Dahl Salt-sensitive Rats Research Articles

Effect of SGLT2 Inhibition on the Development of Salt‐sensitive Hypertension, RAAS, and Sodium Channels and Transporters in Dahl SS Rats

Hypertension is common among patients with type 2 diabetes mellitus (T2DM). The combination of diabetes and hypertension is associated with high morbidity and mortality because of an increased risk of cardiovascular and renal complications. Effective control of blood glucose levels and blood pressure is essential to prevent the development and progression of diabetic nephropathy in T2DM. Sodium‐Glucose Cotransporter‐2 (SGLT2) inhibitors are the new highly effective drugs for the treatment of T2DM and some cardiovascular diseases. Despite all the remarkable benefits on cardiovascular and renal systems, the molecular mechanisms of how SGLT2 inhibition affects water and electrolyte balance are incompletely understood. We hypothesized that SGLT2 inhibition alters blood pressure via affecting Renin‐Angiotensin‐Aldosterone System (RAAS), sodium channels and transporters. Dahl Salt‐Sensitive (SS) rat, a model for salt‐induced hypertension, was used to test this hypothesis in a hypertensive, non‐diabetic environment. We found that administration of dapagliflozin at a concentration of 2 mg/kg/day via drinking water during 3 weeks blunts the development of salt‐induced hypertension with no changes in the heart rate. Mean arterial pressure in the dapagliflozin treated group compared to the vehicle‐treated group during high salt (4% NaCl) challenge was significantly lower (136 ± 4 vs. 157 ± 6 mmHg; P<0.0001 on 21st day of the experiment). SGLT2 inhibition caused a significant diuretic effect (67 ± 6 vs. 40 ± 4 ml/day, P<0.001 on the 21st day of the experiment). Glucose excretion was dramatically increased in inhibitor‐treated rats compared to vehicle‐treated (747 ± 44 vs. 12 ± 2 mg/dL, P<0.0001 on the 21st day). We also observed increased Na+/creatinine ratios (138 ± 10 vs. 110 ± 6, P<0.004 on the 21st day) and Cl‐/creatinine ratios (133 ± 9 vs. 108 ± 6, P<0.03 on the 21st day). To define potential mechanisms, we first measured levels of hormones involved in the RAAS by using LC‐MS/MS. Despite effects on Na+/creatinine and Cl‐/creatinine ratios, dapagliflozin treatment did not affect RAAS metabolites. Subsequently, we assessed the effects of SGLT2 inhibition on renal Na+ channels and transporters using patch‐clamp, Western blotting, and RT‐PCR. Neither mRNA nor protein expressions levels of all tested Na+ transporters (SGLT2, SGLT1, NHE3, NKCC2, NCC, and α‐, β‐, γ‐ ENaC subunits) were different between groups. Electrophysiological studies did not reveal any difference of SGLT2 inhibition on the conductance and activity of ENaC. Therefore, our data suggest that SGLT2 inhibition blunts the development of salt‐induced hypertension, causes glucosuria and natriuresis but does not affect RAAS and expression and activity of Na+ channels and transporters in a model of non‐diabetic, salt‐sensitive hypertension.

Beneficial effects of AT1 antagonists treatment, losartan, on cardiovascular and cognitive function on aged male Dahl salt sensitive rats

Introduction and objectiveCardiovascular diseases and hypertension are the major contributors to the pathogenesis of vascular dementia. The Dahl salt‐sensitive (DSS) rat model is characterized by age‐associated cardiovascular remodeling and the development of central arterial stiffness (CAS) in the presence of renin‐angiotensin system disbalance, accompanied by cognitive decline. We hypothesized, that the anti‐hypertensive treatment, antagonist for angiotensin II receptors losartan (LOS), via reducing CAS, will improve cardiovascular parameters and cognitive function in aged DSS male rats.MethodsMale DSS rats (n=30) were kept on a normal salt diet (0.5% NaCl) for the duration of the study. At 6 months of age, rats were given either LOS in drinking water (30mg/kg/day, n=14) or control treatment (n=16) for 6 months. Measurements, including systolic and diastolic BP (SBP, DBP) and pulse wave velocity (PWV), a measure of CAS (by echocardiology), were taken after 6 months of LOS treatment. Rats were tested in an operant chambers (OCh) attention challenge to assess attention and impulsivity; elevated plus maze (EPM) was performed to estimate anxiety‐like behavior. Statistical analyses were performed using 1‐way ANOVA, t‐test and Pearson correlation. Data are presented as mean ± SEM.Resultsfollowing 6 months of treatment, at 12‐mo of age, LOS treated rats had lower BP and PWV vs. control (Table 1). Performance on the OCh indicated that LOS rats had more correct responses vs. control rats (Fig. 1). The LOS group exhibited lower anxiety levels, because they spent less time on the closed shoulder in EPM (Table 1) and demonstrate 1.5‐fold increase of time in the open shoulder (p < 0.01). PWV and SBP was negatively correlated with time on open shoulder, total distance traveled and total correct answers at 12‐mo of age (Fig.2).ConclusionAged male DSS rats show beneficial effects of LOS on cardiovascular and cognitive function. The CAS and BP improvement by LOS was associated with improved attention and lower anxiety levels. The mechanistic basis of LOS effects on cognition via cerebrovascular and brain changes will be further investigated.

What Doesn't Kill You Makes You Stronger—Even Heart Radiation?

