Development Of Salt-induced Hypertension Research Articles

Effect of SGLT2 and dual SGLT1/2 Inhibition on the Development of Salt-induced Hypertension

In recent years, sodium-glucose co-transporter inhibitors (SGLT2i) have become the new mainstay in treating diabetes mellitus and, lately, cardiovascular diseases. Large clinical trials have shown remarkable benefits of SGLT2i on renal and cardiovascular outcomes. SGLT2i have been reported to slow the progression of chronic kidney disease by reducing hyperfiltration and glomerular capillary pressure, kidney hypertrophy, and albuminuria. Recently, the dual SGLT1/2 inhibitor sotagliflozin (Sota) was approved by the FDA for the treatment of heart failure independent of ejection fraction. However, despite these promising outcomes, the effects of SGLTi, particularly in salt-sensitive hypertension, requires further investigation. The main goal of this study was to define the effects of SGLT inhibition on the development of salt-induced hypertension and characterize potential contributing mechanisms. In our recent studies, we showed that SGLTi dapagliflozin (Dapa) caused an increase of diuresis in 8-weeks old male and female Dahl SS rats when fed a high salt (HS, 4% NaCl for 3 weeks) diet. Additionally, Dapa increased glucose and Na+ excretion. Chronic Dapa treatment also resulted in inhibition of the development of hypertension. Interestingly, male rats had no changes in kidney damage, whereas in females Dapa attenuated kidney fibrosis. To define potential molecular mechanisms involved in the beneficial effects of SGLT2i, we performed additional analyses of tissues collected from SS rats on a HS diet treated with either vehicle or Dapa. Transcriptomics analyses revealed that in the kidney cortex, top biological functions affected by SGLT2 inhibition were related to lipid and amino acid metabolism, molecular transport, as well as cell function. Preliminary studies were performed to test lipid mediators in the kidney cortex of male Dahl SS rats fed 3 weeks with HS diet and treated with Dapa. We found that several tested bioactive lipids, specifically pro-inflammatory mediators, were elevated in Dahl SS rats following HS diet, and these increased levels were completely blocked in Dapa-treated rats. Dual SGLT1/2 inhibition by Sota (30 mg/kg/day in food, n=6 rats/group) resulted in rapid attenuation of salt-induced hypertension. Starting from day 5 of HS challenge, MAP of vehicle-treated Dahl SS male rats was significantly higher compared to Sota-treated rats (~33 mmHg MAP difference on day 21), whichout differences in heart rate. In response to Sota treatment, SS rats had ~1.6-fold greater diuresis (at day 21: vehicle 37±2 vs. treated 61±6 ml/day) and glucosuria (values?). Further studies are ongoing to define the effects on GFR, kidney, and heart damage, and the potential resulting mechanisms. Our preliminary data indicate that dual SGLT1/2 inhibition with Sota had a significant impact on salt-induced hypertension in the Dahl SS rats. Further research will provide a more comprehensive understanding of the mechanisms. This research was supported by National Institutes of Health grant R35 HL135749 (to AS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Alterations of Mitochondrial Respiratory Function in Isolated Nephron Segments of Dahl Salt-Sensitive Rats in Salt-Induced Hypertension

