Salt-sensitive Rats Research Articles

PGI2 Analog Attenuates Salt-Induced Renal Injury through the Inhibition of Inflammation and Rac1-MR Activation.

Renal inflammation is known to be involved in salt-induced renal damage, leading to end-stage renal disease. This study aims to evaluate the role of inflammation in anti-inflammatory and renoprotective effects of beraprost sodium (BPS), a prostaglandin I2 (PGI2) analog, in Dahl salt-sensitive (DS) rats. Five-week-old male DS rats were fed a normal-salt diet (0.5% NaCl), a high-salt diet (8% NaCl), or a high-salt diet plus BPS treatment for 3 weeks. BPS treatment could inhibit marked proteinuria and renal injury in salt-loaded DS rats with elevated blood pressure, accompanied by renal inflammation suppression. Notably, high salt increased renal expression of active Rac1, followed by increased Sgk1 expressions, a downstream molecule of mineralocorticoid receptor (MR) signal, indicating salt-induced activation of Rac1-MR pathway. However, BPS administration inhibited salt-induced Rac1-MR activation as well as renal inflammation and damage, suggesting that Rac1-MR pathway is involved in anti-inflammatory and renoprotective effects of PGI2. Based upon Rac1 activated by inflammation, moreover, BPS inhibited salt-induced activation of Rac1-MR pathway by renal inflammation suppression, resulting in the attenuation of renal damage in salt-loaded DS rats. Thus, BPS is efficacious for the treatment of salt-induced renal injury.

1,3-Butanediol attenuates hypertension and suppresses kidney injury in female rats

Thirty-seven million people in the United States are estimated to have chronic kidney disease (CKD). Hypertension (HTN) is the second leading risk factor for developing kidney disease. A recent study reported that increasing levels of β-hydroxybutyrate levels by administration of its precursor, 1,3-butanediol, decreased salt-induced HTN in male Dahl salt-sensitive (S) rats. The effect of 1,3-butanediol on hypertensive kidney disease in female rats or the absence of high salt has not been investigated. This study tested the hypothesis that 1,3-butanediol attenuates HTN and the progression of CKD in female S-SHR(11) rats. The S-SHR(11) strain is a congenic rat strain generated from genetic modification of the Dahl S rat, previously characterized as a model of accelerated renal disease. Rats received 1,3-butanediol (20% via drinking water) or control for 10 wk and were maintained on a 0.3% NaCl rodent diet (n = 12-14 rats/group). Blood pressure was measured after 6 and 9 wk of treatment by tail-cuff plethysmography; after 10 wk, urine and tissues were collected. Activity of the treatment was confirmed by measuring plasma β-hydroxybutyrate levels, which were greater in the treated group. The 1,3-butanediol-treated group had lower systolic blood pressure, proteinuria, plasma creatinine, and renal fibrosis after 9 wk of treatment compared with controls. The treated group had significantly smaller spleens and increased the renal anti-inflammatory molecules interleukin-10 and granulocyte-macrophage colony-stimulating factor, suggesting reduced inflammation. The present data demonstrate that 1,3-butanediol lowers blood pressure and renal injury in female rats and could be a novel nutritional intervention for the treatment of CKD.

Endogenous miR-204 Protects the Kidney against Chronic Injury in Hypertension and Diabetes.

Aberrant microRNA (miRNA) expression affects biologic processes and downstream genes that are crucial to CKD initiation or progression. The miRNA miR-204-5p is highly expressed in the kidney but whether miR-204-5p plays any role in the development of chronic renal injury is unknown. We used real-time PCR to determine levels of miR-204 in human kidney biopsies and animal models. We generated Mir204 knockout mice and used locked nucleic acid-modified anti-miR to knock down miR-204-5p in mice and rats. We used a number of physiologic, histologic, and molecular techniques to analyze the potential role of miR-204-5p in three models of renal injury. Kidneys of patients with hypertension, hypertensive nephrosclerosis, or diabetic nephropathy exhibited a significant decrease in miR-204-5p compared with controls. Dahl salt-sensitive rats displayed lower levels of renal miR-204-5p compared with partially protected congenic SS.13BN26 rats. Administering anti-miR-204-5p to SS.13BN26 rats exacerbated interlobular artery thickening and renal interstitial fibrosis. In a mouse model of hypertensive renal injury induced by uninephrectomy, angiotensin II, and a high-salt diet, Mir204 gene knockout significantly exacerbated albuminuria, renal interstitial fibrosis, and interlobular artery thickening, despite attenuation of hypertension. In diabetic db/db mice, administering anti-miR-204-5p exacerbated albuminuria and cortical fibrosis without influencing blood glucose levels. In all three models, inhibiting miR-204-5p or deleting Mir204 led to upregulation of protein tyrosine phosphatase SHP2, a target gene of miR-204-5p, and increased phosphorylation of signal transducer and activator of transcription 3, or STAT3, which is an injury-promoting effector of SHP2. These findings indicate that the highly expressed miR-204-5p plays a prominent role in safeguarding the kidneys against common causes of chronic renal injury.

