Salt-sensitive Rats Research Articles

A Biopolymer‐delivered MMP‐2 Inhibitory for Treatment of Renal Fibrosis

Renal fibrosis, the buildup of extracellular matrix (ECM) components and scar tissue leading to organ failure, is the final common pathway of chronic kidney disease (CKD). There are currently no pharmacological treatments for CKD that specifically target the fibrosis process. Matrix metalloproteinases (MMPs) are involved in ECM homeostasis and degrade ECM components during tissue repair. Although an increase in MMPs can have anti-fibrotic effects, some MMPs can also process and activate chemokines and cytokines that are pro-inflammatory. Studies have shown that in the initial stages of CKD, there is an increase in the expression and activity of MMP-2, a gelatinase known to activate pro-TGF-β, TNF-α, and TNF- β, which are known to be pro-inflammatory and profibrotic. Furthermore, MMP-2 can promote epithelial to mesenchymal transition (EMT), a process in which epithelial cells lose their cell-to-cell adhesion and gain the ability to migrate, proliferate, and differentiate to profibrotic mesenchymal cells. Our goal is to develop an MMP-2 inhibitor that specifically targets the renal cells to reduce or reverse the effects of fibrosis. We created a chimeric fusion protein between an MMP-2 inhibitory peptide derived from beta amyloid precursor peptide (APP-IP) and an elastin like polypeptide (ELP) drug carrier. ELP is has been shown to accumulate in the kidney and has a long and tunable plasma half-life, which would allow for the therapeutic to have a sustained activity with minimal off target effects. An MMP-2 fluorometric assay revealed that the ELP – MPP-2 inhibitory peptide fusion protein (ELP-APP-IP) maintained potent MMP-2 inhibitory activity (IC50 = 1 mM, Figure panel A). Biodistribution of ELP-APP-IP or ELP control was determined in Sprague Dawley rats following bolus intravenous injection (30 mg/kg). Pharmacokinetic analysis revealed a terminal half-life approximately 90 minutes for both ELP and ELP-APP-IP. Ex vivo imaging of the organs showed that both ELP and ELP-APP-IP localized to the kidneys at levels approximately 16 times higher than in other major organs (brain, liver spleen, heart and lungs, Figure panel B,C). Histological analysis revealed that ELP and ELP-APP-IP accumulated primarily in the renal cortex (Figure panel D). A pilot efficacy study was conducted in Dahl Salt Sensitive Rats (S-Rats) on a high salt diet (8% NaCl) as a model of hypertension induced glomerulosclerosis. S-Rats (male, 12 weeks of age) on high salt diet were treated with ELP or ELP-APP-IP (n = 5 per group) for 3 weeks via osmotic minipumps (10 mg/kg/day). Urine was collected via metabolic caging 24 hours prior to initiation of treatment and high salt diet, then again weekly throughout the study. 21 days after initiation of treatment, the rats were euthanized, and blood and major organs were harvested for analysis. Rats on a high salt diet treated with ELP-APP-IP had similar urine production to rats treated with ELP. However, there was a trend for reduced onset of proteinuria in rats treated with ELP-APP-IP (two way ANOVA, treatment factor p = 0.088, Figure panel E), suggesting ELP-APP-IP has the potential to reduce kidney damage and slow the progression of renal disease.

Repeated Seizure Exposure in the SS Kcnj16‐/‐ Rat Causes Progressive Respiratory Suppression and Associated Brainstem Pathology

Kir5.1 is an inwardly rectifying potassium (Kir) channel subunit highly expressed in the kidney and brain and encoded by the Kcnj16 gene. Knockout of Kcnj16 in a Dahl salt-sensitive rat (SSKcnj16-/-) results in susceptibility to audiogenic, generalized tonic-clonic seizures (GTCSs), which when repeated lead to spontaneous mortality. Patients with uncontrolled epilepsy are at high risk for Sudden Unexplained Death in Epilepsy (SUDEP), hypothesized to be a result of severe cardiorespiratory suppression and/or failure. We hypothesized that repeated audiogenic seizures in SSKcnj16-/- rats lead to progressively worsening cardiorespiratory suppression and associated brainstem pathology. SSKcnj16-/- and wild-type (WT) rats (10-12 weeks of age) were exposed to the seizure-inducing acoustic stimulus 1/day (d) for 10d and breathing measurement were made before, during and after exposure. Solitary GTSCs acutely led to apnea and decreases in breathing frequency for ~5 min postictally, with no change or an increase in total ventilation due to an increase in tidal volume. We also found a significant reduction in breath-to-breath variability throughout the postictal period. After exposure to repeated seizures, postictal frequency suppression became more pronounced and compensatory increases in tidal volume were reduced, such that total ventilation was also significantly reduced. Repeated seizures also further attenuated the ventilatory response to hypercapnia (p=0.0073). To gain insights into the effects of repeated seizures on cardiorespiratory circuits in the brainstem, we used immunofluorescence staining/confocal imaging to label and quantify total serotonin (5-HT) immunoreactivity (ir), serotonergic neurons (tryptophan hydroxylase+, TPH), microglia (Iba1), astrocytes (GFAP), total neurons (NeuN), and total nuclei (DAPI) in respiratory brainstem nuclei of SSkcnj16-/- rats exposed to 0, 3, or 10 seizures (n=5/group). Repeated seizures resulted in reduced 5-HT-ir in multiple cardiorespiratory brainstem nuclei including the pre-Bötzinger Complex (pre-BötC), nucleus ambiguous (NA), and nucleus tractus solitarious (NTS) in addition to a decrease in 5-HT neurons. We also found increased Iba1-ir selectively within the pre-BötC and NA, whereas GFAP-ir was increased after 10 seizures in these same regions. This data suggests that inflammation and/or serotonergic dysregulation in cardiorespiratory brainstem nuclei may mediate progressive respiratory suppression following repeated seizures in this rat model.