Rationale: Mammalian kidneys actively reabsorb nearly 99% of the glomerular filtrate to maintain body fluid and solute homeostasis which requires a great amount of ATP generation and O2 consumption to meet these demands. A high salt diet increases glomerular filtration of Na+ and places a high metabolic demand on the renal tubules to reabsorb more Na+in order for the organism to maintain Na+ balance. Dahl salt-sensitive (SS) rats which mimic the human condition become hypertensive when fed a high salt diet. They are also known to reabsorb greater amounts of Na+ in the proximal tubules (PT) and medullary thick ascending limbs (mTAL) thereby requiring greater ATP production and O2consumption. We hypothesized that the mitochondrial respiratory function of the PT and mTAL would be altered when SS rats are fed a high salt diet in ways that reduce the ability of the kidney to function efficiency thereby leading to salt-induced hypertension. Method: Agilent Seahorse XF96 Extracellular Flux Analyzer was used to assess mitochondrial respiratory function in freshly bulk isolated PT and mTAL from age-matched SS rats during the development of salt-induced hypertension fed with 0.4% NaCl diet (low salt; LS) or 4.0% NaCl diet (high salt; HS) for 7, 14, and 21 days. The Seahorse “Cell Mito Stress Test Protocol” was slightly modified with a substrate addition step to assess the segment-specific and substrate-dependent tubular O2 consumption rate (OCR) under different perturbations, which was used to determine alterations in different mitochondrial respiratory parameters, including H+ leak and ATP production dependent OCR, in the PT and mTAL during the development of salt-induced hypertension. Results: Renal tubular OCR data show that PT of SS rats preferred to use lactate, glutamine, and palmitate, while mTAL preferred to use glucose, pyruvate, and lactate for ATP production under LS diet condition. HS diet resulted in significantly reduced substrate-induced OCR in PT but increased OCR in mTAL. In addition, HS induced a higher level of OCR in the baseline as well as when oligomycin (an inhibitor of F1F0-ATP synthase) was added to both PT and mTAL suspensions indicating that an increased proton leakage/uncoupling of mitochondria occurs in SS rats during the development of salt-induced hypertension. Conclusion: Our emergent data demonstrated that the mitochondrial respiratory function is differentially altered in the PT and mTAL of SS rats in response to different circulatory substrates during the development of salt-induced hypertension. These changes in metabolism appear to be necessary to meet the increased tubular workloads and need for greater ATP production and O2 utilization in both the PT and mTAL of SS rats. NIH R01-HL151587. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

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.

Metabolomic Kidney Input and Output Analyses in Salt‐Sensitive Hypertension

RationaleSalt‐sensitive hypertension is globally widespread and associated with increased risk of cardiovascular eventsand kidney disease. Although excess dietary salt intake is a major cause of these damaging effects, many of the underlying mechanisms remain poorly defined. Specifically, it is not understood how the critical energy needs of the kidney are met in face of constantly changing environmental nutrients and how this affects metabolic homeostasis in the kidney.MethodsWe hypothesized that a high salt diet in normal salt‐insensitive Sprague Dawley rat would lead to alterations of renal tubular bioenergetics and substrate metabolism. To test this, metabolomic analysis of arterial plasma (input) and renal venous plasma (filtered output) obtained from unanesthetized instrumented rats was performed using tandem mass spectrometry for comparison on a Q‐Exactive Orbitrap mass spectrometer coupled to a dual‐channel Vanquish Ultra‐Performance Liquid Chromatography system. Plasma metabolites were extracted from 20 µL of plasma from SD rats consuming low salt (0.4% NaCl) or high salt (4% NaCl) for 7, 14, or 21 days. These samples were then analyzed with separation over a hydrophobic (C18) and hydrophilic (HILIC) column under both positive and negative polarity to maximize identification of metabolites identified. Varying amounts of quality control plasma sample pools were used to determine injection sample concentration to analyze for each sample and remaining pools were used for normalization of the runs over time.ResultsA total of 4,603 metabolite features were detected, with 1,205 being identified named compounds. After applying further stringency filters to these named metabolites, 350 were significantly different in response to the varying high salt diets (p <0.05; Benjamini‐Hochberg correction) in at least one of the group comparisons performed (versus low salt and artery versus vein). Differential metabolites include numerous lipids, amino acids, and bioenergetic compounds, among others. There appeared to be a shift in these differential compounds between 14 and 21 days of high salt diet, suggesting a potential metabolic shift between initial responses and prolonged high salt responses. Complementary studies are also underway to compare urine metabolite flux, outer medulla, and cortex in conjunction with the plasma analysis to produce a complete computational prediction model.ConclusionTogether, implementation of this methodology will provide key insights and be valuable in further predicting the emergent properties of complex metabolic interactions in the kidney of rats during the development of salt‐induced hypertension.

Arcuate NPY is involved in salt-induced hypertension via modulation of paraventricular vasopressin and brain-derived neurotrophic factor.