Radiation therapy (RT) is a commonly used treatment for thoracic cancers. However, the risk of coronary events increase by 4‐16% per Gy of heart dose. Our prior studies show that localized heart RT leads to radiation‐induced heart disease (RIHD) and increased mortality in Dahl Salt Sensitive (SS) rats starting at 3mo post‐RT. We have also shown that whole thoracic RT increases susceptibility to ischemia‐reperfusion (IR) injury in Wistar rats. Taken together, we hypothesized that 10weeks after targeted cardiac RT (prior to RIHD) SS rat hearts would have worsened cardiac function as measured by echocardiography and be more susceptible to IR injury. Echocardiograms were performed 10weeks after 24Gy dose of CT‐guided localized cardiac RT and in age‐matched controls (C). Next, hearts were isolated and perfused ex vivo using the Langendorf method ‐ without ischemia reperfusion (time controls; TC; n=3 in C and RT) or exposed to 25min of global ischemia followed by 60min reperfusion (IR; n=6 in C and RT). During TC and IR, cardiac function, mitochondrial redox state, and vascular reactivity were assessed; the hearts were then sectioned and stained to assess infarct size. Echocardiography of the RT group when compared to C showed an increase in interventricular septal thickness (1.32±0.1 vs 0.78±0.07 cm, p<0.0001; mean±SD) and left ventricular posterior wall thickness (1.03±0.08 vs 0.75±0.06 cm, p<0.005). Accordingly, a decrease in end diastolic (1.79±0.46 vs 2.38±0.35 ml/kg, p<0.05) and end systolic (0.10±0.04 vs 0.23±0.08 ml/kg, p<0.05) volume was also seen, with no significant differences in ejection fraction (94.5±2.1 vs 90.3±3.9 %) or stroke volume (1.69±0.44 vs 2.15±0.27 ml; p>0.05). Interestingly, in the ex vivo perfused hearts, TC RT had a 2 times higher rate‐pressure product – a measure of cardiac work – compared to C (p<0.05), and following IR, the RT group recovered to 2.5x higher levels to C (p<0.0001). Following IR, the RT group compared to C also showed a greater recovery of coronary flow rate (1.6x, p<0.05) and rates of contractility (2.25x) and relaxation (lusitropy; 2.05x) (p<0.0001). No difference in mitochondrial redox state, infarct size, and endothelium‐dependent and independent vascular reactivity were seen between the groups. In conclusion, our results show for the first time that 10weeks post‐targeted cardiac RT, ex vivo hearts show better recovery and cardiac function compared to C after IR through mechanisms other than differences in redox state and vascular reactivity.

Beyond the Gastrointestinal Tract: Sex‐Specific Skin Microbiota Are Associated With Hypertension in Rats With Genetic Disparities

Current knowledge of microbiota in hypertension is restricted to the gut and mostly to males. Other than the kidney, the skin is the largest organ playing a role in sodium homeostasis and the mouth is the first site exposed to dietary salt, whereby, we hypothesized that microbiota in these sites associate with salt‐sensitive blood pressure (BP). To test this hypothesis, we compared microbiota from the skin, mouth and feces of both male and female young Dahl salt‐sensitive (S) rats and 4 genetically disparate congenic strains (RNO1, RNO5, RNO9 and RNO10). Skin microbiota was from 4‐6 days old rats before they developed their coats. Oral and fecal samples were collected at 4 weeks old. Microbiota were profiled by 16S rRNA sequencing. Compared to the gut, which is an anerobic site, microbiota from the aerobic skin and oral sites were consistently different in composition across all the strains. Unlike in the fecal samples where Firmicutes and Bacteroidetes were enriched, Cyanobacteria and Actinobacteria were enriched in the skin and oral samples, respectively. Female S rats had greater abundance of skin Cyanobacteria than male S rats. Strain effects were not observed in any of the strains or sites except for RNO5, where a strain × sex × site interactive effect was specifically noted for skin Cyanobacteria. Cyanobacteria contain sodium transporters and are salt‐tolerant microbiota, whereby they could potentially regulate sodium excretion and contribute to BP regulation. In summary, our study is the first to discover microbiota beyond the gut as being associated with BP.

Contribution of Adenosine A2A Receptors in the Rostral Ventrolateral Medulla to Acupuncture Modulation of Hypertension

Acupuncture is used to manage high blood pressure (BP) in clinical practice. However, the mechanisms underlying its hypotensive effects remain unclear. Our previous studies have shown that electroacupuncture (EA) at the ST36‐37 acupoints, overlying the deep peroneal nerve, attenuates pressor responses through adenosine A2A receptors (A2AR) in the rostral ventrolateral medulla (rVLM). A2AR activation in the rVLM is associated with the upregulation of nitric oxide synthase (NOS) that contributes to sympathoinhibition and BP reduction. However, it is uncertain whether rVLM A2AR contributes to EA’s action in lowering high BP in sustained hypertension (HTN) in the rVLM. We hypothesized that EA lowers BP in HTN through adenosine A2AR mechanisms associated with the upregulation of neuronal NOS (nNOS) in the rVLM. The nNOS derived NO has been shown to exert central effects on BP. To mimic essential HTN in the clinic, we performed EA in conscious Dahl salt‐sensitive hypertensive rat (DSHR). EA (0.1‐0.4 mA, 2 Hz) was applied at ST36‐37 for 30 min twice weekly for four weeks while sham‐EA was conducted with the same procedures as EA except for no electrical stimulation. Elevated BP was reduced by EA (n=10) but not sham‐EA (n=8) and untreated ones (n=6). The A2AR level in the rVLM after EA treatment was increased (n=5; P < 0.05) compared to the sham‐EA (n=5). EA’s inhibitory effect on elevated BP was reversed transiently by unilateral microinjection of SCH 58261 (1 mM in 50 nl; an A2AR antagonist; n=7; P < 0.05) but not the vehicle (n=5) into the rVLM in EA‐treated DSHRs under anesthesia. Activation of rVLM A2AR in DSHRs treated with sham‐EA using an A2AR agonist, CGS‐21680 (0.4 mM in 50 nl; n=6), decreased BP. Furthermore, we found a co‐localization of A2AR with nNOS in the rVLM of DSHRs. The expression of rVLM nNOS was enhanced in DSHRs treated with EA compared to those treated with sham‐EA (n=5 in each group; P < 0.05). These data suggest that adenosine‐ A2AR in the rVLM contributes to EA’s inhibitory effect on elevated BP in sustained HTN, which is likely relevant to the upregulation of nNOS.