A Potential Regulatory Role of Xanthine Dehydrogenase (XDH) in the Kidney Development and Damage

There is a clear association of uric acid (UA) dysregulation in cardiovascular and chronic kidney diseases elucidated in the literature. Xanthine dehydrogenase (XDH) encodes for XDH enzyme that catalyzes hypoxanthine oxidation to xanthine and xanthine to UA, the end-product of purine catabolism in humans. UA can act as both an antioxidant scavenging reactive oxygen species (ROS) and conditionally a pro-oxidant. The lack of understanding of UA metabolism and its impact on CKD progression reflects the current controversy regarding the clinical management of the related pathologies. To explore mechanistic insights into UA affecting the kidney, we created a genetic ablation of the Xdh gene in the Dahl salt-sensitive rat background (SSXdh-/-). The absence of Xdh protein expression in SSXdh-/- was confirmed by Western blotting and immunohistochemistry analyses. The knockout rat is underdeveloped (179 ± 6 and 70 ± 7 g: N=9 for both male SSXdh+/+ and SSXdh-/-) and hypouricemic (UA in plasma below the level of detection). RNA-Seq analysis of kidney tissue of the SSXdh-/- and SSXdh+/+ 6-week-old rats revealed 4738 differentially expressed genes. The functional analyses of the obtained data were performed using the ingenuity pathway analysis (IPA) software. The changes in oxidative phosphorylation and the presence of mitochondrial dysfunction were among the top canonical pathways associated with the differentially expressed genes. The cardiac hypertrophy signaling pathway was enhanced the most (z-score 6.5). IPA toxic function analysis suggested the presence of renal damage. Among the upregulated genes were transforming growth factor-β (Tgf-β) and connective tissue growth factor (Ccn2), previously reported to drive the tissue fibrosis. Indeed, SSXdh-/- rats show increased levels of fibrosis in the kidneys. Western blot analysis revealed Ctgf lacking in the SSXdh-/- rats despite the increased mRNA levels. Ctgf is essential for kidney development, and the lack of this protein might explain the underdeveloped kidneys seen in the SSXdh-/- rat. Furthermore, we analyzed the expression of the primary transporters for UA in the kidney: Urat1 and Glut9, encoded by Slc22a12 and Slc2a9 genes, respectively. Western blot analysis revealed that Urat1 is not detectable and Glut9 expression is decreased in SSXdh-/- rats; potential glycosylation of Glut9 in SSXdh-/- rats is also observed. Catalase enzyme encoding Cat gene is also downregulated (log FC=-3.05) in SSXdh-/- rats, indicating the potential ROS production increase. These results provide insights into the regulatory role of the Xdh gene beyond purine catabolism that involves kidney tissue development and kidney damage.

Effect of a Combined Treatment with Ivabradine and Spironolactone on Cardiac Structure and Function in Hypertensive Dahl Salt‐Sensitive Rats

Introduction Hypertension-induced left ventricular (LV) hypertrophy and myocardial fibrosis provide structural basis for diastolic dysfunction. It has been previously reported that aldosterone receptor antagonist spironolactone (SL) can prevent myocardial fibrosis and, hence, alleviate hypertension-induced diastolic dysfunction, whereas treatment with heart rate lowering drug ivabradine (IVA) can improve cardiac function during development of hypertension-induced hypertrophy. Hypothesis We hypothesize that a combination of a heart rate-lowering drug IVA and a competitive mineralocorticoid receptor antagonist SL would alleviate adverse LV structural alterations and diastolic dysfunction in animals with chronic hypertension. Methods Seven-week-old, male Dahl salt-sensitive rats were fed a high-salt diet (8% NaCl) to induce sustained hypertension. After 7 weeks on a high-salt diet, in order to reduce the risk of death by stroke, the hypertensive (HT) rats were switched to a low-salt diet (0.3% NaCl) and randomly divided into two experimental groups: 1) treated with IVA (10 mg/kg/day) and SL (20 mg/kg/day) (HT-T) and 2) subjected to a vehicle only (HT-V). A mixture of dimethyl sulfoxide and propylene glycol (50%:50%, v/v) was used as a vehicle to deliver the drugs for 8 weeks by intraperitoneal ALZET osmotic pumps. The rats fed a low-salt diet only were used as an age-matched normotensive controls (NT). Heart rate and blood pressure were evaluated every week using the CODA tail-cuff plethysmography system. At the end of experiment, the hemodynamic parameters and LV function were assessed using a Millar micro-tip pressure catheter and a PowerLab data acquisition system. Subsequently, rats were euthanized, and their hearts were processed to paraffin for morphological evaluation. Serial sections from each heart were stained with various histological techniques. Images were captured using either a MicroScan D2 slide scanner or a Leica DM IRE2 microscope and analyzed by Image-Pro Analyzer v.7.0 software. Statistical analysis was performed using Prism 6 software. Results At the beginning of treatment, HT rats demonstrated a 42.3% increase in mean arterial blood pressure (MAP) and a 17.1% rise in heart rate compared to NT rats. During 8 weeks of treatment, the MAP remained comparably elevated in both HT groups, whereas heart rate had been persistently reduced in treated rats on average by 37.5% and 26.9% in comparison with HT-V and NT rats, respectively. At the end of a treatment period, rats in both HT groups had a significantly increased Tau constant, suggesting an analogous impairment of active relaxation. At the same time, all groups showed a similar level of LV end-diastolic pressure, indicating a preservation of passive myocardial stiffness. The examination of hearts has confirmed that chronic hypertension caused significant LV hypertrophy (P≤0.001) and myocardial fibrosis (P≤0.01) compared to NT rats. More important, HT rats in treated group revealed more pronounced concentric hypertrophy than animals in HT-V group. Conclusion Our findings establish that a combined treatment with IVA and SL augmented concentric LV hypertrophy and did not alleviate the hypertension-induced diastolic dysfunction.

