Salt-sensitive Rats Research Articles

Abstract MP21: Suppression Of Reactive Oxygen Species Protects The Dahl Salt-sensitive Rat From Developing Maternal Syndrome

The Dahl Salt-Sensitive (SS) rat maintained on a low salt diet exhibits a differential response to pregnancy; approximately half develop maternal syndrome (hypertension and proteinuria) while the other half are protected. The exact mechanisms of maternal syndrome are unclear, but it is proposed that reactive oxygen species (ROS) are involved. Previous studies from our laboratory demonstrated the importance of NADPH oxidase (NOX) 2-derived ROS produced by immune cells in the development of salt-sensitive hypertension and renal disease. Given the role of NOX2-derived ROS in the Dahl SS rat, we tested the hypothesis that genetic deletion of the p67phox subunit of NOX2 (SS Ncf2-/- ) will result in the protection from developing maternal syndrome. To test this hypothesis, 8 week old female Dahl SS and SS Ncf2-/- were instrumented with radio-telemeters for continuous measurement of mean arterial pressure (MAP). After a recovery period, baseline MAP was measured as well as an overnight urine collection for markers of renal damage before females were mated with males of the same strain and age. Virgin litter-mate controls were run in parallel. As expected, MAP and protein excretion levels were comparable at baseline between virgin and mated SS Ncf2-/- rats. These parameters remained comparable at the late pregnancy time point (108±3 vs 112±1mmHg, 9±3 vs 16±5mg/day, p >0.05). In contrast to the SS preeclamptic pregnancy, the SS Ncf2-/- mated rats are protected from the development of maternal syndrome (141±8 vs 112±1mmHg, p <0.001; and 98±16 vs 16±5mg/day, p <0.001; in the SS vs SS Ncf2-/- , respectively). This protection is accompanied with a significant reduction in uterine artery resistance index which is a marker of impaired blood flow, in the SS Ncf2-/- (0.85±0.03 vs 0.59±0.02, p <0.01). Despite these phenotypic differences, the number of infiltrating immune cells into kidneys or placentas do not differ between these two groups. These results suggest that NOX2-derived ROS contribute to the pathogenesis of maternal syndrome in the Dahl SS rat, and further investigations are necessary to clarify the source of the ROS within key organs such as the kidney and placenta.

Abstract P148: Sex-dependent Colonic Immune Responses In Salt-sensitive Hypertension

Introduction: Hypertension affects almost half of the adults in the US. Overactivation of immune system results in pathogenesis of hypertension. Given that sex differences are reported in salt-sensitive hypertension and that the gut is an important organ at the interface between gut microbiota and the host, we hypothesized that differential colonic immunity exists between male and female rats on high and low salt diets. Methods: Total RNA was isolated from the colons of male and female Dahl Salt-Sensitive (S) rats fed with either high-salt (2% NaCl) or low-salt (0.3% NaCl) diet at 9-10 weeks old (n=3/sex/diet) and subjected to transcriptome followed by pathway analyses to detect differentially enriched transcripts and biological processes (FDR<0.05). The transcripts overexpressed in the sex-matched S rats on high salt diets were defined as hypertension-associated genes. Among them, the transcripts upregulated in both sexes were inferred as sex-independent, while those upregulated exclusively in either sex were considered as sex-dependent. Results: Upregulation of colonic immunity was evident through the transcriptomic signatures of both sexes with hypertension. Among the upregulated 38 male sex-dependent transcripts, 20 were related to T cell activation including co-receptor Cd4 , co-stimulators Cd80 and Cd86 , and Th1 and Treg cells-related receptors and cytokines Tgfbr2 , Il1r1 , Il18r1 and Lif , whereas 17 out of 27 upregulated female sex-dependent transcripts were related to T cell activation including co-receptor Cd8 , co-stimulators Cd40 , Icoslg , Cd27 , and Tnfsf9 , and Th2 and Th17-related cytokines Tgfb1 , Tgfb3 , Il1b , Il23a and Gata3 . In addition, antigen presenting cells were observed with upregulated MHC II in males, whereas upregulated both MHC I and II in females with hypertension. Further, 12 out of 20 sex-independent transcripts were suggestive of B cell activation. Conclusion: Marked sex differences were noted in the levels of colonic immune function- related transcripts of S rats on high salt diet. In the colonic T cell activation related transcripts, Cd4 and MHC II were predominant in males, while Cd8 and both MHC I and II were more prevalent in females. Additionally, colonic B cell activation was relatively less dependent on sex.