Effects of Potassium Supplementation and Kir7.1 Knockout on Renal Function During the Progression of Salt‐Sensitive Hypertension

The inward‐rectifying potassium channel (Kir) family has been shown to play an important role in the kidney’s ability to maintain electrolyte homeostasis as well as blood pressure. Kir7.1 (encoded by the Kcnj13 gene) is expressed in the kidney, though its role in renal function is rudimentary. To examine the role of Kir7.1 in renal electrolyte handling and the development of salt‐induced hypertension, we generated a knockout of Kcnj13 on the Dahl salt‐sensitive (SS) rat background. Rats with the knockout of Kcnj13 were embryonically lethal; therefore, to study the Kir7.1 channel, we used heterozygous (SSKcnj13+/‐) rats. Immunohistochemistry staining for Kir7.1 revealed basolateral staining in the cortical sections of the kidneys in wild‐type littermates (WT), while SSKcnj13+/‐ rats showed little to no staining. In addition, immunofluorescence analysis confirmed that Kir7.1 was localized on the basolateral side in tubules and revealed that Kir7.1 co‐localized with aquaporin 2 and sodium‐chloride cotransporter (NCC). This data indicates that Kir7.1 is expressed in the cortical collecting duct (CCD) and the distal convoluted tubule (DCT). SSKcnj13+/‐ rats were further used to measure blood pressure and electrolyte handling changes during the development of salt‐induced hypertension. DSI telemeters were implanted in animals at 8 weeks of age; after a week to recover from surgery, animals were fed a high salt 4% NaCl diet (HS) for three weeks. Both WT and SSKcnj13+/‐rats become hypertensive after three weeks of HS; no difference between mean arterial pressures (153.1 ± 5.7 vs. 159.2 ± 6.9 mmHg for WT and SSKcnj13+/‐rats, respectively) was identified. Serum electrolytes were measured and also showed no difference in the amount of potassium (3.4 ± 0.1 vs. 3.5 ± 0.1 mmol/L) or sodium (142 ± 0.8 vs. 142 ± 0.4 mmol/L). Furthermore, urinary electrolyte excretion was measured and revealed no differences between WT and SSKcnj13+/‐ animals for potassium (14.0 ± 0.6 vs. 15.6 ± 1.9 K+/Cre) or sodium excretion (91 ± 11 vs. 117 ± 16 Na+/Cre). Potassium supplementation has previously been shown to augment renal Kir7.1 expression. Thus, we repeated the previous protocol but instead using a high salt and high potassium 4% NaCl and 2% KCl (HSHK) diet. After three weeks on this diet, animals exhibited no difference in blood pressure (154.2 ± 9.1 vs 145.6 ± 4.1 mmHg) and no differences in plasma potassium (4.0 ± 0.1 vs 4.0 ± 0.1 mmol/L) or sodium (139 ± 1 vs 139 ± 1 mmol/L). Urinary electrolyte excretion showed no differences in potassium excretion (60.9 ± 3.3 vs 54.8 ± 1.1 K+/Cre), but SSKcnj13+/‐ animals did have a significant decrease in sodium excretion (130 ± 8 vs 103 ± 3 Na+/Cre). Altogether, we have confirmed the presence of Kir7.1 in the CCD and DCT, found that heterozygous knockout of Kir7.1 does not affect the development of blood pressure on a HS or HSHK diet. However, we do see a reduction in sodium excretion in SSKcnj13+/‐rats on the HSHK diet, suggesting that Kir7.1’s role may be more pronounced under conditions of higher potassium.

Lack of Xdh Leads to Alterations in Renin‐Angiotensin‐Aldosterone System and Kidney Injury