Single Nuclear RNA Sequencing Reveals Activation of Neuroinflammation Within the Pre‐Bötzinger Complex Following Repeated Seizures

Epilepsy is one of the most common neurological diseases in the world. Treatment with anti-epileptic drugs (AEDs) prevents recurrent seizures in ~70% of patients with epilepsy, but the remaining 30% of patients with refractory epilepsies continue to experience seizures. The consequences of repeated seizures include but are not limited to memory impairments and a high risk of post-ictal cardiorespiratory failure, and Sudden Unexpected Death in Epilepsy (SUDEP). Fundamental gaps exist in understanding how repeated seizures disrupt the vital cardiorespiratory control system and neural networks facilitating memory formation in refractory epilepsy. A factor commonly identified in many neurological conditions is neuroinflammation, which may support beneficial health functions but is dysregulated in neurological disease. Key cells within the CNS that mediate beneficial and/or pathological neuroinflammation are resident microglia and astrocytes, which have been shown to be dysfunctional in human epilepsy and alter neuronal function. However, it is not known what mechanistic role they play in contributing to epilepsy-related memory impairment, impairment in cardiorespiratory control, or increased SUDEP risk. Here, we tested the hypothesis that repeated seizures lead to activation of neuroinflammation mediated by microglia and astrocytes in CNS regions of memory and ventilatory control, leading to a progressive decline in neurologic function. Preliminary data in our novel rat model with genetic mutations in Kcnj16, a gene encoding an inwardly-rectifying potassium channel Kir5.1 in Dahl salt-sensitive rat (SSkcnj16-/) show that repeated sound-induced seizures lead to a progressively more severe post-ictal suppression of breathing frequency and heart rate, and impaired memory. Single nuclear RNA sequencing of pre-Bötzinger Complex/nucleus ambiguous cells was performed from rats exposed to 0, 3, 7, or 10 seizures to measure cell type-specific gene expression changes induced by seizures. Bioinformatic analyses of transcriptomic changes in microglia, neuronal, and glial cells within these regions point to activation of specific inflammatory pathways following repeated seizures such as IL-1Beta signaling and Toll-like Receptor signaling. Identifying neuroinflammatory signals/pathways induced by repeated seizures within distinct neural circuits will enhance understanding of pathophysiological consequences of uncontrolled seizures in patients with refractory epilepsy and hold the potential for identifying new therapeutic targets aimed at improving memory and cardiorespiratory function to reduce the risk of SUDEP.

DNA Methylation of the Angiotensinogen Gene, AGT, and the Aldosterone Synthase Gene, CYP11B2 in Cardiovascular Diseases.

Angiotensinogen (AGT) and aldosterone play key roles in the regulation of blood pressure and are implicated in the pathogenesis of cardiovascular diseases. DNA methylation typically acts to repress gene transcription. The aldosterone synthase gene CYP11B2 is regulated by angiotensin II and potassium. DNA methylation negatively regulates AGT and CYP11B2 expression and dynamically changes in response to continuous promoter stimulation of each gene. High salt intake and excess circulating aldosterone cause DNA demethylation around the CCAAT-enhancer-binding-protein (CEBP) sites of the ATG promoter region, thereby converting the phenotype of AGT expression from an inactive to an active state in visceral adipose tissue and heart. A close association exists between low DNA methylation at CEBP-binding sites and increased AGT expression in salt-sensitive hypertensive rats. Salt-dependent hypertension may be partially affected by increased cardiac AGT expression. CpG dinucleotides in the CYP11B2 promoter are hypomethylated in aldosterone-producing adenomas. Methylation of recognition sequences of transcription factors, including CREB1, NGFIB (NR4A1), and NURR1 (NR4A2) diminish their DNA-binding activity. The methylated CpG-binding protein MECP2 interacts directly with the methylated CYP11B2 promoter. Low salt intake and angiotensin II infusion lead to upregulation of CYP11B2 expression and DNA hypomethylation in the adrenal gland. Treatment with the angiotensin II type 1 receptor antagonist decreases CYP11B2 expression and leads to DNA hypermethylation. A close association between low DNA methylation and increased CYP11B2 expression are seen in the hearts of patients with hypertrophic cardiomyopathy. These results indicate that epigenetic regulation of both AGT and CYP11B2 contribute to the pathogenesis of cardiovascular diseases.