Abstract MP20: Prepartum 1,3-butanediol Treatment Improves Placental Perfusion In The Preeclamptic Dahl Salt-sensitive Rat

Chronic hypertension increases the risk of developing superimposed preeclampsia (PE). Previous reports showed that 1,3-Butanediol (BD) lowers blood pressure (BP) in male Dahl salt-sensitive (S) rats and female congenic S.SHR(11) rats which exhibit increased kidney injury over Dahl S rats. The goal of this study was to test if attenuating hypertension before pregnancy through the placentation period via BD prevents the onset of PE. Female Dahl S rats (a spontaneous model of superimposed PE, 11-16 weeks old) were divided into two groups: BD treated (20% via drinking water) and control (ad libitum water). Animals received BD for 7 weeks, baseline BP measurements (telemetry) were taken, and both groups were then mated. On gestation day (GD) 12, treatment was stopped because pilot studies showed that treatment reduced water intake during late pregnancy. Both groups were maintained on low-salt rodent chow (Teklad 7034, 0.3% NaCl; n=8/group). At GD18 (late pregnancy), uterine artery resistance index (UARI) was measured via Doppler ultrasound, 24h urine was collected on GD19, and tissues were harvested on GD20. Mean arterial pressure was lower in the treated group at baseline (141.9 ± 4.1 vs. 165.7 ± 4.5 mmHg, p= 0.008), early (135.9 ± 3.4 vs. 168.9 ± 4.6 mmHg, p= 0.0003), mid (142.0 ± 5.2 vs. 170.8 ± 4.61, p= 0.0048) but not late pregnancy (144.9 ± 5.9 vs. 161.9 ± 4.5 mmHg, p= 0.165). Late pregnancy maternal body weight was similar between groups (299.3 ± 5.6 vs. 311.3 ±7.0 g, p=0.201). Treated dams had a lower UARI (0.71 ± 0.02 vs. 0.81 ± 0.02, p=0.008) and less fetal resorptions (1.12 ± 0.29 vs. 2.25 ± 0.41, p= 0.043). No harm to the fetus was noted as no differences in pup weight (2.20 ±0.05 vs. 2.20 ± 0.09, p= 0.994) and pup length (30.19 ±0.24 vs. 29.45 ± 0.28 cm, p=0.072) were observed. Placentas from treated dams had decreased VEGF levels via ELISA (276.2 ± 33.4 vs. 498.9 ± 16.8 pg/mL, p= 0.001), suggesting reduced placental ischemia. In this study, we observed slightly improved placental perfusion and lower fetal demise following prepartum BD treatment; however, the antihypertensive effects of BD were not sustained through late pregnancy when supplementation was stopped at mid-pregnancy.

Abstract 15: The Role Of Opioid Receptors In Podocytes In The Development Of Hypertension In Dahl Salt-sensitive Rats

The rise in opioid use underscores the importance to better understand the direct and indirect effects of opioids on renal function and blood pressure. Although opioid use is associated with predictors of cardiovascular diseases, these drugs are common analgesics for hypertensive patients. We hypothesize that stimulation of opioid receptors (ORs) leads to elevated intracellular calcium level in podocytes ultimately leading to cell apoptosis, development of albuminuria and consequent progression of hypertension. Live calcium imaging experiments on freshly isolated glomeruli from rat and human kidneys, as well as human immortalized podocyte cell line, was performed to test the effect of specific ORs agonists. Following experiments assessed the effect of opioid signaling on the development of hypertension and kidney function in Dahl salt-sensitive (SS) rats, which were fed a 0.4% (LS) or 8% (HS) NaCl diets for 14 days with or without a daily i.v. bolus infusion of BRL52537, a potent and selective kappa-OR agonist. Stimulation of kappa-ORs, but not mu-ORs or delta-ORs, mediated calcium influx in podocytes through activation of TRPC6 channels. The effect of BRL52537 was completely abolished when we used the 0 mM calcium media or when a TRPC6 channel inhibitor (SAR7334) was applied. Triggering the kappa-OR/TRPC6 pathway induced podocyte cell shape changes via actin cytoskeleton remodeling. In vivo studies revealed that rats chronically treated with BRL52537 exhibited augmented blood pressure (MAP was 179 ± 15 vs. 151 ± 11 mmHg), albuminuria, and elevation in podocyte calcium. Western blot analysis revealed elevated levels of nephrin in urine samples and pro-caspase-3 in renal cortex. Moreover, TRPC6 expression was elevated under hypertensive conditions and further promoted pathological increase in calcium influx in response to kappa-OR stimulation. Summarizing the data, the opioid-induced increase in the calcium flux in podocytes is expected to contribute to kidney injury leading to progression of salt-induced hypertension. These data demonstrate that the kappa-OR/TRPC6 signaling pathway directly influences podocyte calcium handling, provoking the development of kidney injury in the opioid treated hypertensive cohort.

Abstract P150: Concerted Diurnal Rhythms Of Gut Microbiota With Salt-sensitive Hypertension And Renal Damage

