Clarice Kazue Fujihara

Effect of Salt Intake and Inhibitor Dose on Arterial Hypertension and Renal Injury Induced by Chronic Nitric Oxide Blockade

Long-term nitric oxide blockade by N omega -nitro-L-arginine methyl ester (L-NAME) leads to severe and progressive hypertension. The role of salt intake in this model is unclear. To verify whether salt dependence in this model is related to the extent of nitric oxide inhibition, we gave adult male Munich-Wistar rats a low salt, standard salt, or high salt diet and oral L-NAME treatment at either 3 or 25 mg/kg per day. At 10 to 15 days of treatment, the slope of the pressure-natriuresis line was decreased in rats receiving low-dose L-NAME compared with untreated controls. In rats treated with the higher dose, the line was shifted to the right but remained parallel to that obtained in untreated controls. Renal vascular resistance was moderately increased in rats receiving low-dose L-NAME, whereas high-dose L-NAME induced a marked vasoconstriction that was aggravated by salt overload. Low-dose L-NAME treatment induced hypertension only when associated with sodium overload. In rats receiving high-dose L-NAME, hypertension was aggravated by sodium excess but was not ameliorated by sodium restriction. Long-term (6 weeks) L-NAME treatment was associated with progressive hypertension, which was aggravated by salt overload, and with the development of albuminuria, focal glomerular collapse, glomerulosclerosis, and renal interstitial expansion. These abnormalities were worsened by salt overload and largely prevented by salt restriction. In the model of chronic nitric oxide blockade, salt dependence is a function of the inhibitor dose, and renal injury varies directly with the level of salt intake.

Renal effects of acute and chronic nitric oxide inhibition in experimental diabetes

We investigated whether nitric oxide (NO) contributes to glomerular hyperfiltration in experimental diabetes. Thirty-five adult male Munich-Wistar streptozocin-diabetic rats and 39 nondiabetic controls were distributed among 4 groups: C, normal control; C + L-NAME, controls receiving the NO inhibitor N omega-nitro-L-arginine methyl ester (L-NAME), 40 mg/dl in drinking water; DM, diabetic rats; DM + L-NAME, diabetic rats receiving L-NAME, 15 mg/dl in drinking water. After 1 month of treatment, the DM + L-NAME group exhibited renal vasoconstriction and lacked hyperfiltration. Acute administration of L-NAME, 2.5 mg/kg, depressed the glomerular filtration rate and promoted renal vasoconstriction to a much greater extent in the DM than in the C group. Acute administration of endothelin 1 (600 ng/kg, bolus) or angiotensin II (25 micrograms/kg/min, continuous infusion) exerted similar hemodynamic effects in the C and DM groups, suggesting that the enhanced response of DM to L-NAME reflected specific sensitivity to NO inhibition. Urinary excretion of nitrites and nitrates was fourfold higher in DM compared to C. These results support the notion that augmented NO production may contribute to renal hyperfiltration and hyperperfusion in diabetes.

Nitroflurbiprofen, a new nonsteroidal anti-inflammatory, ameliorates structural injury in the remnant kidney

Cyclooxygenase derivatives and nitric oxide (NO) may influence the pathogenesis of progressive nephropathies. We investigated the effect of nitroflurbiprofen (NOF), a NO-releasing nonsteroidal anti-inflammatory drug (NSAID) without gastrointestinal toxicity, in rats with 5/6 ablation (NX). The following four groups were studied: Sham, sham-operated rats; Sham + NOF, Sham receiving oral NOF two times daily; NX, rats subjected to NX; and NX + NOF, NX receiving NOF. NOF was barely detected in plasma but released the parent compound flurbiprofen. At 30 days, glomerular hydraulic pressure (PGC) was 76 ± 3 mmHg in NX (52 ± 1 in Sham, P < 0.05). NOF slightly reduced PGC to 69 ± 2 mmHg in NX + NOF ( P > 0.05 vs. NX). Glomerular volumes behaved similarly. At 60 days, tail cuff pressure was 152 ± 6 mmHg, glomerulosclerosis index was 22.1 ± 9.5, and interstitial fractional area was 9.9 ± 1.2% in NX. NOF reduced these parameters to 137 ± 4 mmHg, 3.5 ± 0.7, and 6.4 ± 0.8%, respectively ( P < 0.05), without causing growth stunting or anemia. These beneficial effects could not be ascribed to NO donation and may reflect cyclooxygenase inhibition. This is the first evidence that chronic NSAID treatment may ameliorate progressive nephropathies.