Potential mechanisms of Guizhi decoction against hypertension based on network pharmacology and Dahl salt-sensitive rat model

BackgroundGuizhi decoction (GZD), a classical Chinese herbal formula, has been widely used to treat hypertension, but its underlying mechanisms remain elusive. The present study aimed to explore the potential mechanisms and therapeutic effects of GZD on hypertension by integrating network pharmacology and experimental validation.MethodsThe active ingredients and corresponding targets were collected from the Traditional Chinese Medicine Systems Pharmacology database and Analysis Platform (TCMSP). The targets related to hypertension were identified from the CTD, GeneCards, OMIM and Drugbank databases. Multiple networks were constructed to identify the key compounds, hub targets, and main biological processes and pathways of GZD against hypertension. The Surflex-Dock software was used to validate the binding affinity between key targets and their corresponding active compounds. The Dahl salt-sensitive rat model was used to evaluate the therapeutic effects of GZD against hypertension.ResultsA total of 112 active ingredients, 222 targets of GZD and 341 hypertension-related targets were obtained. Furthermore, 56 overlapping targets were identified, five of which were determined as the hub targets for experimental verification, including interleukin 6 (IL-6), C–C motif chemokine 2 (CCL2), IL-1β, matrix metalloproteinase 2 (MMP-2), and MMP-9. Pathway enrichment analysis results indicated that 56 overlapping targets were mainly enriched in several inflammation pathways such as the tumor necrosis factor (TNF) signaling pathway, Toll-like receptor (TLR) signaling pathway and nuclear factor kappa-B (NF-κB) signaling pathway. Molecular docking confirmed that most active compounds of GZD could bind tightly to the key targets. Experimental studies revealed that the administration of GZD improved blood pressure, reduced the area of cardiac fibrosis, and inhibited the expression of IL-6, CCL2, IL-1β, MMP-2 and MMP-9 in rats.ConclusionThe potential mechanisms and therapeutic effects of GZD on hypertension may be attributed to the regulation of cardiac inflammation and fibrosis.

Dietary influences on the Dahl SS rat gut microbiota and its effects on salt-sensitive hypertension and renal damage.

Our previous studies have demonstrated the importance of dietary factors in the determination of hypertension in Dahl salt-sensitive (SS) rats. Since the gut microbiota has been implicated in chronic diseases like hypertension, we hypothesized that dietary alterations shift the microbiota to mediate the development of salt-sensitive hypertension and renal disease. This study utilized SS rats from the Medical College of Wisconsin (SS/MCW) maintained on a purified, casein-based diet (0.4% NaCl AIN-76A, Dyets) and from Charles River Laboratories (SS/CRL) fed a whole grain diet (0.75% NaCl 5L79, LabDiet). Faecal 16S rDNA sequencing was used to phenotype the gut microbiota. Directly examining the contribution of the gut microbiota, SS/CRL rats were administered faecal microbiota transfer (FMT) experiments with either SS/MCW stool or vehicle (Vehl) in conjunction with the HS AIN-76A diet. SS/MCW rats exhibit renal damage and inflammation when fed high salt (HS, 4.0% NaCl AIN-76A), which is significantly attenuated in SS/CRL. Gut microbiota phenotyping revealed distinct profiles that correlate with disease severity. SS/MCW FMT worsened the SS/CRL response to HS, evidenced by increased albuminuria (67.4±6.9 vs 113.7±25.0mg/day, Vehl vs FMT, P=.007), systolic arterial pressure (158.6±5.8 vs 177.8±8.9mmHg, Vehl vs FMT, P=.09) and renal T-cell infiltration (1.9-fold). Amplicon sequence variant (ASV)-based analysis of faecal 16S rDNA sequencing data revealed taxa that significantly shifted with FMT: Erysipelotrichaceae_2, Parabacteroides gordonii, Streptococcus alactolyticus, Bacteroidales_1, Desulfovibrionaceae_2, Ruminococcus albus. These data demonstrate that dietary modulation of the gut microbiota directly contributes to the development of Dahl SS hypertension and renal injury.