Circadian, diurnal rhythm is a vital physiological feature of life forms, which enables holobionts to adapt to the day and night cycles. Evidence suggests that both factions of the holobiont, i.e, the host and its microbiota demonstrate physiological circadian rhythms. Blood pressure is a good example of a host physiological feature with a well-defined diurnal rhythm. In a healthy human, blood pressure (BP) rises to its peak during awakening morning hours and declines to the lowest level during night. In salt-sensitive hypertension, aberrant diurnal rhythms of BP and gut dysbiosis have been demonstrated. Given the critical role of gut microbiota in BP regulation, our current objective was to investigate whether there are synchronous rhythms of holobiont in rodents on low salt and high salt diets and if inflammation pattern also changes diurnally. We examined Dahl Salt-Sensitive (S) rats on low (0.3%) and high (2%) salt diets and BP and inflammation pattern was checked. As hypothesized, both microbiota and kidney inflammation showed diurnal rhythm in response to low salt and high salt diet. Major shifts in diurnal patterns of specific groups of microbiota were observed between the dark (active) and light (rest) phases, which correlated with the diurnal rhythmicity of BP. Diurnal rhythms 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, 3) amplitude of BP. PICRUSt analysis revealed diurnal rhythmicity of microbial pathways, characterized by microbiota upregulated biosynthetic processes during active phase and upregulated degradation pathways of metabolites in resting phase. These diurnal changes in microbiota, their functional pathways and BP amplitude were associated with concerted rhythmicity of renal Lipocalin 2 and Kim1 expression and circulating β-hydroxybutyrate in high salt S rats. Such concerted rhythmicity of holobiont with peak of changes at active phase of salt hypertension suggests that targeting this timepoint to reshape microbiota and/or intervene with medication could efficiently benefit the hypertensives

Abstract 22: Formyl Peptide Receptor-1 Activation Is Crucial For The Cause Of Spontaneous Hypertension In Dahl Salt Sensitive Rats

Mitochondria evolved from bacteria and use N-formylated peptides (NFPs) to synthetize protein. Bacterial and mitochondrial NFPs activate formyl peptide receptor 1 (FPR-1) and lead to vascular injury. We previously observed that Dahl Salt Sensitive rats (S) fed a low-salt (LS, 0.3% NaCl) diet presented spontaneous hypertension, vascular dysfunction, and overexpression of FPR-1 in arteries when compared to Dahl Salt Resistant (R) rats. High salt (HS, 2% NaCl) diet worsened these phenotypes in S rats. Interestingly, HS diet induced leaky gut and amoxicillin (AMO) treatment decreased BP in S-HS. Due to the dual sources of NFPs (microbiota and host mitochondria), we hypothesized that cell death-derived mitochondria and/or leaky gut-derived bacterial NFPs lead to FPR-1 activation, vascular injury and elevated BP in S rats independent of HS diet. For this, we used flow cytometry to measure cell necrosis and early and late apoptosis in kidney, bone marrow-derived macrophages and mesenteric resistance arteries (MRA) from male S and R rats (8-week old) on a LS diet. Zonulin, a biomarker for leaky gut, was measured in plasma. In another group, rats were treated with FPR-1 antagonist [Cyclosporin H (CsH), 0.3 mg/kg/day, osmotic mini-pump, 14 days], vehicle (VEH) or received water with AMO (5 mg/kg/day) for 21 days to deplete bacteria. BP was measured by telemetry and vascular function and structure were assessed in MRA. S rats presented increased kidney cell necrosis (R: 3.8±0.3 vs. S: 5.3±0.5* %). CsH decreased spontaneous elevation of BP [Diastolic: R+VEH: 77±2.7 vs. R+CsH: 81±1.2 vs. S+VEH: 126±3.0* vs. S+CsH:115±2.7 # ] and vascular hypercontractility [KCl (120mM): R+VEH: 9.4±1 vs. R+CsH: 10.2±0.4; S+VEH: 15.5±0.9* vs. S+CsH:11.7±0.8 # mN; Phenylephrine (10μM): R+VEH: 9.3±1 vs. R+CsH: 9.7±1; S+VEH: 14.5±1*vs. S+CsH: 11.4±0.6 # mN) in S-LS rats. AMO did not change vascular contraction or BP. Leaky gut was not observed in Dahl S-LS diet. In conclusion, FPR-1 can serve as a causative agent for the spontaneous elevation of BP and kidney-derived mitochondria, but not gut-derived microbiota, are the main source for NFPs.

Abstract P029: Aspartate And Potassium Aspartate May Contribute To A Mechanism By Which Saccharina Japonica Extract Attenuates Hypertension In 2-kidney, 1-clip Renovascular Hypertensive Rats.

Introduction: Saccharina japonica (SJ) is one of brown algae. The extract of SJ (SJE) is often used for soup stock in Japanese cuisine. We recently observed the decreases in blood pressure (BP) by intake of SJ or SJE diet in 2-kidney, 1-clip renovascular hypertensive (2K1C) rats. SJ is rich in aspartate (Asp), which is the umami taste of amino acid. The intake of Asp was reported to reduce BP in Dahl salt sensitive rats. As well, the potassium Asp (AspK) intake was reported to improve BP in hypertensive patients. 70% (70 of 100) of amino acids such as Asp are eluted into the SJE from SJ. Therefore, we hypothesized that Asp in SJE contributes to the mechanism of BP reduced by dietary SJE in 2K1C rats. In this study, we observed BP in 2K1C rats fed a diet containing Asp or AspK and investigated whether those diets alleviate hypertension to the same extent of the diet containing SJE. Methods: Male Sprague-Dawley rats (6 wks) were treated with sham operation (SHAM) or clipping the left renal artery (2K1C). After surgery, the rats fed a control diet (C), a diet containing Asp, AspK or SJE for 6 weeks. The content of SJE was set to be the same as that in our previous study. The amount of Asp in diet was set to be the same as that in our previous study which showed the antihypertensive effect of SJ. The amount of AspK in diet was set to be equimolar to Asp. The systolic BP (SBP) was measured by a tail-cuff method every week. At the end of the procedure, mean arterial BP (MAP) was measured in each rat under anesthesia. Results: Six weeks after the surgery, 2K1C-C significantly increased in SBP compared with SHAM-C (126±2 vs 159±4 mmHg, p<0.05). However, 2K1C-Asp (138±3), -AspK (142±4), and -SJE (135±4) decreased compared with 2K1C-C (p<0.05) respectively. MAP was significantly higher in 2K1C-C than in SHAM-C (162±3 vs 131±5, p<0.05). It showed a significant decrease in 2K1C-Asp (142±3), -AspK (148±3) and -SJE (144±4), compared with 2K1C-C (p<0.05), respectively. There were no significant differences among 2K1C-Asp, -AspK and -SJE, in either SBP or MAP. Conclusions: Asp and AspK may attenuate hypertension in 2K1C renovascular hypertension as much as SJE which contains similar quantities of Asp. It is possible that Asp and/or AspK eluted into SJE contributes to the mechanism of alleviating hypertension by SJE in 2K1C.