Mycophenolate Mofetil Reduces Renal Injury in the Chronic Nitric Oxide Synthase Inhibition Model

We and others have recently shown that mycophenolate mofetil (MMF) reduces renal inflammation and glomerular and interstitial injury in the 5/6 renal ablation model. In the present study, we investigated whether MMF limits renal injury in a model of chronic nitric oxide (NO) inhibition associated with a high-salt diet and characterized by progressive systemic hypertension, albuminuria, glomerular sclerosis and ischemia, interstitial expansion, and progressive macrophage infiltration. Adult male Münich-Wistar rats were distributed among 3 groups: HS, rats receiving a high-salt diet (3.2% Na); HS+N, HS rats orally treated with the NO inhibitor N&ohgr;-nitro-l-arginine methyl ester (L-NAME), 25 mg · kg−1 · d−1; and HS+N+MMF, HS+N rats orally treated with MMF, 10 mg · kg−1 · d−1. Renal hemodynamics were studied after 15 days of treatment; histological and immunohistochemical studies were conducted after 30 days of treatment. MMF treatment did not reverse the hemodynamic alterations characteristic of this model. Renal injury in the HS+N group was associated with macrophage and lymphocyte infiltration. Treatment with MMF reduced glomerular and interstitial injury and limited macrophage and lymphocyte infiltration. These results suggest that renal inflammation is a strong independent factor in the pathogenesis of the nephropathy associated with the HS+N model.

Inhibition of angiotensin II receptor 1 limits tumor-associated angiogenesis and attenuates growth of murine melanoma

PurposeWe evaluated the involvement of angiotensin II (AngII)-dependent pathways in melanoma growth, through the pharmacological blockage of AT1 receptor by the anti-hypertensive drug losartan (LOS).ResultsWe showed immunolabeling for both AngII and the AT1 receptor within the human melanoma microenvironment. Like human melanomas, we showed that murine melanomas also express the AT1 receptor. Growth of murine melanoma, both locally and at distant sites, was limited in mice treated with LOS. The reduction in tumor growth was accompanied by a twofold decrease in tumor-associated microvessel density and by a decrease in CD31 mRNA levels. While no differences were found in the VEGF expression levels in tumors from treated animals, reduction in the expression of the VEGFR1 (Flt-1) at the mRNA and protein levels was observed. We also showed downregulation of mRNA levels of both Flt-4 and its ligand, VEGF-C.ConclusionsTogether, these results show that blockage of AT1 receptor signaling may be a promising anti-tumor strategy, interfering with angiogenesis by decreasing the expression of angiogenic factor receptors.

Regression of glomerular injury by losartan in experimental diabetic nephropathy.

Many features of chronic kidney disease may be reversed, but it is unclear whether advanced lesions, such as adhesions of sclerotic glomerular tufts to Bowmans capsule (synechiae), can resolve during treatment. We previously showed, using a renal ablation model, that the renoprotective effect of the AT-1 receptor blocker, losartan, is dose-dependent. Here we determined if moderate and advanced glomerular lesions, associated with streptozotocin-induced diabetes, regress with conventional or high-dose losartan treatment. Using daily insulin injection for 10 months, we maintained diabetic adult male Munich-Wistar rats in a state of moderate hyperglycemia. Following this period, some rats continued to receive insulin with or without conventional or high-dose losartan for an additional 2 months. Diabetic rats pretreated with insulin for 10 months and age-matched non-diabetic rats served as controls. Mesangial expansion was found in the control diabetic rats and was exacerbated in those rats maintained on only insulin for an additional 2 months. Conventional and high-dose losartan treatments reduced this mesangial expansion and the severity of synechiae lesions below that found prior to treatment; however, the frequency of the latter was unchanged. There was no dose-response effect of losartan. Our results show that regression of mesangial expansion and contraction of sclerotic lesions is feasible in the treatment of diabetes, but complete resolution of advanced glomerulosclerosis may be hard to achieve.