Fumarate exerted an antihypertensive effect and reduced kidney injury molecule (KIM)-1 expression in deoxycorticosterone acetate-salt hypertension

ABSTRACT Background: The tricarboxylic (TCA) acid cycle provides the energy needed for regulatory functions in the cardio-renal system. Recently, a genetic defect in the TCA cycle enzyme, fumarase hydratase, altered L-arginine metabolism and exacerbated hypertension in salt-sensitive rats. This study evaluated the effect of fumarate and its possible link to L-arginine metabolism in deoxycorticosterone (DOCA)-salt hypertension, a non-genetic model of hypertension.Method: Hypertension was induced with DOCA (25 mg/kg s.c, twice weekly) + 1% NaCL in uninephrectomised rats placed on fumarate (1 g/L, ad libitum). Blood pressure was measured in conscious rats via carotid cannulation. Biochemical and western blot analyses were carried out on kidney fractions.Results: Fumarate reduced mean blood pressure (198 ± 5 vs 167 ± 7 mmHg, p < .01), increased nitric oxide levels in the renal cortex (36.1 ± 2 vs 61.3 ± 4 nM/µg) and medulla (27.4 ± 1 vs 54.1 ± 2 nM/µg) of DOCA-salt rats (p < .01). Consistent with this, arginase activity was reduced (threefold) in the renal medulla but not cortex of DOCA-salt rats. Fumarate increased superoxide dismutase activity in the medulla (p < .001) of DOCA-hypertensive rats. However, catalase activity was exacerbated by fumarate in both renal cortex (4.5 ± 1 vs 11.2 ± 1) and medulla (3.7 ± 1 vs 16.3 ± 1 units/mg) of DOCA-salt rats (p < .001). Proteinuria (64.6%), kidney injury molecule-1 expression and kidney weight were reduced in DOCA-hypertensive rats treated with fumarate (p< .05). However, there was a paradoxical increase in TGF-β expression in fumarate-treated DOCA-salt rats. Conclusion: These data show that fumarate attenuated hypertension, renal injury and improved the redox state of the kidney in DOCA/salt hypertension by mechanisms involving selective reduction of L-arginine metabolism.

Cooperation of augmented calcium sensitization and increased calcium entry contributes to high blood pressure in salt-sensitive Dahl rats.

Salt hypertensive Dahl rats are characterized by sympathoexcitation and relative NO deficiency. We tested the hypothesis that the increased blood pressure (BP) response to fasudil in salt hypertensive Dahl rats is due to augmented calcium sensitization in the salt-sensitive strain and/or due to their decreased baroreflex efficiency. BP reduction after acute administration of nifedipine (an L-type voltage-dependent calcium channel blocker) or fasudil (a Rho kinase inhibitor) was studied in conscious intact rats and in rats subjected to acute NO synthase inhibition or combined blockade of the renin-angiotensin system (captopril), sympathetic nervous system (pentolinium), and NO synthase (L-NAME). Intact salt-sensitive (SS) Dahl rats fed a low-salt diet had greater BP responses to nifedipine (-31 ± 6 mmHg) or fasudil (-34 ± 7 mmHg) than salt-resistant (SR) Dahl rats (-16 ± 4 and -17 ± 2 mmHg, respectively), and a high-salt intake augmented the BP response only in SS rats. These BP responses were doubled after acute NO synthase inhibition, indicating that endogenous NO attenuates both calcium entry and calcium sensitization. Additional pentolinium administration, which minimized sympathetic compensation for the drug-induced BP reduction, magnified the BP responses to nifedipine or fasudil in all groups except for salt hypertensive SS rats due to their lower baroreflex efficiency. The BP response to the calcium channel blocker nifedipine can distinguish SS and SR rats even after calcium sensitization inhibition by fasudil, which was not seen when fasudil was administered to nifedipine-pretreated rats. Thus, enhanced calcium entry (potentiated by sympathoexcitation) in salt hypertensive Dahl rats is the abnormality that is essential for their BP increase, which was further augmented by increased calcium sensitization in salt-sensitive Dahl rats.

URINARY PLASMIN PLAYS PATHOGENIC ROLES FOR THE DEVELOPMENT OF HYPERTENSION IN SALT-SENSITIVE HYPERTENSION WITH PROTEINURIA

Objective: The epithelial sodium channel (ENaC) in the renal collecting duct plays pivotal roles in the regulation of sodium homeostasis and blood pressure. The proteolytic cleavage of γENaC by extracellular serine proteases is an important process for the full activation of this channel and is physiologically regulated by aldosterone. In the setting of proteinuria, plasminogen/plasmin filtered through damaged glomeruli could activate ENaC leading to hypertension, independently of aldosterone. We reported that Dahl salt-sensitive (DS) rats with high-salt (HS) diet developed severe hypertension together with aberrant activation of γENaC by serine proteases. However, the role of plasmin in the hypertension of DS rats remain to be elucidated. In this study, we evaluated the relationship of proteinuria, urinary plasmin activity and hypertension, and the antihypertensive effect of a serine protease inhibitor camostat mesilate (CM) in DS rats. Design and method: Five-week-old DS rats were divided into normal-salt (NS) diet, HS and HS+CM (0.1% diet) groups. After systolic BP (SBP) measurement and 24h urine collection were performed for 5 weeks, rats were sacrificed for biochemical examination. Urinary plasmin activities were evaluated by zymography and Western blotting (WB). The proteolytic cleavage of γENaC in urinary exosome was evaluated by WB. Results: HS diet induced severe hypertension, marked proteinuria and urinary plasmin activation, as well as the cleavage of urinary exosomal γENaC. The treatment with CM suppressed these changes, together with significantly reduced BP and proteinuria [SBP (mmHg): NS, 141.1 ± 5.8; HS, 222.3 ± 15.4; HS + CM, 199.5 ± 5.0. urinary protein (mg/day): NS, 23.8 ± 12.1; HS 272.3 ± 79.9; HS ± CM, 135.1 ± 27.4]. Conclusions: In conclusion, plasmin is associated with the pathogenesis of hypertension in DS rats, and serine protease inhibition could be a potential therapeutic strategy against salt-sensitive hypertension with proteinuria.