Abstract P124: Blood Pressure Characterization In Btg2 Mutant Rat

Hypertension (HTN) is a complex disease influenced by heritable genetic elements and environmental interactions. Dietary salt is among the most influential modifiable factors contributing to increased blood pressure (BP). It is well established that men and women develop BP impairment in different patterns and a recent emphasis has been placed on identifying mechanisms leading to the differences observed between the sexes in HTN development. The current work reported here builds on an extensive genetic mapping experiment which sought to identify genetic determinants of salt sensitive (SS) HTN using the Dahl SS rat. BTG anti-proliferation factor 2 ( Btg2 ) was previously isolated by our group using congenic breeding experiments which identified it as a female causative SS HTN candidate gene. In the current study, Btg2 was mutated using TALEN targeted gene disruption on the SSBN congenic rat background. The Btg2 mutated rats exhibited impaired BP and proteinuria responses to a high salt diet compared to wild type littermates. Differences in body weight, mutant pup viability, skeletal morphology, and adult nephron density suggest a potential role for Btg2 in developmental signaling pathways. Subsequent cell cycle gene expression assessment provides several additional signaling pathways that Btg2 may function through during salt handling in the kidney. The expression analysis also indicates several upstream targets that can be explored to further isolate therapeutic approaches for SS HTN.

Abstract P056: Voltage-gated Chloride Channel 6 Regulates Intracellular Calcium Signaling In Vascular Smooth Muscle Cells And Prevents Arterial Stiffening

Hypertension is a global health problem that increases the risk of stroke, heart failure, and kidney disease. GWAS have linked the CLCN6 gene to blood pressure regulation. CLCN6 encodes the voltage-sensitive chloride channel 6 (ClC-6), an intracellular protein of little-known function. Due to its high expression in vascular smooth muscle cells (VSMCs) and its localization in the Golgi, we hypothesized that ClC-6 is involved in Ca 2+ uptake in the Golgi and that reducing Golgi Ca 2+ levels changes VSMC function and therefore blood pressure, potentially by altering protein trafficking or cell proliferation. Using a knock-out of Clcn6 on the Dahl salt-sensitive rat background, we determined that KO rats have impaired vasodilation in response to 10 μM acetylcholine in mesenteric arteries (82 ± 6% vs 41 ± 13%, N=6 rats, n ≥ 9 vessels, p<0.01). Two-Photon microscopy in mesenteric arteries determined that KO VSMCs continue to release intracellular Ca 2+ in response to acetylcholine at a greater frequency than WT. Additionally, arterial stiffness, determined by pulse-wave velocity (PWV) measurements, was greater in KO rats at baseline (4.5 ± 0.3 vs. 3.6 ± 0.2 mm/msec, N≥6 rats, p<0.05); however, after 3 weeks on a 4% NaCl diet to develop hypertension, the WT rat PWVs increased significantly (4.6 ± 0.1 vs. 3.6 ± 0.2 mm/sec, N=6, p<0.05) whereas the KO rat PWVs did not change (4.6 ± 0.1 vs. 4.5 ± 0.3 mm/msec, N=6, p=0.91). To assess the effect of ClC-6 on Golgi Ca 2+ , confocal microscopy with an organelle-specific Ca 2+ dye and pharmacological agents was used to measure Ca 2+ . SR Ca 2+ stores were depleted with thapsigargin + ATP, followed by release of Golgi Ca 2+ stores by emetine. Total Golgi calcium levels were determined by area under the curve measurements of recorded fluorescence changes. Emetine-sensitive Golgi Ca 2+ stores were significantly reduced in KO rat VSMCs compared to WT. In conclusion, these results suggest that ClC-6 regulates Golgi Ca 2+ levels, and alteration to these stores results in cellular changes that slow or prevent arterial stiffness during the development of hypertension. Future studies will assess vessel fibrosis, cell proliferation, and apoptosis to elucidate the underlying molecular mechanisms impacting vessel stiffness and Ca 2+ signaling.