Uric acid (UA) homeostasis has been elucidated as a key player in chronic kidney disease and cardiovascular diseases, yet definitive mechanisms are unknown. Previous studies in rodents and humans have shown that increased levels of serum UA predict an activated intrarenal Renin‐Angiotensin‐Aldosterone System (RAAS). In hyperuricemic rats, nephropathy was reported with glomerular hypertrophy and interstitial inflammation accompanied by increased renal renin expression. To explore whether hypouricemia leads to RAAS changes and kidney damage, we utilized a novel rat model with genetic ablation of the Xdh gene in the Dahl salt‐sensitive rat background (SSXdh‐/‐). The Xdh gene encodes for the Xanthine Oxidoreductase enzyme group, which produces UA from xanthine and hypoxanthine. We have shown that the SSXdh‐/‐model is hypouricemic and has severe kidney damage. The SSXdh‐/‐ rats demonstrated significant kidney function decline (SSXdh+/+& SSXdh‐/‐respectively, diuresis: 2.7 ± 0.9 & 14.4 ± 5.1 ml; creatinine clearance: 0.51 ± 0.19 & 0.12 ± 0.04 ml/min; and plasma Na+: 136 ± 1.8 & 150 ± 3.7 mmol/l). Quantification of RAAS components was done using liquid chromatography‐tandem mass spectrometry. The comparison of plasma from SSXdh+/+vs SSXdh‐/‐rats revealed significantly lower levels of angiotensin I (650 ± 109, 178 ± 25 pmol/l), Ang II (583 ± 70, 171 ± 16 pmol/l), and renin (indirect measurements using Ang I + Ang II: 1233 ± 172, 349 ± 40 pmol/l). Aldosterone level was increased in SSXdh‐/‐rats (870 ± 115 vs. 4106 ± 1038 pmol/l). The aldosterone/ Ang II ratio was significantly increased in the homozygous rats suggesting enhanced adrenal function (2 ± 0.1, 25 ± 6.3). To further evaluate the contribution of RAAS and its downstream signaling mechanisms, we performed RNA‐Seq analysis on kidney cortex tissue, which showed a number of RAAS‐related genes being differentially expressed between the SSXdh+/+and SSXdh‐/‐ rats. In hypouricemic rats, there was an increase in expression levels of Ace, Ccl2, Fos, Map3k1, Ptger2,and Stat3,and a decrease of Agtr1a, Hsd11b1,and Renb. Therefore, our results show that lack of Xdh/UA leads to kidney injury and functional decline with substantial remodeling in RAAS.

SK Channel Dysfunction in the Hypothalamic Paraventricular Nucleus Contributes to Sympathoexcitation in Dahl Salt‐Sensitive Rats

Salt‐sensitive hypertension is characterized by marked activation of the sympathetic nervous system. Previous studies have consistently demonstrated that increased excitability or activity of pre‐sympathetic neurons in the hypothalamic paraventricular nucleus (PVN) contribute to the increased blood pressure and sympathetic nerve activity (SNA) in salt‐sensitive hypertension; however, the underlying mechanisms have not been fully elucidated. Small conductance calcium‐activated potassium (SK) channels act as negative feedback regulators of neuronal excitability in PVN neurons, and inhibition of SK channel function in the PVN is known to augment SNA and arterial blood pressure. In the present study, we tested the hypothesis that reduced SK channel function in the PVN contributes to sympathoexcitation in Dahl salt‐sensitive rats fed a high salt (HS, 4%) diet compared to a normal salt (NS, 0.4%) diet. Sympathoexcitatory responses to bilateral microinjection of the SK channel blocker apamin into the PVN were assessed in anesthetized 12–14‐week‐old male Dahl salt‐sensitive rats fed a high salt (n=7) or a normal salt (n=6) diet for 4 weeks. Baseline mean arterial pressure (MAP) was significantly elevated in rats fed a HS diet compared to NS (HS 129±4 vs. NS 114±4 mmHg, p<0.05), however baseline heart rate (HR) was not different between groups (HS 411±10 vs. NS 385±13 beats/min, p=0.07). Bilateral microinjection of apamin (12.5pmol, 50nL) into the PVN significantly increased splanchnic SNA in both HS and NS rats compared to baseline, however responses were significantly attenuated in HS rats compared to NS (HS 97±22% vs. NS 249±57%, p<0.01). In contrast, increases in MAP in response to PVN microinjection of apamin were similar between HS and NS rats (HS 24±4 vs. NS 29±4 mmHg, p=0.25), whereas HR responses were variable but not significantly different between groups (HS ‐8.5±9 vs. NS 7.5±7 beats/min, p=0.11). Next, we utilized qPCR to assess mRNA expression of SK1, SK2, and SK3 channels in PVN tissue from HS (n=5) and NS (n=6) rats. Interestingly, there was no significant difference in mRNA levels of either SK1, SK2, or SK3 channels between HS and NS fed rats. In summary, increases in splanchnic SNA due to PVN SK channel blockade with apamin were significantly attenuated in Dahl salt‐sensitive rats fed a HS diet, whereas there was no difference in SK channel expression between rats fed a HS vs. NS diet. These results suggest that dysfunction of SK channels in the PVN, but not decreased expression, contributes to sympathoexcitation Dahl salt‐sensitive rats fed a HS diet.

Assessment of Mitochondrial Respiratory Function in Isolated Nephron Segments of Dahl SS Rats in Salt Induced Hypertension