Chronic high salt intake is one of the leading causes of hypertension. Salt activates the release of the key neurotransmitters in the hypothalamus such as vasopressin to increase blood pressure, and neuropepetide Y (NPY) has been implicated in the modulation of vasopressin levels. NPY in the hypothalamic arcuate nucleus (Arc) is best known for its control in appetite and energy homeostasis, but it is unclear whether it is also involved in the development of salt‐induced hypertension. Here, we demonstrate that wild‐type mice given 2% NaCl salt water for 8 weeks developed hypertension which was associated with marked downregulation of NPY expression in the hypothalamic Arc as demonstrated in NPY‐GFP reporter mice as well as by in situ hybridization analysis. Furthermore, salt intake activates neurons in the hypothalamic paraventricular nucleus (PVN) where mRNA expression of brain‐derived neurotrophic factor (BDNF) and vasopressin was found to be upregulated, leading to elevated serum vasopressin levels. This finding suggests an inverse correlation between the Arc NPY level and expression of vasopressin and BDNF in the PVN. Specific restoration of NPY by injecting AAV‐Cre recombinase into the Arc only of the NPY‐targeted mutant mice carrying a loxP‐flanked STOP cassette reversed effects of salt intake on vasopressin and BDNF expression, leading to a normalization of salt‐dependent blood pressure. In summary, our study uncovers an important Arc NPY‐originated neuronal circuitry that could sense and respond to peripheral electrolyte signals and thereby regulate hypertension via vasopressin and BDNF in the PVN.

Na+/K+-ATPase Alpha 2 Isoform Elicits Rac1-Dependent Oxidative Stress and TLR4-Induced Inflammation in the Hypothalamic Paraventricular Nucleus in High Salt-Induced Hypertension.

Numerous studies have indicated that a high salt diet inhibits brain Na+/K+-ATPase (NKA) activity, and affects oxidative stress and inflammation in the paraventricular nucleus (PVN). Furthermore, Na+/K+-ATPase alpha 2-isoform (NKA α2) may be a target in the brain, taking part in the development of salt-dependent hypertension. Therefore, we hypothesized that NKA α2 regulates oxidative stress and inflammation in the PVN in the context of salt-induced hypertension. Part I: We assessed NKA subunits (NKA α1, NKA α2, and NKA α3), Na+/K+-ATPase activity, oxidative stress, and inflammation in a high salt group (8% NaCl) and normal salt group (0.3% NaCl). Part II: NKA α2 short hairpin RNA (shRNA) was bilaterally microinjected into the PVN of salt-induced hypertensive rats to knockdown NKA α2, and we explored whether NKA α2 regulates downstream signaling pathways related to protein kinase C γ (PKC γ)-dependent oxidative stress and toll-like receptor 4 (TLR4)-induced inflammation in the PVN to promote the development of hypertension. High salt diet increased NKA α1 and NKA α2 protein expression in the PVN but had no effect on NKA α3 compared to the normal salt diet. Na+/K+-ATPase activity and ADP/ATP ratio was lower, but NAD(P)H activity and NF-κB activity in the PVN were higher after a high salt diet. Bilateral PVN microinjection of NKA α2 shRNA not only improved Na+/K+-ATPase activity and ADP/ATP ratio but also suppressed PKC γ-dependent oxidative stress and TLR4-dependent inflammation in the PVN, thus decreasing sympathetic activity in rats with salt-induced hypertension. NKA α2 in the PVN elicits PKC γ/Rac1/NAD (P)H-dependent oxidative stress and TLR4/MyD88/NF-κB-induced inflammation in the PVN, thus increasing MAP and sympathetic activity during the development of salt-induced hypertension.

Commentary: Gut Microbiota-Related Evidence Provides New Insights Into the Association Between Activating Transcription Factor 4 and Development of Salt-Induced Hypertension in Mice.

Commentary: Gut Microbiota-Related Evidence Provides New Insights Into the Association Between Activating Transcription Factor 4 and Development of Salt-Induced Hypertension in Mice.

Gut Microbiota-Related Evidence Provides New Insights Into the Association Between Activating Transcription Factor 4 and Development of Salt-Induced Hypertension in Mice.