Abstract MP39: Autophagy Regulates β-hydroxybutyrate Synthesis To Prevent High-salt Induced Vascular Damage

Overconsumption of dietary salt is unequivocally linked with end organ damage. We have recently observed that a high salt (HS) diet induces liver dysfunction in the form of impaired synthesis of ketone body, β-hydroxybutyrate (βOHB), in Dahl salt-sensitive rats. However, the molecular mechanisms as to why βOHB is decreased after consuming a HS diet remains unknown, as well as if it contributes to vascular dysfunction. We hypothesized that a HS diet (2%) for 8 weeks would decrease systemic levels of βOHB in Dahl salt-resistant (R) rats and this would be associated with endothelial dysfunction. Furthermore, reconstitution of βOHB bioavailability by giving 1,3-Butanediol (1,3-BD; 20% v/v ) in the drinking water would improve endothelial function in HS fed Dahl R. As hypothesized, a HS diet decreased serum concentration (mM) of βOHB [Low salt (LS), non-fasting (NF): 0.31±0.02 vs. fasting (F): 0.96±0.04*; HS, NF: 0.33±0.02 vs. F: 0.77±0.04*#, *p<0.05 vs. NF, #p<0.05 vs. LS-F]. A HS diet decreased the sensitivity of isolated mesenteric resistance arteries (MRA) to acetylcholine (ACh), and 1,3-BD treatment prevented this endothelial dysfunction (LogEC50, Dahl R-LS: -7.35±0.05 vs. Dahl R-HS: -7.19±0.07* vs. Dahl R-HS+1,3-BD: -7.42±0.10, p<0.05). Furthermore, when MRA were incubated with indomethacin, only Dahl R-HS presented with a decreased sensitivity to ACh (LogEC50, HS: -7.19±0.07 vs. HS+indomethacin: -7.66±0.08*, p<0.05), indicating that 1,3-BD treatment decreased cyclooxygenase-induced vascular inflammation (LogEC50, 1,3-BD -7.42±0.10 vs. 1,3-BD +indomethacin: -7.61±0.09, p>0.05). Evolutionarily, autophagy functions to mobilize nutrients in times of starvation, including ketone bodies such as βOHB. Therefore, we hypothesized that inhibition of autophagy would decrease βOHB. Treatment with lysosomal alkalizer chloroquine (CQ), impeded the starvation-induced increase in βOHB (NF: 0.38±0.02 vs. F: 1.11±0.09* vs. F+CQ: 0.78±0.19*#, *p<0.05 vs NF, # p<0.05 vs F). In summary, these data reveal that a HS diet decreases liver-derived βOHB, independent of high blood pressure, and is protective against HS-induced vascular damage. We suggest that autophagy could be a novel mechanism underlying the vascular protective effects of βOHB.

Abstract P014: 1,3-butanediol Induces Weight Loss But No Antihypertensive Effects In Male S.SHR(11) Rats, An Accelerated Model Of Renal Injury

The ketone body precursor 1,3-Butanediol (BD) has been reported to lower blood pressure in male Dahl Salt-sensitive rats fed a 2% NaCl diet and female S.SHR(11) rats on a 0.3% NaCl diet. Treatment in female S.SHR(11) was also associated with weight loss and suppression of renal injury. The present study tested whether BD attenuates hypertension and renal injury in male S.SHR(11) rats. The S.SHR(11) rat is a congenic strain generated from genetic modification of the Dahl S rat. Previously, we demonstrated that the S.SHR(11) rat fed a 0.3% NaCl diet exhibits earlier onset of kidney injury, a greater decline in kidney function compared to the Dahl S rat despite having similar blood pressure, thus providing a better model to test potential therapeutic interventions. Rats (7 -18 weeks old) were divided into two groups: the treatment group that received BD (20%) for 10 weeks via drinking water and controls on ad libitum water and both maintained on a low-salt rodent chow (Teklad 7034, 0.3% NaCl; n=5-6/group). Systolic blood pressure was measured after 9 weeks by tail-cuff plethysmography. After 10 weeks of BD treatment, 24h urine was collected and tissues were harvested. Treated rats were smaller than controls (330.2 ± 14.2 vs. 449.8 ± 22.9 g, p= 0.001). Urine excretion rate was lower in treated rats (11.3± 1.1 vs. 19.6 ± 3.8 ml/day, p=0.047) whereas kidney weight normalized to body weight was higher in this group (0.95 ± 0.04 vs. 0.77 ± 0.03 %, p=0.011). BD did not lower systolic blood pressure (157.0 ±14.0 vs. 163.9 ±14.6 mmHg, p=0.740). Proteinuria was diminished in the treated group, although not significant (135 ± 51 vs. 311 ± 67 mg/day, p= 0.064). Renal fibrosis (7.6 ± 0.61 vs. 9.4 ± 0.95 %, p=0.139), plasma creatinine (0.46 ±0.03 vs. 0.49 ± 0.01 mg/dL, p=0.466), and creatinine clearance normalized to kidney weight (0.57 ±0.05 vs. 0.67 ± 0.02 ml/min, p= 0.138) were not changed by treatment. Blood urea nitrogen was similar between groups (30.7 ± 2.57 vs. 27.4 ± 2.25mg/dL, p= 0.367). The current data demonstrate that BD does not mitigate hypertension nor improve renal function in male S.SHR(11) rats suggesting potential sex differences in the antihypertensive effects of BD. However, weight loss which has been associated with ketogenic interventions such as BD was still observed in this study.