RationaleKidney nephrons actively reabsorb 99% of the filtrate to maintain body fluid and solute homeostasis. A large amount of ATP synthesis and O2 consumption must work coordinal to support this demand. We hypothesized that mitochondrial function of proximal tubules (PT) and medullary thick ascending limbs (mTAL) is altered during the development of salt induced hypertension in the Dahl salt‐sensitive (SS) rat.MethodThe mitochondrial oxygen consumption rates (OCR) were determined in freshly bulk isolated PT and mTAL segments from SS rats fed 0.4% NaCl diet (low salt; LS) or 4% NaCl diet (high salt; HS) for 7 days. A protocol was developed to determine basal levels of OCR and the effects of various key circulating substrates (glucose, lactate, pyruvate, glutamine, and palmitate) upon mitochondrial respiration (OCR) using the Agilent Seahorse XF96 Extracellular Flux Analyzer combined with the Agilent Seahorse XF Cell Mito Stress Test protocol.ResultsAnalysis of the renal tubular mitochondrial OCR data for five different circulating substrates confirmed that there were clear differences in the substrate preference between the PT and mTAL which were altered by HS feeding. It was found that PT preferred lactate and glutamine while mTAL preferred glucose and pyruvate and that both segments could effectively utilize palmitate as a mitochondrial substrate. PT isolated from SS rats fed HS displayed higher OCR levels compared to PT from LS fed rats, but HS diet did not result in significant differences in individual substrate effects. In contrast, isolated mTAL from HS fed rats exhibited increased OCR in response to glutamine and palmitate compared to mTAL from LS fed rats although no basal differences in OCR were observed.ConclusionThese observations demonstrated that renal tubular mitochondrial respiratory function is altered in SS rats during the development of salt‐induced hypertension and that both PT and mTAL exhibit an increased respiratory capacity in response to HS feeding. These changes in metabolism appear necessary to meet the increased tubular workloads and need for greater ATP production and O2 utilization in both PT and mTAL. We conclude, however, that different metabolic strategies are utilized by each of these tubular segments to meet these increased metabolic requirements.

Paraoxonase-1 Regulation of Renal Inflammation and Fibrosis in Chronic Kidney Disease.

Papraoxonase-1 (PON1) is a hydrolytic lactonase enzyme that is synthesized in the liver and circulates attached to high-density lipoproteins (HDL). Clinical studies have demonstrated an association between diminished PON-1 and the progression of chronic kidney disease (CKD). However, whether decreased PON-1 is mechanistically linked to renal injury is unknown. We tested the hypothesis that the absence of PON-1 is mechanistically linked to the progression of renal inflammation and injury in CKD. Experiments were performed on control Dahl salt-sensitive rats (SSMcwi, hereafter designated SS rats) and Pon1 knock-out rats (designated SS-Pon1em1Mcwi, hereafter designated SS-PON-1 KO rats) generated by injecting a CRISPR targeting the sequence into SSMcwi rat embryos. The resulting mutation is a 7 bp frameshift insertion in exon 4 of the PON-1 gene. First, to examine the renal protective role of PON-1 in settings of CKD, ten-week-old, age-matched male rats were maintained on a high-salt diet (8% NaCl) for up to 5 weeks to initiate the salt-sensitive hypertensive renal disease characteristic of this model. We found that SS-PON-1 KO rats demonstrated several hallmarks of increased renal injury vs. SS rats including increased renal fibrosis, sclerosis, and tubular injury. SS-PON-1 KO also demonstrated increased recruitment of immune cells in the renal interstitium, as well as increased expression of inflammatory genes compared to SS rats (all p < 0.05). SS-PON-1 KO rats also showed a significant (p < 0.05) decline in renal function and increased renal oxidative stress compared to SS rats, despite no differences in blood pressure between the two groups. These findings suggest a new role for PON-1 in regulating renal inflammation and fibrosis in the setting of chronic renal disease independent of blood pressure.

SGLT2 inhibition effect on salt-induced hypertension, RAAS, and Na+ transport in Dahl SS rats.

Na+-glucose cotransporter-2 (SGLT2) inhibitors are the new mainstay of treatment for diabetes mellitus and cardiovascular diseases. Despite the remarkable benefits, the molecular mechanisms mediating the effects of SGLT2 inhibitors on water and electrolyte balance are incompletely understood. The goal of this study was to determine whether SGLT2 inhibition alters blood pressure and kidney function via affecting the renin-angiotensin-aldosterone system (RAAS) and Na+ channels/transporters along the nephron in Dahl salt-sensitive rats, a model of salt-induced hypertension. Administration of dapagliflozin (Dapa) at 2 mg/kg/day via drinking water for 3 wk blunted the development of salt-induced hypertension as evidenced by lower blood pressure and a left shift of the pressure natriuresis curve. Urinary flow rate, glucose excretion, and Na+- and Cl--to-creatinine ratios increased in Dapa-treated compared with vehicle-treated rats. To define the contribution of the RAAS, we measured various hormones. Despite apparent effects on Na+- and Cl--to-creatinine ratios, Dapa treatment did not affect RAAS metabolites. Subsequently, we assessed the effects of Dapa on renal Na+ channels and transporters using RT-PCR, Western blot analysis, and patch clamp. Neither mRNA nor protein expression levels of renal transporters (SGLT2, Na+/H+ exchanger isoform 3, Na+-K+-2Cl- cotransporter 2, Na+-Cl- cotransporter, and α-, β-, and γ-epithelial Na+ channel subunits) changed significantly between groups. Furthermore, electrophysiological experiments did not reveal any difference in Dapa treatment on the conductance and activity of epithelial Na+ channels. Our data suggest that SGLT2 inhibition in a nondiabetic model of salt-sensitive hypertension blunts the development of salt-induced hypertension by causing glucosuria and natriuresis without changes in the RAAS or the expression or activity of the main Na+ channels and transporters.NEW & NOTEWORTHY The present study indicates that Na+-glucose cotransporter-2 (SGLT2) inhibition in a nondiabetic model of salt-sensitive hypertension blunts the development and magnitude of salt-induced hypertension. Chronic inhibition of SGLT2 increases glucose and Na+ excretion without secondary effects on the expression and function of other Na+ transporters and channels along the nephron and hormone levels in the renin-angiotensin-aldosterone system. These data provide novel insights into the effects of SGLT2 inhibitors and their potential use in hypertension.

Effect of Cardiotonic Steroid Marinobufagenin on Vascular Remodeling and Cognitive Impairment in Young Dahl-S Rats.