AMILORIDE ATTENUATES ALDOSTERONE-INDUCED RENAL INJURIES VIA THE SUPPRESSION OF PLASMIN ACTIVITY IN THE KIDNEY

Objective: Aldosterone has been known to exert direct detrimental effects on the kidney independently of its hemodynamic effects. We have reported that a serine protease plasmin was highly activated in the kidney of aldosterone/salt-treated rats and that plasmin caused kidney injuries through the induction of inflammatory and profibrotic cytokines. Amiloride is one of the potassium-sparing diuretics and it inhibits epithelial sodium channel (ENaC). On the other hand, it was reported that lower dose amiloride attenuated renal injuries without blood pressure (BP) lowering effect in salt-sensitive hypertension model rats. Because amiloride has a pleiotropic effect to inhibit urokinase-type plasminogen activator (uPA), we evaluated whether amiloride could attenuate aldosterone-induced renal injuries via the suppression of uPA-plasmin cascade in the kidney. Design and method: Nine-week old male uninephrectomized Sprague-Dawley rats were divided into following groups (1) normal salt diet (0.3%NaCl; NS), (2) aldosterone + high salt diet (0.75 μg/kg/h, subcutaneously + 8.0% NaCl; Ald+HS), and (3) aldosterone + high salt diet + amiloride (1 mg/kg/day, subcutaneously; Ami). Systolic BP (SBP) measurement and 24 h urine collection were performed every second week, and rats were sacrificed at 4 week. Renal plasmin activities were evaluated by zymography. Results: Amiloride did not affect either electrolytes in serum and urine nor blood pressure, indicating that the current dose of amiloride did not inhibit ENaC in the distal nephron [SBP (mmHg): NS, 115 ± 11; Ald+HS, 169 ± 17; Ami, 161 ± 21.]. Amiloride suppressed renal plasmin activation, and mitigated urinary protein excretion, renal dysfunction and histological damages as well as mRNA expression of kidney injury markers induced by aldosterone/salt [urinary protein (mg/day): NS, 10 ± 3; Ald + HS, 381 ± 61; Ami, 161 ± 22.]. Conclusions: Our findings indicate that pleiotropic effects of amiloride to inhibit uPA-plasmin cascade may be beneficial for the treatment of aldosterone-associated renal injuries.

RENALASE PROTECTS AGAINST SALT-INDUCED KIDNEY INJURY AND INFLAMMATION BY INHIBITING THE ACTIVATION OF THE AKT-1 SIGNALING PATHWAYS

Objective: An excess of dietary salt is the most common environmental factor that contributes to the development of hypertension leading to renal pathologies. Renalase (RNLS), a novel secreted flavoprotein with oxidoreductase activity, functions as a survival factor and promotes cell and organ survival. Previous evidence showed that recombinant renalase exerts powerful hypotensive effects and ameliorates renal interstitial fibrosis in rodents. In this study, we evaluated the potential protective role of renalase and underlying mechanisms in salt-induced kidney injury and inflammation. Design and method: In vitro, human proximal renal tubular epithelial (HK-2) cells were treated with normal salt or high salt (40 mM NaCl) for 24 hours, receiving recombinant renalase (25ug/mL) or vehicle. In vivo, Dahl salt-sensitive (DS) rats and renalase knockout mice were placed on normal (0.8% NaCl) or high salt (8% NaCl) diet with or without renalase (1 mg/kg) for 4 weeks. Results: In vitro, renalase decreased the phosphorylation levels of protein kinase B (Akt-1), leading to a reduction in the mRNA expression of pro-inflammatory cytokines and oxidative stress induced by high salt. And when knockdown the channel plasma membrane calcium ATPase (PMCA4b) in HK-2 cells, the inflammation and oxidative stress levels induced by high-salt decreased. In DS rats, high salt diet significantly increased the blood pressure and the 24 h urinary sodium excretion. Furthermore, renal inflammation and fibrosis of high salt group were more severe compared to normal salt diet group. Administration of recombinant human renalase reduced kidney inflammation through inhibition of Akt-1 pathway. Similarly, genetic depletion of renalase in mice (renalase knockout) exacerbated kidney injury in animals with high salt diet. Treatment with recombinant renalase significantly decreased the kidney inflammation through inhibition Akt-1 expression. Conclusions: Our study provides the first evidence that renalase can ameliorate kidney injury and inflammation through inhibition of the Akt-1 pathway. These results suggest that renalase has potential renoprotective effects in renal injury and inflammation and may be an effective agent for slowing kidney damage.