NOX4/H2O2/mTORC1 Pathway in Salt-Induced Hypertension and Kidney Injury.

We have reported that a high-salt (4.0% NaCl) dietary intake activates mTORC1 and inhibition of this pathway with rapamycin blunts the chronic phase of salt-induced hypertension and renal injury in Dahl salt-sensitive (SS) rats. In SS rats, high-salt intake is known to increase the renal production of H2O2 by NOX4, the most abundant NOX isoform in the kidney, and the global knockout of NOX4 blunts salt-sensitivity in these rats. Here, we explored the hypothesis that elevations of H2O2 by NOX4 in high-salt fed SS rat stimulate mTORC1 for the full development of salt-induced hypertension and renal injury. Our in vitro studies found that H2O2 activates mTORC1 independent of PI3K/AKT and AMPK pathways. To determine the in vivo relevance of NOX4/H2O2/mTORC1 in the salt-induced hypertension, SS-Nox4 knockout (SSNox4-/-) rats were daily administrated with vehicle/rapamycin fed a high-salt diet for 21 days. Rapamycin treatment of SSNox4-/- rats had shown no augmented effect on the salt-induced hypertension nor upon indices of renal injury. Significant reductions of renal T lymphocyte and macrophage together with inhibition of cell proliferation were observed in rapamycin treated rats suggesting a role of mTORC1 independent of NOX4 in the proliferation of immune cell. Given the direct activation of mTORC1 by H2O2 and absence of any further protection from salt-induced hypertension in rapamycin-treated SSNox4-/- rats, we conclude that NOX4-H2O2 is a major upstream activator of mTORC1 that contributes importantly to salt-induced hypertension and renal injury in the SS rat model.

MO059CONTRIBUTION OF OPIOID RECEPTOR SIGNALING IN PODOCYTES TOWARDS THE DEVELOPMENT OF SALT-SENSITIVE HYPERTENSION AND KIDNEY INJURY

Abstract Background and Aims The global increase in opioid use requires practitioners to be aware of the side effects and contraindications of these medications in order to avoid complications, especially in patients with organ dysfunction such as cardiovascular disease and chronic kidney disease. It has been reported that opioid use is strongly associated with predictors of poor cardiovascular outcomes such as increased albuminuria and reduced GFR. Opioids work by binding to kappa-, mu- and delta-opioid receptors (ORs). We hypothesize that stimulation of ORs modulates calcium flux in podocytes ultimately leading to cell apoptosis, development of albuminuria, glomeruli damage and progression of hypertension. Method To test this hypothesis, we applied specific OR agonists to: 1) in vitro - immortalized cultured human podocytes (hPod), 2) ex vivo - freshly isolated human and rat glomeruli; and 3) in vivo - Dahl salt-sensitive (SS) rats. Calcium response in the podocytes was analyzed via ratiometric confocal fluorescent microscopy using calcium fluorescent dyes. For chronic studies Dahl SS rats were fed a high salt (HS; 8%) NaCl diet for 14 days with or without a daily i.v. bolus infusion of BRL52537, a potent and selective kappa-OR agonist. Results The hPods displayed a fast increase in fluorescence following an addition of BRL52537 (EC50 = 162 ± 2 µM) in a 2 mM calcium solution. A pre-application of kappa-OR antagonist (NorBNI) disrupted the effect of BRL52537 on calcium flux. The effect of BRL52537 on calcium in podocytes was completely abolished when we used the calcium-free solution or a pre-application of SAR7334 (a TRPC6 channel inhibitor), which revealed that calcium flux is mediated by activation of TRPC6 channels rather than other plasma membrane channels or depletion of intracellular calcium stores. Immunofluorescent analysis of human kidney validated kappa-OR protein expression in podocytes. Podocytes of the freshly isolated human glomeruli also revealed robust increases in fluorescent intensity following a single application of BRL52537, which was abolished by a pre-application of SAR7334. Effect of kappa-OR agonist was specific since application of Damgo (mu-OR agonist) or DPDPE (delta-OR agonist) had either no or minimal effect in normotensive rats. Chronic studies in Dahl SS rats showed that in BRL-treated group animals had elevation of microalbuminuria (13 ± 3 vs. 3 ± 1 Alb/Cre at day 7 of HS), higher mean arterial pressures (179 ± 15 mmHg vs. 151 ± 11 mmHg), and nephrinuria (50,232 ± 22,254 vs. 15,230 ± 4,647 a.u./Cre) compared to control rats. Glomeruli isolated from the hypertensive opioid treated rats had a significantly higher level of basal calcium (245 ± 23 vs. 139 ± 9 nM) in podocytes compared to non-treated group. Additionally, acute application of BRL52537 to podocytes of hypertensive animals resulted in a sustained increase in total [Ca2+]i influx. Conclusion Our data establish that stimulation of kappa-ORs are specifically involved in increasing of intracellular calcium in podocytes through the TRPC6 pathway, which causes calcium overload and cellular damage. Observed increase in the calcium flux in podocytes most likely further contribute to the kidney injury and progression of salt-induced hypertension.