The hypertensive response in Dahl salt-sensitive (DSS) rats on a high-salt (HS) diet is accompanied by central arterial stiffening (CAS), a risk factor for dementia, and heightened levels of a prohypertensive and profibrotic factor, the endogenous Na/K-ATPase inhibitor marinobufagenin (MBG). We studied the effect of the in vivo administration of MBG or HS diet on blood pressure (BP), CAS, and behavioral function in young DSS rats and normotensive Sprague–Dawley rats (SD), the genetic background for DSS rats. Eight-week-old male SD and DSS rats were given an HS diet (8% NaCl, n = 18/group) or a low-salt diet (LS; 0.1% NaCl, n = 14–18/group) for 8 weeks or MBG (50 µg/kg/day, n = 15–18/group) administered via osmotic minipumps for 4 weeks in the presence of the LS diet. The MBG-treated groups received the LS diet. The systolic BP (SBP); the aortic pulse wave velocity (aPWV), a marker of CAS; MBG levels; spatial memory, measured by a water maze task; and tissue collection for the histochemical analysis were assessed at the end of the experiment. DSS-LS rats had higher SBP, higher aPWV, and poorer spatial memory than SD-LS rats. The administration of stressors HS and MBG increased aPWV, SBP, and aortic wall collagen abundance in both strains vs. their LS controls. In SD rats, HS or MBG administration did not affect heart parameters, as assessed by ECHO vs. the SD-LS control. In DSS rats, impaired whole-heart structure and function were observed after HS diet administration in DSS-HS vs. DSS-LS rats. MBG treatment did not affect the ECHO parameters in DSS-MBG vs. DSS-LS rats. The HS diet led to an increase in endogenous plasma and urine MBG levels in both SD and DSS groups. Thus, the prohypertensive and profibrotic effect of HS diet might be partially attributed to an increase in MBG. The prohypertensive and profibrotic functions of MBG were pronounced in both DSS and SD rats, although quantitative PCR revealed that different profiles of profibrotic genes in DSS and SD rats was activated after MBG or HS administration. Spatial memory was not affected by HS diet or MBG treatment in either SD or DSS rats. Impaired cognitive function was associated with higher BP, CAS, and cardiovascular remodeling in young DSS-LS rats, as compared to young SD-LS rats. MBG and HS had similar effects on the cardiovascular system and its function in DSS and SD rats, although the rate of change in SD rats was lower than in DSS rats. The absence of a cumulative effect of increased aPWV and BP on spatial memory can be explained by the cerebrovascular and brain plasticity in young rats, which help the animals to tolerate CAS elevated by HS and MBG and to counterbalance the profibrotic effect of heightened MBG.

Blockade of Microglial Activation in Hypothalamic Paraventricular Nucleus Improves High Salt-Induced Hypertension.

It has been shown that activated microglia in brain releasing proinflammatory cytokines (PICs) contribute to the progression of cardiovascular diseases. In this study, we tested the hypothesis that microglial activation in hypothalamic paraventricular nucleus (PVN), induced by high-salt diet, increases the oxidative stress via releasing PICs and promotes sympathoexcitation and development of hypertension. High-salt diet was given to male Dahl salt-sensitive rats to induce hypertension. Those rats were bilaterally implanted with cannula for PVN infusion of minocycline, a selective microglial activation blocker, or artificial cerebrospinal fluid for 4 weeks. High-salt diet elevated mean arterial pressure of Dahl salt-sensitive rats. Meanwhile, elevations of renal sympathetic nerve activity and central prostaglandin E2, as well as increase of plasma norepinephrine, were observed in those hypertensive rats. Tumor necrosis factor-α, interleukin-1β (IL-1β), and IL-6 increased in the PVN of those rats, associated with a significant activation of microglia and prominent disruption of redox balance, which was demonstrated by higher superoxide and NAD(P)H oxidase 2 (NOX-2) and NAD(P)H oxidase 4 (NOX-4), and lower Cu/Zn superoxide dismutase in PVN. PVN infusion of minocycline attenuated all hypertension-related alterations described above. This study indicates that high salt leads to microglial activation within PVN of hypertensive rats, and those activated PVN microglia release PICs and trigger the production of reactive oxygen species, which contributes to sympathoexcitation and development of hypertension. Blockade of PVN microglial activation inhibits inflammation and oxidative stress, therefore attenuating the development of hypertension induced by high-salt diet.

Acute and long-term effects of cannabinoids on hypertension and kidney injury

Cannabinoids and their endogenous and synthetic analogs impact blood pressure and contribute to the incidence of hypertension. It was previously reported that the endocannabinoid system plays an important role in developing hypertension; however, it was also shown that cannabinoids elicit profound hypotension associated with hemorrhagic, cardiogenic, and endotoxic shock. This study aimed to test acute and chronic effects of an endogenous ligand of cannabinoid receptor anandamide (AEA) on blood pressure and kidney injury in vivo in conscious Dahl salt-sensitive (SS) rats. We demonstrated that acute i.v. bolus administration of a low or a high doses (0.05 or 3 mg/kg) of AEA did not affect blood pressure for 2 h after the injection in Dahl SS rats fed a normal salt diet (0.4% NaCl). Neither low nor high doses of AEA had any beneficial effects on blood pressure or kidney function. Furthermore, hypertensive rats fed a HS diet (8% NaCl) and chronically treated with 3 mg/kg of AEA exhibited a significant increase in blood pressure accompanied by increased renal interstitial fibrosis and glomerular damage at the late stage of hypertension. Western blot analyses revealed increased expression of Smad3 protein levels in the kidney cortex in response to chronic treatment with a high AEA dose. Therefore, TGF-β1/Smad3 signaling pathway may play a crucial role in kidney injury in SS hypertension during chronic treatment with AEA. Collectively, these data indicate that prolonged stimulation of cannabinoid receptors may result in aggravation of hypertension and kidney damage.