Abstract MP12: Increasing Circulating Anp Levels With Sacubitril Without Ras Blockage Aggravates Renal Disease In Dahl Ss Rats

Salt-sensitive (SS) hypertension is accompanied with severe cardiorenal complications. In this condition, elevated blood pressure (BP) resulting from high salt intake can be partially associated with a lower level of the atrial natriuretic peptide (ANP). In plasma, ANP is quickly degraded by neprilysin; therefore, pharmacological inhibition of this metalloprotease (f.i. with sacubitril) can increase ANP level. We have shown earlier that lower dose of sacubitril in combination with valsartan (75 μg/day) has beneficial effects on renal function in SS hypertension. The goal of this study was to evaluate the effects of sacubitril administered at a higher dose on renal damage of Dahl SS rats. Sacubitril (125 μg/day) or vehicle were administered to male Dahl SS rats (Charles River) via s.c. osmotic pumps. To induce hypertension, both groups were fed a purified 4% NaCl diet (HS, Dyets Inc) for 21 days. Glomerular filtration rate (GFR, FITC-inulin) and BP (tail cuff) values were obtained before and after the HS challenge. Data was analyzed with 1-way ANOVA. When compared to vehicle treated rats, 125 μg/day of sacubitril insignificantly increased systolic BP measured at the end of the 21-day HS challenge; no changes in GFR, heart weight, plasma electrolytes, BUN and creatinine were observed. However, sacubitril caused kidney hypertrophy (two kidneys to body weight: 8.8±0.4 vs 10.4±0.4 mg/g, p=0.04,), but did not affect renal medullary of cortical fibrosis. We also observed aggravated glomerular lesions in the sacubitril-treated animals compared to controls (glomerular injury score: 1.6±0.02 vs 0.9±0.08 au, p=0.034), and increased formation of protein casts (1.8±0.3 vs 1.3±0.2 au, p=0.034). Thus, in Dahl SS rats, administration of sacubitril at 125 μg/day had adverse effects on renal disease progression, especially glomerular damage and protein casts formation. In our prior study we showed that the combination of sacubitril at 75 μg/day with the angiotensin receptor blocker (ARB), valsartan, is effective to alleviate these outcomes. It is likely that the inhibition of neprilysin by sacubitril results in accumulation of Ang II and/or bradykinin and/or RAAS activation, which masks the beneficial effects of ANP level increase.

NOX4-dependent regulation of ENaC in hypertension and diabetic kidney disease.

NADPH oxidase 4 (NOX4) is the most abundant NOX isoform in the kidney; however, its importance for renal function has only recently emerged. The NOX4-dependent pathway regulates many factors essential for proper sodium handling in the distal nephron. However, the functional significance of this pathway in the control of sodium reabsorption during the initiation of chronic kidney disease is not established. The goal of this study was to test Nox4-dependent ENaC regulation in two models: SS hypertension and STZ-induced type 1 diabetes. First, we showed that genetic ablation of Nox4 in Dahl salt-sensitive (SS) rat attenuated a high-salt (HS)-induced increase in epithelial Na+ channel (ENaC) activity in the cortical collecting duct. We also found that H2 O2 upregulated ENaC activity, and H2 O2 production was reduced in both the renal cortex and medulla in SSNox4-/- rats fed an HS diet. Second, in the streptozotocin model of hyperglycemia-induced renal injury ENaC activity in hyperglycemic animals was elevated in SS but not SSNox4-/- rats. NaCl cotransporter (NCC) expression was increased compared to healthy controls, while expression values between SS and SSNox4-/- groups were similar. These data emphasize a critical contribution of the NOX4-mediated pathway in maladaptive upregulation of ENaC-mediated sodium reabsorption in the distal nephron in the conditions of HS- and hyperglycemia-induced kidney injury.

Macrophage Depletion Improves Endothelial Insulin Resistance and Protects against Cardiovascular Injury in Salt-Sensitive Hypertension.

Vascular endothelial insulin signaling is critical for the maintenance of vascular and metabolic homeostasis. We have previously shown that in hypertensive Dahl rats, impaired vascular insulin action is linked to angiotensin II activation of the NFκB inflammatory pathway. Macrophage polarization (M1) has implicated in hypertensive and metabolic diseases. Here, we investigated the effect of macrophage depletion using liposome-encapsulated clodronate (LEC) on endothelial insulin resistance and cardiovascular remodeling in Dahl salt-sensitive (DS) rats. High salt intake (HS) for 5 weeks increased systolic blood pressure (SBP: 192 ± 5 vs. 144 ± 4 mmHg in NS, p < 0.05), aortic and cardiac hypertrophy, cardiac fibrosis, and impaired acetylcholine- and insulin-induced vasorelaxation, accompanied by impaired insulin activation of endothelial nitric oxide synthases (eNOS)/NO signaling. HS rats had a significant increase in CD68 (a monocyte/macrophage marker) expression in the aorta and the heart. LEC reduced SBP (168 ± 5 mmHg, p < 0.05) and cardiovascular injury and improved acetylcholine- and insulin-mediated vasorelaxation and insulin signaling molecules with a reduction in the macrophage infiltration in the aorta and the heart. HS rats also manifested an increase in the aortic expressions of inflammatory cytokines, including the ratio of phosphorylated inhibitory kappa B (Iκb)/Iκb, tumor necrosis factor α, and phosphorylated c-Jun N-terminal kinase (JNK) and oxidative stress, which were reduced in HS/LEC rats. Our results suggest that in salt-sensitive hypertension, macrophage may importantly contribute to endothelial insulin resistance, vascular inflammation, and injury. These findings support the idea that macrophages may be a new target for immunotherapy of vasculopathy in hypertensive and metabolic disorders.