DIURNAL RHYTHM AND SALT RESPONSIVE RESHAPING OF GUT MICROBIOTA CORRELATES WITH HYPERTENSION

Objective: Aberrant diurnal rhythms of Blood Pressure (BP) are well known to be associated with hypertension, however, whether rhythmicity of microbiotal composition is associated with salt sensitive hypertension remains unknown. We hypothesized that BP and gut microbiotal composition follow synchronous rhythms in high salt fed Dahl Salt Salt-Sensitive (S) rats and contribute to the progression of hypertension. Design and method: To test this hypothesis, we examined groups of Dahl S rats for their diurnal rhythms of both the gut microbiota and blood pressure in low salt (0.3%) and high salt (2%) conditions. Results: In high salt fed animals, Firmicutes/Bacteroidetes (F/B) ratio significantly changed between the dark (active) and light (rest) phases, which correlated with the diurnal rhythmicity of BP suggesting salt mediated rhythmic regulation of microbiota. Diurnal rhythms of Firmicutes, Bacteroidetes and Actinobacteria were independently associated with BP and significant in high salt fed animals. Discrete genera were observed to correlate independently or interactively with one or more of the following 3 factors- 1) BP rhythm, 2) dietary salt, 3) amplitude of BP. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUST) analysis revealed diurnal rhythmicity of microbiotal pathways. During the active phase of the host, microbiota upregulated biosynthetic processes whereas during the resting phase of the host, microbiota upregulated degradation pathways of metabolites. These diurnal changes in microbiota, their functional pathways and BP response were prominently associated with a concerted rhythmicity of renal Lipocalin 2 and Endothelin1. Conclusions: Collectively, these data demonstrated the existence of synchronous diurnal rhythms of BP and renal inflammation with diurnal reshaping of gut microbiota in salt-sensitive hypertension. Such a concerted rhythmicity with peaks observed at the mid-active phase suggests that targeting this timepoint to reshape microbiota and/or intervene with medication could benefit hypertensives.

INTROGRESSION OF A BROWN NORWAY CHROMOSOME 2 FRAGMENT INTO DAHL SALT-SENSITIVE RATS EXERTS ANTI- AND PRO-INFLAMMATORY EFFECTS UNDER A NORMAL AND HIGH-SALT DIET, RESPECTIVELY

Objective: Chromosome 2 (Chr2) introgression from normotensive Brown Norway (BN) rats into hypertensive Dahl salt-sensitive (SS) background (SB2) reduced blood pressure (BP) and vascular inflammation under normal-salt diet (NSD). We hypothesized that BN Chr2 contains anti-inflammatory genes that could reduce BP elevation and vascular inflammation in rats fed NSD and high-salt diet (HSD). Design and method: Four- to 6-week-old male SS and congenic rats containing the BN Chr2 distal (SB2a) and middle segment (SB2b) were fed NSD or HSD (4% NaCl) for 8 weeks. BP was determined by telemetry, reactive oxygen species generation using dihydroethidium staining, and vascular cell adhesion molecule 1 and monocyte chemoattractant protein-1 expression and monocyte/macrophage infiltration by immunofluorescence in aorta or perivascular fat. RNA was extracted from aorta and used for small and total RNA-sequencing. Differentially expressed genes (DEGs) were identified with fold change &gt; 1.3 and fold discovery rate &lt; 0.05 and some of them were validated with reverse transcription-quantitative PCR (RT-qPCR). Results: SS Systolic BP was 145 ± 2 mm Hg under NSD and 168 ± 1 mm Hg under HSD, which was lower in SB2a and SB2b SBP (125 ± 3 and 127 ± 6, P &lt; 0.05) under NSD but similar under HSD. Examination of inflammation markers revealed that SB2a presented less and more inflammation under NSD and HSD, respectively, compared to SS. DEGs were identified in SB2a vs. SS uniquely under NSD (27) and HSD (608) and under both diet (7), and in SB2b vs. SS uniquely under NSD (133) and HSD (9) and under both diet (12). DEGs encoded within BN Chr2a were identified uniquely under NSD (2) and HSD (8), and in both diets (3). RNA-sequencing and RT-qPCR validation identified two DEGs encoded within BN Chr2 distal segment that could play a role in the vascular inflammation. These were down-regulated glutamyl aminopeptidase (Enpep) that was anti-inflammatory under NSD and up-regulated heparan sulfate 2-O-sulfotransferase 1 (Hs2st1) that was pro-inflammatory under HSD. Conclusions: Two DEGs encoded within introgressed BN Chr2 distal segment were identified: Enpep associated with decreased vascular inflammation under NSD, and Hs2st1, associated with elevated vascular inflammation under HSD.