Pial Vessel-Associated Microglia/Macrophages Increase in Female Dahl-SS/Jr Rats Independent of Pregnancy History.

As the resident immune cells of the central nervous system, microglia have a wide range of functions such as surveillance, phagocytosis, and signaling through production of chemokines and cytokines. Recent studies have identified and characterized macrophages residing at the meninges, a series of layers surrounding the brain and spinal cord. While perivascular microglia within the brain parenchyma increase following chronic hypertension, there are no reports of changes at the meninges, and specifically, associated with the pial vasculature. Thus, we used female Sprague Dawley and Dahl salt-sensitive (SS/Jr) rat brains, stained for ionized calcium-binding adapter molecule (Iba1), and characterized microglia/macrophages associated with pial vessels in the posterior brain. Results indicate that Iba1+ pial vessel-associated microglia (PVAM) completely surrounded the vessels in brains from the Dahl-SS/Jr rats. PVAM density was significantly higher and distance between PVAMs lower in Dahl-SS/Jr compared to the Sprague Dawley rat brains. Pregnancy history did not affect these findings. While the functional role of these cells are not known, we contextualize our novel findings with that of other studies assessing or characterizing myeloid cells at the borders of the CNS (meninges and choroid plexus) and perivascular macrophages and propose their possible origin in the Dahl-SS/Jr model of chronic hypertension.

High Salt Upregulates Ca2+-Sensing Receptor Expression and Ca2+-Induced Relaxation of Contracted Mesenteric Arteries from Dahl Salt-Sensitive Rats.

High Ca2+ lowers blood pressure in hypertension, but the mechanism is not clear. The missing link may be the perivascular sensory nerve Ca2+-sensing receptor (CaSR) that mediates a vasodilator system after activation by interstitial Ca2+ Our results show that high salt increased CaSR expression in mesenteric arteries as well as Ca2+ relaxation of contracted mesenteric arteries from salt-sensitive (SS) rats. The CaSR was expressed as a doublet (≈120-150 kDa) in arteries from animals fed a high-salt diet for 1-4 weeks. The higher molecular weight glycosylated protein increased in arteries from SS animals; however, expression of the low molecular mass high-mannose protein decreased over 4 weeks of feeding the diet. In tissues from salt-resistant (SR) rats, the diet decreased CaSR expression after 4 weeks. Ca2+ relaxation of mesenteric arteries under phenylephrine tone increased in SS rats but decreased in arteries from SR rats fed the high-salt diet. Ca2+-activated K+ channels have a larger role in Ca2+ relaxation of arteries in SR than SS rats. The data suggest that high salt epigenetically regulates the receptor at the translational level in vivo and that the in vitro effect of Ca2+ is on receptor trafficking and signaling. In conclusion, upregulated expression of the CaSR in salt sensitivity increased receptor-mediated vascular relaxation. These findings show that CaSR signaling may compensate for changes in the vasculature in salt-sensitive hypertension. SIGNIFICANCE STATEMENT: The perivascular sensory nerve Ca2+-sensing receptor (CaSR) mediates Ca2+ relaxation of isolated mesenteric arteries under tension. This receptor may therefore play a significant role in relaxation of resistance arteries in vivo, thus explaining the blood pressure-lowering effect of dietary Ca2+. The present studies describe the effect of high salt-induced upregulation of the CaSR in salt-sensitive rats and the roles played by Ca2+-activated K+ channels and nitric oxide in Ca2+ responses.

Cardiorenal protective effects of sodium-glucose cotransporter 2 inhibition in combination with angiotensin II type 1 receptor blockade in salt-sensitive Dahl rats.

The kidney plays a central role in regulating the salt sensitivity of blood pressure (BP) by governing sodium excretion and reabsorption via renal sodium transporters. We hypothesized that sodium-glucose cotransporter 2 (SGLT2) inhibition and angiotensin II type 1 receptor (AT1R) blockade can synergistically reduce renal sodium reabsorption by beneficially effects on these transporters, leading to lower BP and ameliorating renal and cardiac damage. Dahl salt-sensitive rats were treated orally for 8weeks with a normal salt diet (0.3% NaCl), a high-salt diet (8% NaCl), high-salt diet with ipragliflozin (0.04%), high-salt diet with losartan (0.05%) or high-salt diet with a combination of ipragliflozin and losartan. The combination treatment significantly reduced BP and increased daily urine sodium excretion compared with losartan or ipragliflozin monotherapy, leading to greater improvement in BP salt sensitivity than ipragliflozin monotherapy. The combination treatment significantly ameliorated glomerulosclerosis and reduced cardiomyocyte hypertrophy compared with losartan or ipragliflozin monotherapy. The protein expression levels of Na+/H+ exchanger isoform 3 (NHE3) and Na+-K+-CI- cotransporter 2 (NKCC2) in the kidney were significantly decreased with losartan monotherapy and combination treatment, but not with ipragliflozin monotherapy. Inhibition of SGLT2 in combination with an angiotensin II receptor blocker effectively improved BP salt sensitivity by reducing renal expression levels of sodium transporters including NHE3 and NKCC2, which eventually led to improvement of BP salt sensitivity and cardiorenal protection.