Fetal programming effects of pentaerythritol tetranitrate in a rat model of superimposed preeclampsia

Preeclampsia is a common medical condition during pregnancy and a major cause of maternal and prenatal mortality. The present study was conducted to investigate the effects of maternal treatment with pentaerythritol tetranitrate (PETN) in Dahl salt-sensitive rats (DSSR), a model of superimposed preeclampsia. F0 parental DSSR were treated with PETN (50 mg/kg) from the time point of mating to the end of lactation. Maternal PETN treatment improved fetal growth and had no effect on blood pressure in DSSR offspring fed with normal chow or high-salt diet. Upon high-fat diet (HFD) feeding, offspring from PETN-treated mother showed improved glucose tolerance despite similar weight gain. Unexpectedly, maternal PETN treatment significantly potentiated the HFD-induced blood pressure elevation in male DSSR offspring. Endothelium-derived hyperpolarization factor (EDHF)-mediated vasodilation was similar between NCD-fed and HFD-fed control offspring but was markedly reduced in HFD-fed PETN offspring. EDHF genes were downregulated in the vasculature of HFD-fed PETN offspring, which was associated with epigenetic changes in histone modifications. In conclusion, maternal PETN treatment in DSSR shows both beneficial and unfavorable effects. It improves fetal growth and ameliorates glucose tolerance in the offspring. Although maternal PETN treatment has no effect on blood pressure in offspring fed with normal chow or high-salt diet, the offspring is at higher risk to develop HFD-induced hypertension. PETN may potentiate the blood pressure response to HFD by epigenetic modifications of EDHF genes.Key messagesThe core findings of this article suggest that maternal PETN treatment of DSSR, a rat model of a spontaneous superimposed preeclampsia, leads to• Improvement of fetal growth;• No changes of maternal blood pressure or markers of preeclampsia;• Amelioration of HFD-induced glucose intolerance in adult offspring;• No changes in blood pressure development of the offspring on normal chow or high salt-diet;• Potentiation of blood pressure elevation of the offspring on HFD.

Conserved mammalian modularity of quantitative trait loci revealed human functional orthologs in blood pressure control.

Genome-wide association studies (GWAS) have routinely detected human quantitative trait loci (QTLs) for complex traits. Viewing that most GWAS single nucleotide polymorphisms (SNPs) are found in non-coding regions unrelated to the physiology of a polygenic trait of interest, a vital question to answer is whether or not any of these SNPs can functionally alter the phenotype with which it is associated. The study of blood pressure (BP) is a case in point. Conserved mechanisms in controlling BP by modularity is now unifying differing mammalian orders in that understanding mechanisms in rodents is tantamount to revealing the same in humans, while overcoming experimental limitations imposed by human studies. As a proof of principle, we used BP QTLs from Dahl salt-sensitive rats (DSS) as substitutes to capture distinct human functional orthologs. 3 DSS BP QTLs are located into distinct genome regions and correspond to several human GWAS genes. Each of the QTLs independently exerted a major impact on BP in vivo. BP was functionally changed by normotensive alleles from each of these QTLs, and yet, the human GWAS SNPs do not exist in the rat. They cannot be responsible for physiological alterations in BP caused by these QTLs. These SNPs are genome emblems for QTLs nearby, rather than being QTLs per se, since they only emerged during primate evolution after BP-regulating mechanisms have been established. We then identified specific mutated coding domains that are conserved between rodents and humans and that may implicate different steps of a common pathway or separate pathways.

Clinical Implications

Clinical Implications

High Salt Intake Impairs Erectile Function in Salt-Sensitive Rats Through Mineralocorticoid Receptor Pathway Beyond Its Effect on Blood Pressure