Activating transcription factor 4 (ATF4), which regulates genes associated with endoplasmic reticulum stress, apoptosis, autophagy, the gut microbiome, and metabolism, has been implicated in many diseases. However, its mechanistic role in hypertension remains unclear. In the present study, we investigated its role in salt-sensitive hypertensive mice. Wild-type (WT) C57BL/6J mice were used to establish Atf4 knockout (KO) and overexpression mice using CRISPR-Cas9 and lentiviral overexpression vectors. Then, fecal microbiota transplantation (FMT) from Atf4± mice and vitamin K2 (VK2) supplementation were separately carried out in high-salt-diet (8% NaCl)-induced mice for 4 weeks. We found that Atf4 KO inhibited and Atf4 overexpression enhanced the increase in blood pressure and endothelial dysfunction induced by high salt intake in mice, while regulating the gut microbiota composition and VK2 expression. It was further verified that ATF4 is involved in the regulation of salt-sensitive hypertension and vascular endothelial function, which is achieved through association with gut microbiota and may be related to VK2 and different bacteria such as Dubosiella. In addition, we found that VK2 supplementation prevents the development of salt-sensitive hypertension and maintains vascular endothelial function; moreover, VK2 supplementation increases the abundance of intestinal Dubosiella and downregulates the relative expression of Atf4 in the thoracic aorta of mice. We conclude that ATF4 plays an important role in regulating gut microbiota and VK2 production, providing new insights into the association between ATF4 and development of salt-induced hypertension in mice, meanwhile contributing to the development for a new preventive strategy of hypertension.

Role of opioid signaling in kidney damage during the development of salt-induced hypertension.

Opioid use is associated with predictors of poor cardiorenal outcomes. However, little is known about the direct impact of opioids on podocytes and renal function, especially in the context of hypertension and CKD. We hypothesize that stimulation of opioid receptors (ORs) contributes to dysregulation of intracellular calcium ([Ca2+]i) homeostasis in podocytes, thus aggravating the development of renal damage in hypertensive conditions. Herein, freshly isolated glomeruli from Dahl salt-sensitive (SS) rats and human kidneys, as well as immortalized human podocytes, were used to elucidate the contribution of specific ORs to calcium influx. Stimulation of κ-ORs, but not μ-ORs or δ-ORs, evoked a [Ca2+]i transient in podocytes, potentially through the activation of TRPC6 channels. κ-OR agonist BRL52537 was used to assess the long-term effect in SS rats fed a high-salt diet. Hypertensive rats chronically treated with BRL52537 exhibited [Ca2+]i overload in podocytes, nephrinuria, albuminuria, changes in electrolyte balance, and augmented blood pressure. These data demonstrate that the κ-OR/TRPC6 signaling directly influences podocyte calcium handling, provoking the development of kidney injury in the opioid-treated hypertensive cohort.

Abstract 16: Progressive Alterations Of Mitochondrial Bioenergetics In The Kidney During The Development Of Salt-induced Hypertension

Hypertension is a complex disease and a leading cause of morbidity and mortality globally. Although oxidative stress and mitochondrial dysfunction have been found in the kidney in various models of hypertension, progressive alteration of mitochondrial oxidative phosphorylation (OxPhos) in the kidney during the development of salt-sensitive hypertension has not been characterized. The present study determined changes of OxPhos in kidneys of Dahl salt-sensitive (SS) rats before (0.4% NaCl diet; LS) and after switching to a high salt diet (4.0% NaCl; HS) during the development of hypertension. Mitochondria were isolated from the outer medulla (OM) and cortex of the kidney of SS rats fed a LS diet since weaning and studied at days 3, 7, 14 & 21 of a HS diet feeding. Oxygen consumption rates (OCR) were measured in mitochondria energized with pyruvate + malate as substrates for three different respiratory states using an Oroboros Oxygraph-2k Instrument. This includes i) leak state (in the absence of ADP), ii) ADP-stimulated state, and iii) uncoupled state (in the presence of an uncoupler FCCP). A biphasic pattern of ADP-stimulated OCR with progressive uncoupling was observed in both the renal OM and cortex. Mitochondrial efficiency for ATP synthesis was increased in the early phases of hypertension (3 & 7 days) but was severely compromised in the established phases of hypertension (14 & 21 days). This decreased mitochondrial efficiency was associated with uncoupling of OxPhos and high levels of oxidative stress which we hypothesized were due to mitochondrial ROS stimulation of membrane NOXs. To test this, experiments were performed in SS rats with double knock out (DKO) of the cytosolic subunit of NOX2 (p67 phox ) and NOX4 (SS p67phox-/-/Nox4-/- ). DKO SS rats were fed a HS diet and OCR of renal cortical and OM mitochondria was determined at days 7 and 14. In contrast to SS rats, the DKO SS rats fed a HS diet showed no significant differences in mitochondrial OCR in the cortex or OM, nor to a control group maintained on a LS diet. HS diet in SS rats initially increases the efficiency of renal cortical and medullary mitochondrial ATP production (days 1-7) followed by an enhanced ROS production with mitochondrial uncoupling and reduced efficiency of ATP production by the third week.