An Effective Sodium-Dependent Glucose Transporter 2 Inhibition, Canagliflozin, Prevents Development of Hypertensive Heart Failure in Dahl Salt-Sensitive Rats.

Background: The aim of the study was to investigate the protective effect of canagliflozin (CANA) on myocardial metabolism and heart under stress overload and to further explore its possible molecular mechanism. Methods: High-salt diet was used to induce heart failure with preserved ejection fraction (HFpEF), and then, the physical and physiological indicators were measured. The cardiac function was evaluated by echocardiography and related indicators. Masson trichrome staining, wheat germ agglutinin, and immunohistochemical staining were conducted for histology analysis. Meanwhile, oxidative stress and cardiac ATP production were also determined. PCR and Western blotting were used for quantitative detection of related genes and proteins. Comprehensive metabolomics and proteomics were employed for metabolic analysis and protein expression analysis. Results: In this study, CANA showed diuretic, hypotensive, weight loss, and increased intake of food and water. Dahl salt-sensitive (DSS) rats fed with a diet containing 8% NaCl AIN-76A developed left ventricular remodeling and diastolic dysfunction caused by hypertension. After CANA treatment, cardiac hypertrophy and fibrosis were reduced, and the left ventricular diastolic function was improved. Metabolomics and proteomics data confirmed that CANA reduced myocardial glucose metabolism and increased fatty acid metabolism and ketogenesis in DSS rats, normalizing myocardial metabolism and reducing the myocardial oxidative stress. Mechanistically, CANA upregulated p-adenosine 5′-monophosphate-activated protein kinase (p-AMPK) and sirtuin 1 (SIRT1) and significantly induced the expression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1a). Conclusion: CANA can improve myocardial hypertrophy, fibrosis, and left ventricular diastolic dysfunction induced by hypertension in DSS rats, possibly through the activation of the AMPK/SIRT1/PGC-1a pathway to regulate energy metabolism and oxidative stress.

Lisinopril Mitigates Radiation-Induced Mitochondrial Defects in Rat Heart and Blood Cells.

The genetic bases and disparate responses to radiotherapy are poorly understood, especially for cardiotoxicity resulting from treatment of thoracic tumors. Preclinical animal models such as the Dahl salt-sensitive (SS) rat can serve as a surrogate model for salt-sensitive low renin hypertension, common to African Americans, where aldosterone contributes to hypertension-related alterations of peripheral vascular and renal vascular function. Brown Norway (BN) rats, in comparison, are a normotensive control group, while consomic SSBN6 with substitution of rat chromosome 6 (homologous to human chromosome 14) on an SS background manifests cardioprotection and mitochondrial preservation to SS rats after injury. In this study, 2 groups from each of the 3 rat strains had their hearts irradiated (8 Gy X 5 fractions). One irradiated group was treated with the ACE-inhibitor lisinopril, and a separate group in each strain served as nonirradiated controls. Radiation reduced cardiac end diastolic volume by 9-11% and increased thickness of the interventricular septum (11-16%) and left ventricular posterior wall (14-15%) in all 3 strains (5-10 rats/group) after 120 days. Lisinopril mitigated the increase in posterior wall thickness. Mitochondrial function was measured by the Seahorse Cell Mitochondrial Stress test in peripheral blood mononuclear cells (PBMC) at 90 days. Radiation did not alter mitochondrial respiration in PBMC from BN or SSBN6. However, maximal mitochondrial respiration and spare capacity were reduced by radiation in PBMC from SS rats (p=0.016 and 0.002 respectively, 9-10 rats/group) and this effect was mitigated by lisinopril (p=0.04 and 0.023 respectively, 9-10 rats/group). Taken together, these results indicate injury to the heart by radiation in all 3 strains of rats, although the SS rats had greater susceptibility for mitochondrial dysfunction. Lisinopril mitigated injury independent of genetic background.

Effects of Exercise Training on the Renin-Angiotensin System in the Kidneys of Dahl Salt-Sensitive Rats.

Exercise training (Ex) has antihypertensive and renal protective effects; however, the precise mechanisms remain unclear. The renal renin-angiotensin system (RAS) plays a vital role in renal function and pathology. Therefore, we investigated the effects of Ex on the renal RAS components in Dahl salt-sensitive (Dahl-S) rats. Male Dahl-S rats were divided into four groups: normal salt diet + sedentary, normal salt diet + Ex, high-salt diet (HS, 8% NaCl) + sedentary, and HS + Ex. Treadmill running was performed for 8 wk in the Ex groups. Ex attenuated the HS-induced renal dysfunction and glomerular injury without causing blood pressure alterations. HS increased urinary excretion of both total and intact angiotensinogen. Ex decreased the HS-induced increased urinary excretion of total angiotensinogen. However, it did not change the HS-induced urinary excretion of intact angiotensinogen, indicating reduced intact angiotensinogen cleaving. Ex restored the HS-induced increased angiotensinogen and angiotensin II type 1 receptor expressions in the outer medulla and the HS-induced increased angiotensin-converting enzyme expression in the cortex. Ex restored the HS-induced decreased renin expression in the cortex and outer medulla, and the HS-induced decreased angiotensin-converting enzyme 2, angiotensin II type 2 receptor, and Mas receptor expressions in the outer medulla. Ex attenuates HS-induced renal dysfunction, glomerular injury, and renal RAS dysregulation in Dahl-S rats.