Diurnal Timing Dependent Alterations in Gut Microbial Composition Are Synchronously Linked to Salt-Sensitive Hypertension and Renal Damage.

Alterations of diurnal rhythms of blood pressure (BP) and reshaping of gut microbiota are both independently associated with hypertension. However, the relationships between biorhythms of BP and gut microbial composition are unknown. We hypothesized that diurnal timing-associated alterations of microbial compositions are synchronous with diurnal rhythmicity, dip in BP, and renal function. To test this hypothesis, Dahl salt-sensitive (S) rats on low- and high-salt diets were examined for time of day effects on gut microbiota, BP, and indicators of renal damage. Major shifts in night and day patterns of specific groups of microbiota were observed between the dark (active) and light (rest) phases, which correlated with diurnal rhythmicity of BP. The diurnal abundance of Firmicutes, Bacteroidetes, and Actinobacteria were independently associated with BP. Discrete bacterial taxa were observed to correlate independently or interactively with one or more of the following 3 factors: (1) BP rhythm, (2) dietary salt, and (3) dip in BP. Phylogenetic Investigation of Communities revealed diurnal timing effects on microbial pathways, characterized by upregulated biosynthetic processes during the active phase of host, and upregulated degradation pathways of metabolites in the resting phase. Additional metagenomics functional pathways with rhythm variations were noted for aromatic amino acid metabolism and taurine metabolism. These diurnal timing dependent changes in microbiota, their functional pathways, and BP dip were associated with concerted effects of the levels of renal lipocalin 2 and kidney injury molecule-1 expression. These data provide evidence for a firm and concerted diurnal timing effects of BP, renal damage, and select microbial communities.

Enhanced Cardiomyocyte Function in Hypertensive Rats With Diastolic Dysfunction and Human Heart Failure Patients After Acute Treatment With Soluble Guanylyl Cyclase (sGC) Activator.

AimsOur aim was to investigate the effect of nitric oxide (NO)-independent activation of soluble guanylyl cyclase (sGC) on cardiomyocyte function in a hypertensive animal model with diastolic dysfunction and in biopsies from human heart failure with preserved ejection fraction (HFpEF).MethodsDahl salt-sensitive (DSS) rats and control rats were fed a high-salt diet for 10 weeks and then acutely treated in vivo with the sGC activator BAY 58-2667 (cinaciguat) for 30 min. Single skinned cardiomyocyte passive stiffness (Fpassive) was determined in rats and human myocardium biopsies before and after acute treatment. Titin phosphorylation, activation of the NO/sGC/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) cascade, as well as hypertrophic pathways including NO/sGC/cGMP/PKG, PKA, calcium–calmodulin kinase II (CaMKII), extracellular signal-regulated kinase 2 (ERK2), and PKC were assessed. In addition, we explored the contribution of pro-inflammatory cytokines and oxidative stress levels to the modulation of cardiomyocyte function. Immunohistochemistry and electron microscopy were used to assess the translocation of sGC and connexin 43 proteins in the rat model before and after treatment.ResultsHigh cardiomyocyte Fpassive was found in rats and human myocardial biopsies compared to control groups, which was attributed to hypophosphorylation of total titin and to deranged site-specific phosphorylation of elastic titin regions. This was accompanied by lower levels of PKG and PKA activity, along with dysregulation of hypertrophic pathway markers such as CaMKII, PKC, and ERK2. Furthermore, DSS rats and human myocardium biopsies showed higher pro-inflammatory cytokines and oxidative stress compared to controls. DSS animals benefited from treatment with the sGC activator, as Fpassive, titin phosphorylation, PKG and the hypertrophic pathway kinases, pro-inflammatory cytokines, and oxidative stress markers all significantly improved to the level observed in controls. Immunohistochemistry and electron microscopy revealed a translocation of sGC protein toward the intercalated disc and t-tubuli following treatment in both control and DSS samples. This translocation was confirmed by staining for the gap junction protein connexin 43 at the intercalated disk. DSS rats showed a disrupted connexin 43 pattern, and sGC activator was able to partially reduce disruption and increase expression of connexin 43. In human HFpEF biopsies, the high Fpassive, reduced titin phosphorylation, dysregulation of the NO–sGC–cGMP–PKG pathway and PKA activity level, and activity of kinases involved in hypertrophic pathways CaMKII, PKC, and ERK2 were all significantly improved by sGC treatment and accompanied by a reduction in pro-inflammatory cytokines and oxidative stress markers.ConclusionOur data show that sGC activator improves cardiomyocyte function, reduces inflammation and oxidative stress, improves sGC–PKG signaling, and normalizes hypertrophic kinases, indicating that it is a potential treatment option for HFpEF patients and perhaps also for cases with increased hypertrophic signaling.

High salt-induced weakness of anti-oxidative function of natriuretic peptide receptor-C and podocyte damage in the kidneys of Dahl rats.

