the therapeutic approaches for AKI are disappointing. This deficiency stems from the poor understanding of the pathogenesis of AKI. Recent studies demonstrate that αMUPA, alpha murine urokinase-type plasminogen activator (uPA) transgenic mice, display a cardioprotective pathway following myocardial ischemia. We hypothesize that these mice also possess protective renal pathways. Male and female αMUPA mice and their wild type were subjected to 30 min of bilateral ischemic AKI. Blood samples and kidneys were harvested 48 h following AKI for biomarkers of kidney function, renal injury, inflammatory response, and intracellular pathways sensing or responding to AKI. αMUPA mice, especially females, exhibited attenuated renal damage in response to AKI, as was evident from lower SCr and BUN, normal renal histology, and attenuated expression of NGAL and KIM-1. Notably, αMUPA females did not show a significant change in renal inflammatory and fibrotic markers following AKI as compared with wild-type (WT) mice and αMUPA males. Moreover, αMUPA female mice exhibited the lowest levels of renal apoptotic and autophagy markers during normal conditions and following AKI. αMUPA mice, especially the females, showed remarkable expression of PGC1α and eNOS following AKI. Furthermore, MUPA mice showed a significant elevation in renal leptin expression before and following AKI. Pretreatment of αMUPA with leptin-neutralizing antibodies prior to AKI abolished their resistance to AKI. Collectively, the kidneys of αMUPA mice, especially those of females, are less susceptible to ischemic I/R injury compared to WT mice, and this is due to nephroprotective actions mediated by the upregulation of leptin, eNOS, ACE2, and PGC1α along with impaired inflammatory, fibrotic, and autophagy processes.

Effects of Angiotensin 1-7 and Mas Receptor Agonist on Renal System in a Rat Model of Heart Failure.

Congestive heart failure (CHF) is often associated with impaired kidney function. Over- activation of the renin-angiotensin-aldosterone system (RAAS) contributes to avid salt/water retention and cardiac hypertrophy in CHF. While the deleterious effects of angiotensin II (Ang II) in CHF are well established, the biological actions of angiotensin 1-7 (Ang 1-7) are not fully characterized. In this study, we assessed the acute effects of Ang 1-7 (0.3, 3, 30 and 300 ng/kg/min, IV) on urinary flow (UF), urinary Na+ excretion (UNaV), glomerular filtration rate (GFR) and renal plasma flow )RPF) in rats with CHF induced by the placement of aortocaval fistula. Additionally, the chronic effects of Ang 1-7 (24 µg/kg/h, via intra-peritoneally implanted osmotic minipumps) on kidney function, cardiac hypertrophy and neurohormonal status were studied. Acute infusion of either Ang 1-7 or its agonist, AVE 0991, into sham controls, but not CHF rats, increased UF, UNaV, GFR, RPF and urinary cGMP. In the chronic protocols, untreated CHF rats displayed lower cumulative UF and UNaV than their sham controls. Chronic administration of Ang 1-7 and AVE 0991 exerted significant diuretic, natriuretic and kaliuretic effects in CHF rats, but not in sham controls. Serum creatinine and aldosterone levels were significantly higher in vehicle-treated CHF rats as compared with controls. Treatment with Ang 1-7 and AVE 0991 reduced these parameters to comparable levels observed in sham controls. Notably, chronic administration of Ang 1-7 to CHF rats reduced cardiac hypertrophy. In conclusion, Ang 1-7 exerts beneficial renal and cardiac effects in rats with CHF. Thus, we postulate that ACE2/Ang 1-7 axis represents a compensatory response to over-activity of ACE/AngII/AT1R system characterizing CHF and suggest that Ang 1-7 may be a potential therapeutic agent in this disease state.

Heparanase Inhibition Prevents Liver Steatosis in E0 Mice.

BACKGROUND: Non-alcoholic fatty liver disease affects up to 30% of adults in the USA, and is associated with a higher incidence of chronic liver morbidity and mortality. Several molecular pathways are involved in the pathology of liver steatosis, including lipid uptake, lipogenesis, lipolysis, and beta-oxidation. The enzyme heparanase has been implicated in liver steatosis. Herein, we investigated the effect of heparanase inhibition on liver steatosis in E0 mice. METHODS: In vivo experiments: Male wild-type mice fed with either chow diet (n = 4) or high-fat diet (n = 6), and male E0 mice fed with chow diet (n = 8) or high-fat diet (n = 33) were included. Mice on a high-fat diet were treated for 12 weeks with PG545 at low dose (6.4 mg/kg/week, ip, n = 6) or high dose (13.3 mg/kg/week, ip, n = 7), SST0001 (1.2 mg/mouse/day, ip, n = 6), or normal saline (control, n = 14). Animals were sacrificed two days after inducing peritonitis. Serum was analyzed for biochemical parameters. Mouse peritoneal macrophages (MPMs) were harvested and analyzed for lipid content. Livers were harvested for histopathological analysis of steatosis, lipid content, and the expression of steatosis-related factors at the mRNA level. In vitro experiments: MPMs were isolated from untreated E0 mice aged 8-10 weeks and were cultured and treated with either PG545 or SST0001, both at 50 µg/mL for 24 h, followed by assessment of mRNA expression of steatosis related factors. RESULTS: Heparanase inhibition significantly attenuated the development of liver steatosis, as was evident by liver histology and lipid content. Serum analysis indicated lowering of cholesterol and triglycerides levels in mice treated with heparanase inhibitors. In liver tissue, assessment of mRNA expression of key factors in lipid uptake, lipolysis, lipogenesis, and beta-oxidation exhibited significant downregulation following PG545 treatment and to a lesser extent when SST0001 was applied. However, in vitro treatment of MPMs with PG545, but not SST0001, resulted in increased lipid content in these cells, which is opposed to their effect on MPMs of treated mice. This may indicate distinct regulatory pathways in the system or isolated macrophages following heparanase inhibition. CONCLUSION: Heparanase inhibition significantly attenuates the development of liver steatosis by decreasing tissue lipid content and by affecting the mRNA expression of key lipid metabolism regulators.