BackgroundHigh salt intake is a risk factor for hypertension, which can potentially lead to erectile dysfunction (ED); however, the underlying pathological mechanisms remain unclear. AimTo investigate whether erectile function is directly impaired by high salt intake and whether selective inhibition of mineralocorticoid receptor (MR) could provide protection from ED. Methods6-week-old male Dahl salt-sensitive rats were randomly divided into 3 groups: normal diet (0.3% NaCl; control, n = 8), high-salt diet (8% NaCl; HS, n = 8), and high-salt diet plus eplerenone (HS + EPL, n = 11). HS + EPL rats were orally administered daily doses of EPL (75 mg/kg) for 6 weeks; control and HS rats received purified water on the same schedule. OutcomesAt the end of the study period, erectile function was evaluated by measuring intracavernosal pressure and mean arterial pressure after cavernous nerve stimulation. Serum levels of asymmetric dimethylarginine and L-arginine were determined using ultraperformance liquid chromatography–tandem mass spectrometry. Quantitative PCR was used to assess the expression of MR, inflammation, and oxidative stress markers (nicotinamide adenine dinucleotide phosphate oxidase-1/4, p22phox, interleukin-6, and superoxide dismutase-1), and protein arginine N-methyltransferase-1. ResultsThe intracavernosal pressure/mean arterial pressure ratio was significantly lower, whereas systolic blood pressure, MR expression, serum asymmetric dimethylarginine levels, oxidative stress, and levels of inflammatory biomarkers were significantly higher in HS rats than in control rats (P < .05). EPL administration significantly improved each of these parameters except systolic blood pressure and MR expression. No significant intergroup differences were observed for L-arginine and superoxide dismutase-1 levels. Clinical TranslationOur results provide a rationale for the need of salt restriction and the use of selective MR inhibitors in prophylaxis or treatment of ED in men consuming a high-salt diet. Strengths & LimitationsWe are the first to report that the adverse impact of high salt intake on erectile function is mediated via MR activation, independent of its effect on blood pressure. A major limitation of this study is that responses of salt-resistant rats were not studied. ConclusionsHigh salt intake directly impaired erectile function in Dahl salt-sensitive rats, whereas selective MR inhibition ameliorated this effect.Kishimoto T, Kataoka T, Yamamoto Y, et al. High Salt Intake Impairs Erectile Function in Salt-Sensitive Rats Through Mineralocorticoid Receptor Pathway Beyond Its Effect on Blood Pressure. J Sex Med 2020;17:1280–1287.

Differential effects of low-dose sacubitril and/or valsartan on renal disease in salt-sensitive hypertension.

Diuretics and renin-angiotensin system blockers are often insufficient to control the blood pressure (BP) in salt-sensitive (SS) subjects. Abundant data support the proposal that the level of atrial natriuretic peptide may correlate with the pathogenesis of SS hypertension. We hypothesized here that increasing atrial natriuretic peptide levels with sacubitril, combined with renin-angiotensin system blockage by valsartan, can be beneficial for alleviation of renal damage in a model of SS hypertension, the Dahl SS rat. To induce a BP increase, rats were challenged with a high-salt 4% NaCl diet for 21 days, and chronic administration of vehicle or low-dose sacubitril and/or valsartan (75 μg/day each) was performed. Urine flow, Na+ excretion, and water consumption were increased on the high-salt diet compared with the starting point (0.4% NaCl) in all groups but remained similar among the groups at the end of the protocol. Upon salt challenge, we observed a mild decrease in systolic BP and urinary neutrophil gelatinase-associated lipocalin levels (indicative of alleviated tubular damage) in the valsartan-treated groups. Sacubitril, as well as sacubitril/valsartan, attenuated the glomerular filtration rate decline induced by salt. Alleviation of protein cast formation and lower renal medullary fibrosis were observed in the sacubitril/valsartan- and valsartan-treated groups, but not when sacubitril alone was administered. Interestingly, proteinuria was mildly mitigated only in rats that received sacubitril/valsartan. Further studies of the effects of sacubitril/valsartan in the setting of SS hypertension, perhaps involving a higher dose of the drug, are warranted to determine if it can interfere with the progression of the disease.

The early effects of uninephrectomy on rat kidney metabolic state using optical imaging.

Uninephrectomy (UNX) is known to result in structural and metabolic changes to the remaining kidney, although it is uncertain if this alters the mitochondrial redox state and how soon such changes may occur. A custom-designed fluorescence cryo-imaging technique was used to quantitatively assess the effect of UNX by measuring the levels of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in the remaining kidney. Kidneys were snap-frozen 3 days following UNX, and the intrinsic fluorescence of NADH and FAD were optically acquired. The 3D images were created to characterize the NADH/FAD redox ratios (RR) of the right kidneys, which underwent UNX and the remaining kidneys 3 days following UNX. Both the NADPH-oxidases (Nox2 and Nox4) and the mitochondria are the main sources of reactive oxygen species (ROS) production in tubular epithelial cells. Responses to the UNX were obtained in kidneys of normal Sprague Dawley (SD) rats, Dahl salt-sensitive (SS) rats and SS rats in which NADPH-oxidase isoform 4 (Nox4) was knocked out (SSNox4-/- ). The results found that each of the strains exhibited similar increase in kidney weights averaging 17% after 3 days of UNX. SD and SSNox4-/- rats both exhibited global reductions of the RR (P < .05) with a similar tendency observed in SS rats (P < .08), indicating increased ROS production. The unexpected reduction of the RR in the remnant kidneys of SSNox4-/- rats indicates that mechanisms independent of H2 O2 produced from Nox4 may be responsible for this global increase of ROS. We propose that the reduced RR was largely a consequence of enhanced mitochondrial bioenergetics due to increased tubular workload of the remaining kidney. The data indicate that mitochondria become the dominant source of increased ROS following UNX and could represent an important hypertrophic signaling mechanism.