Background:Atrial natriuretic peptide (ANP) and its natriuretic peptide receptors A (NPR-A) and C (NPR-C) are involved in the regulation of physiological and pathophysiological process of blood pressure. The present study aimed to determine the role of NPR-C in the development of salt-sensitive hypertension.Methods:The Dahl salt-sensitive (DS) and salt-resistant (DR) rats were used in this study. Animals were matched according to their age and weight, and then placed on either a high-salt (HS, 8%) or a normal-salt (NS, 0.4%) diet for 6 weeks randomly using random number table. The systolic blood pressure (SBP), plasmatic sodium concentration (PLNa), urinary sodium excretion (UVNa), and serum creatinine concentration (Scr) were measured. The concentration of ANP in blood and tissues (heart and kidney) was detected by enzyme-linked immunosorbent assay. The expression of ANP, NPR-A, and NPR-C in kidney was evaluated with western blot analysis. Regarding renal redox state, the concentration changes in malondialdehyde (MDA), lipofuscin, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox), and nitric oxide synthase (NOS) in kidney were detected by a spectrophotometric method. The kidney damage was evaluated using pathological techniques and the succinodehydrogenase (SDHase) examination. Furthermore, after an intra-peritoneal injection of C-atrial natriuretic peptide (ANP)4–23 (C-ANP4–23), an NPR-C receptor agonist, the SBP, biochemical values in blood and urine, and renal redox state were evaluated. The paired Student's t test and analysis of variance followed by the Bonferroni test were performed for statistical analyses of the comparisons between two groups and multiple groups, respectively.Results:The baseline SBP in all groups was within the normal range. At the end of the 6-week experiment, HS diet significantly increased the SBP in DS rats from 116.63 ± 2.90 mmHg to 162.25 ± 2.15 mmHg (t = −10.213, P < 0.001). The changes of SBP were not significant in DS rats on an NS diet and DR rats on an NS diet or on an HS diet (all P > 0.05). The significant increase of PLNa, UVNa, and Scr related to an HS diet was found in both DS and DR rats (all P < 0.05). However, significant changes in the concentration (t = −21.915, P < 0.001) and expression of renal ANP (t = −3.566, P = 0.016) and the expression of renal NPR-C (t = 5.864, P = 0.002) were only observed in DS hypertensive rats. The significantly higher desmin immunochemical staining score (t = −5.715, P = 0.005) and mitochondrial injury score (t = −6.325, P = 0.003) accompanied by the lower SDHase concentration (t = 3.972, P = 0.017) revealed mitochondrial pathologic abnormalities in podocytes in DS rats with an HS diet. The distinct increases of MDA (t = −4.685, P = 0.009), lipofuscin (t = −8.195, P = 0.001), and Nox (t = −12.733, P < 0.001) but not NOS (t = −0.328, P = 0.764) in kidneys were also found in DS hypertensive rats. C-ANP4–23 treatment significantly decreased the SBP induced by HS in DS rats (P < 0.05), which was still higher than NS groups with the vehicle or C-ANP4–23 treatment (P < 0.05). Moreover, the HS-induced increase of MDA, lipofuscin, Nox concentrations, and Nox4 expression in DS rats was significantly attenuated by C-ANP4–23 treatment as compared with those with HS diet and vehicle injection (all P < 0.05).Conclusions:The results indicated that the renal NPR-C might be involved in the salt-sensitive hypertension through the damage of mitochondria in podocytes and the reduction of the anti-oxidative function. Hence, C-ANP4–23 might serve as a therapeutic agent in treating salt-sensitive hypertension.

SAT-552 Epigenetic Regulation of 11beta-Hydroxysteroid Dehydrogenase 1 and 2 Gene in Salt-Sensitive Hypertensive Rats

ENDO 2020Epigenetic regulation of 11beta-hydroxysteroid dehydrogenase 1 and 2 gene in salt-sensitive hypertensive rats[Objective]11Beta-hydroxysteroid dehydrogenase type1 (11-HSD1) is the modulator of glucocorticoid hormone and type2 (11-HSD2) is the modulator of mineralocorticoid hormone. We investigated the effect of high salt diet on the methylation of both enzyme gene in salt-sensitive hypertensive (SSH) rats [Methods]SSH rats were fed a high (7% NaCl) or normal (0.45%) salt chow for 4 weeks. Body weight, blood pressure, plasma and urinary aldosterone concentration and PRA were measured. DNA was extracted from kidneys and visceral fats. Bisulfite sequencing and Pyrosequencing were done for the analysis of methylation status of 11-HSD1 and 2 gene. [Results] High salt diet significantly decreased methylation ratio of 11-HSD1 gene in the visceral fats of SSH rats compared with controls (p<0.05). The methylation ratio of 11-HSD2 gene in the kidney of SSH rats was not influenced by high salt diet. [Discussion and Conclusion]11-HSD1 overexpression in visceral fats in mice was reported to show SSH. We reported decreased 11-HSD2 activity in the artery in SSH rats. In this study high salt diet affected methylation status of 11-HSD1 in the adipose tissue but not 11-HSD2 gene in the kidney in SSH. Food intake such as salt may influence the epigenesis of 11-HSD and induce hypertension.