Increased Intra-abdominal Pressure Induces Acute Kidney Injury in an Experimental Model of Congestive Heart Failure.

BACKGROUND: Congestive heart failure (CHF) entails a complex interaction between the heart and the kidney that represents a clinical entity called cardiorenal syndrome (CRS). One of the mechanisms underlying CRS includes increased intra-abdominal pressure (IAP). We examined the effect of elevated IAP on kidney function in rats with low- and high-output CHF. METHODS AND RESULTS: Rats with compensated and decompensated CHF induced by means of aortocaval fistula, rats with myocardial infraction (MI) induced by means of left anterior descending artery ligation, and sham control rats were subjected to either 10 or 14 mm Hg IAP. Urine flow (V), Na+ excretion (UNaV), glomerular filtration rate (GFR), and renal plasma flow (RPF) were determined. The effects of pretreatment with tadalafil (10 mg/kg orally for 4 days) on the adverse renal effects of IAP were examined in decompensated CHF and MI. Basal V and GFR were significantly lower in rats with decompensated CHF compared with sham control rats. Decompensated CHF rats and MI rats subjected to 10 and 14 mm Hg IAP exhibited more significant declines in V, UNaV, GFR and RPF than compensated and sham controls. Elevated IAP also induced tubular injury, as evidenced by significantly increased absolute urinary excretion of neutrophil gelatinase-associated lipocalin. In addition, in a nonquantitative histologic analysis, elevated IAP was associated with increase in necrosis and cell shedding to the tubule lumens, especially in the decompensated CHF subgroup. Pretreatment of decompensated CHF rats and MI rats with tadalafil ameliorated the adverse renal effects of high IAP. CONCLUSIONS: Elevated IAP contributes to kidney dysfunction in high- and low-cardiac output CHF. IAP induces both hemodynamic alterations and renal tubular dysfunction. These deleterious effects are potentially reversible and can be ameliorated with the use of phosphodiesterase-5 inhibition.

Cardiac and renal distribution of ACE and ACE-2 in rats with heart failure.

Congestive heart failure is often associated with impaired kidney function. Over-activation of the renin-angiotensin-aldosterone system (RAAS) contributes to avid salt and water retention in heart failure. While the expression of angiotensin converting enzyme (ACE), a key enzyme in the synthesis of angiotensin II (Ang II), is well established, the expression of angiotensin converting enzyme-2 (ACE-2), an enzyme responsible for angiotensin 1-7 generation, is largely unknown. This issue is of a special interest since angiotensin 1-7 counteracts many of the proliferative and hypertensive effects of angiotensin II. Therefore, the present study was designed to investigate the expression of both enzymes in the kidney and heart of rats with heart failure. Heart failure (CHF) was induced in male Sprague Dawley rats (n=9) by the creation of a surgical aorto-caval fistula. Sham-operated rats served as controls (n=8). Two weeks after surgery, the animals were sacrificed and their hearts and kidneys were harvested for assessment of cardiac remodeling and ACE and ACE-2 immunoreactivity by immunohistochemical staining. ACE immunostaining was significantly increased in the kidneys (4.34 ± 0.39% vs. 2.96 ± 0.40%, P<0.05) and hearts (4.57 ± 0.54% vs. 2.19 ± 0.37%, P<0.01) of CHF rats as compared with their sham controls. In a similar manner, ACE-2 immunoreactivity was also elevated in the kidneys (4.65 ± 1.17% vs. 1.75 ± 0.29%, P<0.05) and hearts (5.48 ± 1.11% vs. 1.13 ± 0.26%, P<0.01) of CHF rats as compared with their healthy controls. This study showed that both ACE and ACE-2 are overexpressed in the cardiac and renal tissues of animals with heart failure as compared with their sham controls. The increased expression of the beneficial ACE-2 in heart failure may serve as a compensatory response to the over-activity of the deleterious isoform, namely, angiotensin converting enzyme 1(ACE-1).

An engineered muscle flap for reconstruction of large soft tissue defects.

Large soft tissue defects involve significant tissue loss, requiring surgical reconstruction. Autologous flaps are occasionally scant, demand prolonged transfer surgery, and induce donor site morbidity. The present work set out to fabricate an engineered muscle flap bearing its own functional vascular pedicle for repair of a large soft tissue defect in mice. Full-thickness abdominal wall defect was reconstructed using this engineered vascular muscle flap. A 3D engineered tissue constructed of a porous, biodegradable polymer scaffold embedded with endothelial cells, fibroblasts, and/or myoblasts was cultured in vitro and then implanted around the femoral artery and veins before being transferred, as an axial flap, with its vascular pedicle to reconstruct a full-thickness abdominal wall defect in the same mouse. Within 1 wk of implantation, scaffolds showed extensive functional vascular density and perfusion and anastomosis with host vessels. At 1 wk posttransfer, the engineered muscle flaps were highly vascularized, were well-integrated within the surrounding tissue, and featured sufficient mechanical strength to support the abdominal viscera. Thus, the described engineered muscle flap, equipped with an autologous vascular pedicle, constitutes an effective tool for reconstruction of large defects, thereby circumventing the need for both harvesting autologous flaps and postoperative scarification.