IDENTIFICATION OF CANDIDATE BIOMARKERS FOR SALT SENSITIVITY OF BLOOD PRESSURE BY INTEGRATED BIOINFORMATICS ANALYSIS

Objective: In the current study, we aimed to identify potential biomarkers for salt sensitivity of blood pressure (SSBP), which may provide a novel insight into the pathogenic mechanisms of salt-sensitive hypertension. Firstly, we conducted weighted gene coexpression network analysis (WGCNA) and selected a gene module and 60 hub genes significantly correlated to SSBP. Then, GO function and KEGG signaling pathway enrichment analysis and protein-protein interaction (PPI) network analysis were performed. Furthermore, we identified a five-gene signature with high connectivity degree in the PPI network and high AUC of ROC curves, which may have high diagnosis value for SSBP. Moreover, through combining two gene screening methods, we identified 23 differentially expressed circRNAs and selected the top 5% circRNAs (1 circRNA) with the highest connectivity degree in the coexpression network as hub circRNA highly associated with SSBP. Finally, we carried out RT-qPCR to validate the expression of five hub genes, and our results showed that the expression of HECTD1 (P = 0.017), SRSF5 (P = 0.003), SRSF1 (P = 0.006), HERC2 (P = 0.004), and TNPO1 (P = 0.002) was significantly upregulated in the renal tissue in salt-sensitive rats compared to salt-resistant rats, indicating that these five hub genes can serve as potential biomarkers for SSBP.

Hydrogen peroxide (H2O2) mediated activation of mTORC2 increases intracellular Na+ concentration in the renal medullary thick ascending limb of Henle

Hydrogen peroxide (H2O2) production in the renal outer medulla is an important determinant of renal medullary blood flow and blood pressure (BP) salt-sensitivity in Dahl salt-sensitive (SS) rats. The mechanisms and pathways responsible for these actions are poorly understood. Recently, we have discovered that the mTOR complex 2 (mTORC2) plays a critical role in BP salt-sensitivity of SS rats by regulating Na+ homeostasis. PP242, an inhibitor of mTORC1/2 pathways exhibits potent natriuretic actions and completely prevented salt-induced hypertension in SS rats. In the present study, we have found that chronic infusion of H2O2 into the single remaining kidney of Sprague Dawley (SD) rats (3 days) stimulated the functional marker (pAKTSer473/AKT) of mTORC2 activity measured by Western Blot analysis. No changes in mTORC1 activity in OM were observed as determined by pS6Ser235/236/S6. Using fluorescent microscopy and the Na+ sensitive dye Sodium Green, we have shown that H2O2 (100 µM added in the bath) increased intracellular sodium concentration ([Na+]i) in renal medullary thick ascending limbs (mTALs) isolated from SD rats. These responses were almost completely abolished by pretreatment of mTAL with 10 µM PP242, indicating that mTORC1/2 pathways were involved in the H2O2 induced increase of [Na+]i. mTAL cell volume remained unchanged (± 1%) by H2O2 as determined by 3D reconstruction confocal laser scanning microscopy techniques. Consistent with the microscopy data, Western Blot analysis of proteins obtained from freshly isolated mTAL treated with 100 µM H2O2 exhibited increased activity/phosphorylation of AKT (pAKTSer473/AKT) that was inhibited by PP242. This was associated with increased protein activity of the apical membrane cotransporter Na+-K+-2Cl− (NKCC2) and the Na/H exchanger (NHE-3). Na+-K+-ATPase activity was increased as reflected an increase in the ratio of pNa+-K+-ATPaseSer16 to total Na+-K+-ATPase. Overall, the results indicate that H2O2 mediated activation of mTORC2 plays a key role in transducing the observed increases of cytosolic [Na+]i despite associated increases of basolateral pump activity.

Transfer RNA Fragments in the Kidney in Hypertension.

[Figure: see text].

Associations of plasma PAPP-A2 and genetic variations with salt sensitivity, blood pressure changes and hypertension incidence in Chinese adults.

Pregnancy-associated plasma protein-A2 (PAPP-A2) is the homolog of PAPP-A in the vertebrate genome and its role in protecting against salt-induced hypertension in salt-sensitive rats has been confirmed. We sought to examine the associations of plasma PAPP-A2 levels and its genetic variants with salt sensitivity, blood pressure (BP) changes and hypertension incidence in humans. Eighty participants (18-65 years old) sequentially consuming a usual diet, a 7-day low-salt diet (3.0 g/day) and a 7-day high-salt diet (18 g/day). In addition, we studied participants of the original Baoji Salt-Sensitive Study, recruited from 124 families in Northern China in 2004 who received the same salt intake intervention, and evaluated them for the development of hypertension over 14 years. The plasma PAPPA2 levels significantly decreased with the change from baseline to a low-salt diet and decreased further when converting from the low-salt to high-salt diet. SNP rs12042763 in the PAPP-A2 gene was significantly associated with systolic BP responses to both low-salt and high-salt diet while SNP rs2861813 showed a significant association with the changes in SBP and pulse pressure at 14-year follow-up. Additionally, SNPs rs2294654 and rs718067 demonstrated a significant association with the incidence of hypertension over the 14-year follow-up. Finally, the gene-based analysis found that Pappa2 was significantly associated with longitudinal SBP changes and the incidence of hypertension over the 14-year follow-up. This study shows that dietary salt intake affects plasma PAPP-A2 levels and that PAPP-A2 may play a role in salt sensitivity, BP progression and development of hypertension in the Chinese populations.

FPR-1 (Formyl Peptide Receptor-1) Activation Promotes Spontaneous, Premature Hypertension in Dahl Salt-Sensitive Rats.

[Figure: see text].