Berry fruits modulate kidney dysfunction and urine metabolome in Dahl salt-sensitive rats

Berries are rich sources of (poly)phenols which have been associated with the prevention of cardiovascular diseases in animal models and in human clinical trials. Recently, a berry enriched diet was reported to decrease blood pressure and attenuate kidney disease progression on Dahl salt-sensitive rats. However, the relationship between kidney function, metabolism and (poly)phenols was not evaluated. We hypothesize that berries promote metabolic alterations concomitantly with an attenuation of the progression of renal disease. For that, kidney and urinary metabolomic changes induced by the berry enriched diet in hypertensive rats (Dahl salt-sensitive) were analyzed using liquid chromatography (UPLC-MS/MS) and 1H NMR techniques. Moreover, physiological and metabolic parameters, and kidney histopathological data were also collected. The severity of the kidney lesions promoted in Dahl rats by a high salt diet was significantly reduced by berries, namely a decrease in sclerotic glomeruli. In addition, was observed a high urinary excretion of metabolites that are indicators of alterations in glycolysis/gluconeogenesis, citrate cycle, and pyruvate metabolism in the salt induced-hypertensive rats, a metabolic profile counteracted by berries consumption. We also provide novel insights that relates (poly)phenols consumption with alterations in cysteine redox pools. Cysteine contribute to the redox signaling that is normally disrupted during kidney disease onset and progression. Our findings provide a vision about the metabolic responses of hypertensive rats to a (poly)phenol enriched diet, which may contribute to the understanding of the beneficial effects of (poly)phenols in salt-induced hypertension.

AJP-Regulatory, Integrative and Comparative Physiology: Looking Toward the Future

<i>AJP-Regulatory, Integrative and Comparative Physiology</i>: Looking Toward the Future

Amelioration of diastolic dysfunction by dapagliflozin in a non-diabetic model involves coronary endothelium

The results of trials with sodium-glucose cotransporter 2 (SGLT2) inhibitors raised the possibility that this class of drugs provides cardiovascular benefits independently from their anti-diabetic effects, although the mechanisms are unknown. Therefore, we tested the effects of SGLT2 inhibitor dapagliflozin on the progression of experimental heart disease in a non-diabetic model of heart failure with preserved ejection fraction. Dahl salt-sensitive rats were fed a high-salt diet to induce hypertension and diastolic dysfunction and were then treated with dapagliflozin for six weeks. Dapagliflozin ameliorated diastolic function as documented by echo-Doppler and heart catheterization, while blood pressure remained markedly elevated. Chronic in vivo treatment with dapagliflozin reduced diastolic Ca2+ and Na+ overload and increased Ca2+ transient amplitude in ventricular cardiomyocytes, although no direct action of dapagliflozin on isolated cardiomyocytes was observed. Dapagliflozin reversed endothelial activation and endothelial nitric oxide synthase deficit, with reduced cardiac inflammation and consequent attenuation of pro-fibrotic signaling. The potential involvement of coronary endothelium was supported by the endothelial upregulation of Na+/H+ exchanger 1in vivo and direct effects on dapagliflozin on the activity of this exchanger in endothelial cells in vitro. In conclusions, several mechanisms may cumulatively play a significant role in the dapagliflozin-associated cardioprotection. Dapagliflozin ameliorates diastolic function and exerts a positive effect on the myocardium, possibly targeting coronary endothelium. The lower degree of endothelial dysfunction, inflammation and fibrosis translate into improved myocardial performance.

High Salt Intake-Increased (Pro)renin Receptor Expression Is Exaggerated in the Kidney of Dahl Salt-Sensitive Rats.

The (P)RR ([pro]renin receptor) was identified as a new component of the renin-angiotensin system. We previously reported that high salt (HS) intake increased the (P)RR expression in several nephron segments of Sprague-Dawley rats. Other studies reported HS intake increased the XO (xanthine oxidase) activity and an MR (mineralocorticoid receptor) antagonist inhibited HS intake-increased (P)RR expression in the kidneys of Dahl salt-sensitive (DS) rats. The present study examined the effects of HS intake on (P)RR expression in the kidney of DS rats. Male DS rats were fed a normal salt diet or an HS diet for 4 weeks. Some of the rats fed on the HS diet were treated with the XO inhibitor, febuxostat, and the MR antagonist, spironolactone. Immunoblot and immunohistochemical analyses showed that HS intake increased (P)RR expression in the renal cortex by 22.6-fold, the proximal tubules by 4.9-fold and the distal tubules, respectively. Both febuxostat and spironolactone inhibited HS intake-increased (P)RR expression in the renal cortex. Febuxostat inhibited HS intake-increased (P)RR expression in the proximal tubules, whereas spironolactone inhibited HS intake-increased (P)RR expression in the distal tubules. Additionally, deoxycorticosterone acetate increased (P)RR expression in the renal cortex and distal tubules but not in the proximal tubules of DS rats fed the normal salt diet. These results indicate that HS intake greatly increases (P)RR expression in the renal cortex of DS rats. The mechanisms of HS intake-increased (P)RR expression may work in an XO-dependent manner in the proximal tubules and an MR-dependent manner in the distal tubules.