Glomerular Abnormalities in Long-Term Experimental Diabetes: Role of Hemodynamic and Nonhemodynamic Factors and Effects of Antihypertensive Therapy

To evaluate the role of glomerular hypertension, glomerular hypertrophy, glomerular lipid deposition, and plasma cholesterol levels in diabetic glomerulopathy, Munich-Wistar rats received streptozocin and daily insulin injections and were assigned to one of three groups: untreated diabetic (DMC), hydralazine-treated diabetic (DMH), and enalapril-treated diabetic (DME). Age-matched control rats were also studied. At 6–10 wk of diabetes, DMC rats showed marked elevations of glomerular pressure and glomerular filtration rate as well as slight glomerular enlargement and cholesterol elevation. DMH and DME rats exhibited arterial hypotension but no change in cholesterol or glomerular volume. Glomerular pressure was normalized by enalapril but not by hydralazine treatment. Additional rats were followed up to 12 mo of diabetes. Slight hypertension was seen in DMC rats, whereas sustained hypotension occurred in DMH and DME rats. Progressive albuminuria occurred in DMC and DMH but not in DME rats. At 12 mo, glomerular hypertension persisted in DMC and DMH rats but was still absent in DME rats. Cholesterol was elevated in DMC and slightly lower in DMH and DME rats. Glomeruli were equally enlarged in the diabetic groups. Glomerular sclerotic lesions and lipid deposits appeared in DMC and DMH but not in DME rats. These findings are consistent with the notion that glomerular hypertension may promote glomerular injury in experimental diabetes. Glomerular lipid deposition may also participate in this process, although a causal relationship was not demonstrated. Glomerular hypertrophy and cholesterol were unrelated to glomerular injury, although they may have exacerbated hemodynamically mediated damage.

Statin Monotherapy Attenuates Renal Injury in a Salt-Sensitive Hypertension Model of Renal Disease

Background: Several salutary biological effects of statins have been described. We sought to investigate more closely the anti-inflammatory and antiproliferative effects of simvastatin (SIMV) in a model of hypertension and progressive renal disease, as well as its effects on the cyclin-cdk inhibitors p21 and p27. Methods: Munich-Wistar rats received the nitric oxide (NO) synthase inhibitor L-NAME (25 mg/kg/day p.o.) for 20 days accompanied by a high-salt diet (HS, 3% Na) and then were kept on HS for 60 days. Animals were then divided into two groups: vehicle (VH) or SIMV 2 mg/kg/day p.o. Albuminuria and tail-cuff pressure were determined at 30 and 60 days. RT-PCR was done to assess renal expression of TGF-β1, collagen I and III, fibronectin, p27, p21 and monocyte chemoattractant protein-1 (MCP-1). Renal protein expression was assessed by Western blot (proliferating cell nuclear antigen (PCNA)) and immunostaining (macrophage, lymphocyte, PCNA). Results: SIMV did not prevent the development of severe hypertension or albuminuria. SIMV-treated animals had less severe renal interstitial inflammation and cell proliferation. MCP-1 expression was significantly diminished in the SIMV-treated animals (55.4 ± 7.3 vs. 84.4 ± 8.2 OD, p = 0.02). mRNA renal expression for p27 and TGF-β did not change between groups, but p21 mRNA renal expression, highly induced in this model, significantly decreased with SIMV treatment (31.6 ± 6.6 vs. 50.2 ± 5.8 OD, p < 0.05). The interstitial fibrosis score significantly decreased with SIMV (2.46 ± 0.40 vs. 4.07 ± 0.38%, p < 0.01), which was confirmed by a decrease in renal collagen I and fibronectin expression. Serum cholesterol level did not change with SIMV. Conclusion: SIMV attenuated interstitial fibrosis associated with this model of hypertensive renal disease. The mechanism involved MCP-1 downregulation. SIMV treatment was also associated with a p21 downregulation in the kidney, which might be involved in the protection of renal scarring.