Potential Therapeutic Value of Urotensin II Receptor Antagonist in Chronic Kidney Disease and Associated Comorbidities.

Chronic kidney disease (CKD) remains a common disorder, leading to growing health and economic burden without curative treatment. In diabetic patients, CKD may result from a combination of metabolic and nonmetabolic-related factors, with mortality mainly driven by cardiovascular events. The marked overactivity of the urotensinergic system in diabetic patients implicates this vasoactive peptide as a possible contributor to the pathogenesis of renal as well as heart failure. Previous preclinical studies with urotensin II (UII) antagonists in chronic kidney disease were based on simple end points that did not reflect the complex etiology of the disease. Given this, our studies revisited the therapeutic value of UII antagonism in CKD and extensively characterized 1-({[6-{4-chloro-3-[3-(dimethylamino)propoxy]phenyl}-5-(2-methylphenyl)pyridin-2-yl]carbonyl}amino) cyclohexanecarboxylic acid hydrochloride (SAR101099), a potent, selective, and orally long-acting UII receptor competitive antagonist, inhibiting not only UII but also urotensin-related peptide activities. SR101099 treatment more than halved proteinurea and albumin/creatinine ratio in spontaneously hypertensive stroke-prone (SHR-SP) rats fed with salt/fat diet and Dahl-salt-sensitive rats, respectively, and it halved albuminuria in streptozotocin-induced diabetes rats. Importantly, these effects were accompanied by a decrease in mortality of 50% in SHR-SP and of 35% in the Dahl salt-sensitive rats. SAR101099 was also active on CKD-related cardiovascular pathologies and partly preserved contractile reserve in models of heart failure induced by myocardial infarction or ischemia/reperfusion in rats and pigs, respectively. SAR101099 exhibited a good safety/tolerability profile at all tested doses in clinical phase-I studies. Together, these data suggest that CKD patient selection considering comorbidities together with new stratification modalities should unveil the urotensin antagonists' therapeutic potential. SIGNIFICANCE STATEMENT: Chronic kidney disease (CKD) is a pathology with growing health and economic burden, without curative treatment. For years, the impact of urotensin II receptor (UT) antagonism to treat CKD may have been compromised by available tools or models to deeper characterize the urotensinergic system. New potent, selective, orally long-acting cross-species UT antagonist such as SAR101099 exerting reno- and cardioprotective effects could offer novel therapeutic opportunities. Its preclinical and clinical results suggest that UT antagonism remains an attractive target in CKD on top of current standard of care.

Interaction between Mas1 and AT1RA contributes to enhancement of skeletal muscle angiogenesis by angiotensin-(1-7) in Dahl salt-sensitive rats.

The heptapeptide angiotensin-(1-7) (Ang-(1-7)) is protective in the cardiovascular system through its induction of vasodilator production and angiogenesis. Despite acting antagonistically to the effects of elevated, pathophysiological levels of angiotensin II (AngII), recent evidence has identified convergent and beneficial effects of low levels of both Ang-(1-7) and AngII. Previous work identified the AngII receptor type I (AT1R) as a component of the protein complex formed when Ang-(1-7) binds its receptor, Mas1. Importantly, pharmacological blockade of AT1R did not alter the effects of Ang-(1-7). Here, we use a novel mutation of AT1RA in the Dahl salt-sensitive (SS) rat to test the hypothesis that interaction between Mas1 and AT1R contributes to proangiogenic Ang-(1-7) signaling. In a model of hind limb angiogenesis induced by electrical stimulation, we find that the restoration of skeletal muscle angiogenesis in SS rats by Ang-(1-7) infusion is impaired in AT1RA knockout rats. Enhancement of endothelial cell (EC) tube formation capacity by Ang-(1-7) is similarly blunted in AT1RA mutant ECs. Transcriptional changes elicited by Ang-(1-7) in SS rat ECs are altered in AT1RA mutant ECs, and tandem mass spectrometry-based proteomics demonstrate that the protein complex formed upon binding of Ang-(1-7) to Mas1 is altered in AT1RA mutant ECs. Together, these data support the hypothesis that interaction between AT1R and Mas1 contributes to proangiogenic Ang-(1-7) signaling.

Recombinant Cellular Repressor of E1A-Stimulated Genes Protects against Renal Fibrosis in Dahl Salt-Sensitive Rats

Background: Human cellular repressor of E1A-stimulated genes (CREG) is a secreted glycoprotein that attenuates angiotensin II-induced hypertension, alleviates myocardial fibrosis, and improves heart function. However, the role of CREG in high-salt (HS) diet-induced hypertensive nephropathy is unclear. Methods: To determine the effects and molecular mechanisms of CREG in HS diet-induced hypertensive nephropathy, we established a hypertensive nephropathy animal model in Dahl salt-sensitive (SS) rats fed a HS diet (8% NaCl, n = 20) for 8 weeks. At week 4 of HS loading, these rats were administered recombinant CREG (reCREG; 35 µg/kg·day, n = 5) and saline (n = 5) via subcutaneously implanted pumps and were also administered the vasodilator hydralazine (20 mg/kg·day, n = 5) in drinking water. We used hematoxylin and eosin staining, Masson’s trichrome staining, immunohistochemical labeling, western blotting, RT-PCR, and Tunel staining to determine the signaling pathways of CREG in HS diet-induced hypertensive nephropathy. Results: After 8 weeks of HS intake, the Dahl SS rats developed renal dysfunction and severe renal fibrosis associated with reductions of 78 and 67% in CREG expression, respectively, at both mRNA and protein levels in the kidney. Administration of reCREG improved renal function and relieved renal fibrosis. Administration of CREG also inhibited monocyte infiltration and reduced apoptosis in the kidney cells. CREG overexpression upregulated forkhead box P1 expression and inhibited the transforming growth factor-β1 signaling pathway. Conclusion: Our study shows that CREG protected the kidney against HS-diet-induced renal damage and provides new insights into the mechanisms underlying kidney injury.

Acquired Immunity Is Not Essential for Radiation-Induced Heart Dysfunction but Exerts a Complex Impact on Injury.

While radiation therapy (RT) can improve cancer outcomes, it can lead to radiation-induced heart dysfunction (RIHD) in patients with thoracic tumors. This study examines the role of adaptive immune cells in RIHD. In Salt-Sensitive (SS) rats, image-guided whole-heart RT increased cardiac T-cell infiltration. We analyzed the functional requirement for these cells in RIHD using a genetic model of T- and B-cell deficiency (interleukin-2 receptor gamma chain knockout (IL2RG−/−)) and observed a complex role for these cells. Surprisingly, while IL2RG deficiency conferred protection from cardiac hypertrophy, it worsened heart function via echocardiogram three months after a large single RT dose, including increased end-systolic volume (ESV) and reduced ejection fraction (EF) and fractional shortening (FS) (p < 0.05). Fractionated RT, however, did not yield similarly increased injury. Our results indicate that T cells are not uniformly required for RIHD in this model, nor do they account for our previously reported differences in cardiac RT sensitivity between SS and SS.BN3 rats. The increasing use of immunotherapies in conjunction with traditional cancer treatments demands better models to study the interactions between immunity and RT for effective therapy. We present a model that reveals complex roles for adaptive immune cells in cardiac injury that vary depending on clinically relevant factors, including RT dose/fractionation, sex, and genetic background.

Autophagy contributes to the development of salt‐induced hypertension and kidney injury in Dahl S rats

Studies were carried to determine the role of autophagy in the development of renal injury in the hypertensive Dahl salt‐sensitive (SS) rat fed a high‐salt (HS; 4.0% NaCl) diet. Autophagy is an essential process (autophagosome formation, maturation, fusion with lysosome, release of macromolecular back into cytosol) required to remove misfolded proteins and damaged organelles (mitochondria, endoplasmic reticulum and peroxisomes) from all cells. It is a dynamic process (“autophagic flux”) and challenged in cells faced with excessive metabolic workloads such as the renal nephrons in kidneys of SS rats fed a HS diet. The tubular mitochondria of SS rats fed a HS produce excess reactive oxygen species (ROS) which we have found can activate mammalian target of rapamycin complex 1 (mTORC1). We have reported that the elevation of renal tissue H2O2 in kidneys of SS rats fed a HS diet can activate mTORC1 and that inhibition of this pathway with rapamycin attenuated salt‐induced hypertension and kidney injury. It is also known that mTORC1 plays a crucial role in regulating autophagy but the extent to which these events contribute to the renal injury observed in the SS rat has not been examined.GoalStudies were carried out in male SS rats to determine the effects of a HS diet (4.0% NaCl) upon the “autophagic flux” of the kidneys. SS rats (n=6 each group) were studied at days 3 or 21 of the HS diet and compared to those receiving a 0.4% NaCl diet. Rat received either the HS diet treated with vehicle, or HS plus daily rapamycin (IP, 1.5 mg/kg per day); Western blot analysis of renal cortical and medullary tissue LCII (microtubule‐associated protein light chain 2) and BECLIN1 protein levels was carried out as an index of autophagic flux.ResultsWhile no changes in the LCII levels were observed at day 3 of HS, a significant reduction was found in both the cortex and medulla after 21 days of HS compared to rats fed 0.4% NaCl. In rapamycin treated SS rats, both the renal cortex and medulla showed significantly greater increases of LCII and BECLIN1 compared with vehicle treated rats suggested an increase of autophagic index. Together, our observations suggest that the attenuation of salt‐induced hypertension and kidney injury in rapamycin treated SS rats may be in part the consequence of increased autophagic influx.Support or Funding InformationHL‐116264

Progressive Alterations of Mitochondrial Function in the Kidney and Heart During the Development of Salt‐Induced Hypertension