Altered KLOTHO and NF-κB-TNF-α Signaling Are Correlated with Nephrectomy-Induced Cognitive Impairment in Rats

Renal insufficiency can have a negative impact on cognitive function. Neuroinflammation and changes in klotho levels associate with chronic kidney disease (CKD) and may play a role in the development of cognitive impairment (CI). The present study evaluates the correlation of cognitive deficits with neuroinflammation and soluble KLOTHO in the cerebral spinal fluid (CSF) and brain tissue of nephrectomized rats (Nx), with 5/6 renal mass ablation. Nx and sham Munich Wistar rats were tested over 4 months for locomotor activity, as well as inhibitory avoidance or novel object recognition, which started 30 days after the surgery. EMSA for Nuclear factor-κB and MILLIPLEXMAP or ELISA kit were used to evaluate cytokines, glucocorticoid and KLOTHO levels. Nx animals that showed a loss in aversive-related memory and attention were included in the CI group (Nx-CI) (n=14) and compared to animals with intact learning (Nx-M n=12 and Sham n=20 groups). CSF and tissue samples were collected 24 hours after the last behavioral test. The results show that the Nx-groups have increased NF-κB binding activity and tumor necrosis factor-alpha (TNF-α) levels in the hippocampus and frontal cortex, with these changes more pronounced in the Nx-CI group frontal cortex. In addition, the Nx-CI group showed significantly increased CSF glucocorticoid levels and TNF-α /IL-10 ratio compared to the Sham group. Klotho levels were decreased in Nx-CI frontal cortex but not in hippocampus, when compared to Nx-M and Sham groups. Overall, these results suggest that neuroinflammation mediated by frontal cortex NF-κB, TNF-α and KLOTHO signaling may contribute to Nx-induced CI in rats.

AT1 blockade during lactation as a model of chronic nephropathy: mechanisms of renal injury

Suppression of the renin-angiotensin system during lactation causes irreversible renal structural changes. In this study we investigated 1) the time course and the mechanisms underlying the chronic kidney disease caused by administration of the AT(1) receptor blocker losartan during lactation, and 2) whether this untoward effect can be used to engender a new model of chronic kidney disease. Male Munich-Wistar pups were divided into two groups: C, whose mothers were untreated, and L(Lact), whose mothers received oral losartan (250 mg.kg(-1).day(-1)) during the first 20 days after delivery. At 3 mo of life, both nephron number and the glomerular filtration rate were reduced in L(Lact) rats, whereas glomerular pressure was elevated. Unselective proteinuria and decreased expression of the zonula occludens-1 protein were also observed, along with modest glomerulosclerosis, significant interstitial expansion and inflammation, and wide glomerular volume variation, with a stable subpopulation of exceedingly small glomeruli. In addition, the urine osmolality was persistently lower in L(Lact) rats. At 10 mo of age, L(Lact) rats exhibited systemic hypertension, heavy albuminuria, substantial glomerulosclerosis, severe renal interstitial expansion and inflammation, and creatinine retention. Conclusions are that 1) oral losartan during lactation can be used as a simple and easily reproducible model of chronic kidney disease in adult life, associated with low mortality and no arterial hypertension until advanced stages; and 2) the mechanisms involved in the progression of renal injury in this model include glomerular hypertension, glomerular hypertrophy, podocyte injury, and interstitial inflammation.