IntroductionHypertension is a multi‐factorial disease and a leading cause of morbidity and mortality globally. Nearly half of these individuals are sensitive to dietary salt intake with the kidney and oxidative stress playing a key role in this disease leading to eventual failure of both the kidney and heart. Although oxidative stress and mitochondrial dysfunction in the kidney and heart have been studied in various models of hypertension, progressive alterations of mitochondrial function and development of oxidative stress in these two organs have not been studies in parallel during the development of hypertension. The goal of the present study was to investigate progressive alterations in mitochondrial energy metabolism in both the kidney and heart concurrently in the Dahl salt‐sensitive (SS) rats during the development of hypertension when rats were switched from a 0.4% NaCl (LS) to a 4% NaCl (HS) diet.MethodMitochondria were isolated from the ventricles of the heart and outer medulla (OM) and cortex of the kidney of SS rats fed a LS diet since weaning and studied at days 3, 7, 14 &amp; 21 of a HS diet feeding. Oxygen consumption rates (OCR) were measured in mitochondria energized with pyruvate + malate as substrates for 3 different respiratory states using an Oroboros Oxygraph‐2k respirometer. This includes a) leak state (energized mitochondria in the absence of ADP), b) ADP‐stimulated state, and c) uncoupled state (energized mitochondria in the presence of an uncoupler FCCP).ResultsA biphasic pattern of ADP‐stimulated OCR with progressive uncoupling was observed in both the kidney (OM &amp; cortex) and heart mitochondria. In kidney, mitochondrial efficiency for ATP synthesis was increased in the early phase of hypertension (3 &amp; 7 days) but was severely compromised in the established phase of hypertension (14 &amp; 21 days). Cardiac mitochondria also showed a similar pattern, however the reduced mitochondrial efficiency for ATP synthesis occurred at a later time, specifically at day 21. This decreased mitochondrial efficiency was hypothesized to be associated with higher levels of uncoupling, ROS production, and oxidative stress in both organs due to mitochondrial ROS‐cellular ROS crosstalk. To test this, experiments were performed in SS rats with double knock out (DKO) of the Nox family of NADPH oxidases: Nox4 and p67phox cytosolic subunit of Nox2 (SSp67phox−/−/Nox4−/−) and determined the role of mitochondrial ROS in stimulating cellular ROS production, particularly by membrane Noxs, and vice versa. Specifically, the DKO SS rats were fed a HS diet and isolated mitochondrial OCR was studied at days 7 and 14. DKO SS rats fed a HS diet showed no significant differences in mitochondrial OCR as compared to the control group (LS diet) in the kidney (OM &amp; cortex) and heart.ConclusionOur data suggest that a HS diet in SS rats results initially in increased efficiency of renal cortical and medullary mitochondrial energy production during the 1st week of a HS diet followed by an enhanced ROS production with mitochondrial uncoupling and reduced efficiency of ATP production by the 3rd week. Similar changes occurred in the heart but 1–2 weeks following the changes observed in the kidney.Support or Funding InformationP01‐GM066730, P01‐HL116264

Selective Phosphodiesterase 1 Inhibitor LY1 Reduces Blood Pressure in Spontaneously Hypertensive and Dahl Salt Sensitive Rats

Regulation of the peripheral vascular resistance via modulating the vessel diameter has been considered as a main determinant of the arterial blood pressure. Phosphodiesterase enzymes (PDE1‐11) hydrolyse cyclic nucleotides, which are key players controlling the vessel diameter and thus peripheral resistance, aiming at achieving and maintaining a homeostatic balance between their production and destruction. On the contrary, pathological shift in the balance to words the destruction leads to depletion of the cyclic nucleotides and dysregulation of the blood pressure. Here, we have tested and reported the effects of a novel selective PDE1 inhibitor (LY1) on the cardiovascular system. In vitro experiments have been conducted to determine the IC50 of LY1 using phosphodiesterase radiometric enzyme assay. We used rat mesenteric artery to conduct myogrphy study to determine the effect of the drug on the vessel diameter. Also, short as well as long term telemetric studies were done on normal Sprague Dawley (SD), spontaneously hypertensive (SHR), and Dahl salt sensitive (SS) rats to document the in vivo efficacy of the compound. LY1 inhibited all three isoforms of PDE1 in nanomolar concentration (1.74 nM, 2.44 nM and 1.20 nM for PDE1A, PDE1B and PDE1C, respectively), while micromolar concentrations were needed to induce effective inhibition for other PDEs. The myography study revealed a potent vasodilatory effect of the drug, which was confirmed in vivo by an increase in the blood flow in the rat ear arteriole reflected by the rise in the temperature. LY1 proved a high efficacy in lowering the blood pressure in two animal models for hypertension i.e. SHR and SS rats. 3 mg/kg of LY1 twice daily through oral gavage could efficiently reduce the blood pressure in both SHR and SS rats approximately 40 mmHg compared to the untreated control group. Therefore, LY1 induced an efficient vasodilation that was accompanied by a significant reduction of blood pressure in different hypertensive rat models. Thus, PDE1 appears to be a tempting therapeutic target for treatment of resistant hypertension.