The Protective Effects of Atrial Natriuretic Peptide Infusion in Salt‐Sensitive Hypertension

Atrial Natriuretic Peptide (ANP) is a hormone secreted by cardiomyocytes in response to increased atrial volume; it stimulates water and sodium excretion by the kidneys and relieves pressure on the circulatory system. In our previous studies we compared wild type Dahl Salt Sensitive (SS) rats to SS rats lacking the Nppa gene which encodes ANP (SSNppa−/−). The data revealed that salt‐induced blood pressure development was significantly exacerbated in the SSNppa−/− rats. Furthermore, SSNppa−/− rats exhibited reduced urinary sodium excretion and diuresis, as well as aggravated kidney damage compared to wild type controls (FASEB J 2017 31:855.8.). These data led to a hypothesis that ANP infusion can have beneficial effects during the development of salt‐induced hypertension.Dahl SS rats were fed a high salt diet (HS, 4% NaCl) for 21 days, with a continuous i.v. infusion of ANP (100 ng/kg/day) administered from day 0 or day 14 onward to test preventive and therapeutic effects of ANP, respectively. Blood pressure was monitored throughout the study, and urine samples were collected in metabolic cages on days 0 and 21. At the end of the protocol animals were sacrificed and kidney tissues were harvested and stained with Masson's trichrome to evaluate renal damage. Fibrosis, glomerular injury, and protein casts formation were assessed; urine and plasma samples were also analyzed to estimate electrolyte homeostasis.ANP infusion had beneficial effects on BP and renal function compared to control animals. We observed a decrease in blood pressure in both groups infused with ANP during 0–21 days (21D) and 14–21 days (7D) of a HS challenge. Kidney to body weight ratio did not differ between the groups; however, we observed a trend for reduced renal hypertrophy in the 21D animals. Metabolic cage studies revealed no changes in diuresis; urinary electrolyte analysis showed a more effective sodium excretion in the 21D rats. Assessment of kidney damage demonstrated a decrease in protein casts in both 7D and 21D rats: 3.8% ± 0.47%, 2.7% ± 0.24%, and 2.0% ± 0.41% of the tissue surface in the control, 7D and 21D groups, respectively. Glomerular injury had a similar pattern: scores were 2.86 ± 0.09, 2.62 ± 0.07, and 2.41 ± 0.06. Renal fibrosis was alleviated in the 21D group (1.4% ± 0.18%, compared to 1.9% ± 0.49% and 3.1% ± 0.31% in the 7D and control groups).Therefore, ANP infusion had both preventive and therapeutic effects. Our data revealed that ANP administration during the HS diet challenge resulted in decreased blood pressure, increased electrolyte excretion, and reduced tissue damage.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 107: Role of Nox4 in the Control of ENaC Activity in Dahl SS Rats During the Development of Salt-induced Hypertension and Diabetic Nephropathy

Dahl salt sensitive (SS) rat is a well-established model for studying salt-induced hypertension and associated kidney injury. We and others have previously shown that salt-sensitive hypertension is accompanied by increased renal production of reactive oxygen species (ROS) and excessive activity of ENaC in the distal nephron. To investigate role of ROS, and specifically NADPH oxidase 4 (Nox4), a primary source of ROS in the kidney, involved in the regulation of ENaC activity during the development of SS hypertension and type 1 diabetes, we performed patch clamp analysis in the cortical collecting ducts of Dahl SS rats and SS rats lacking Nox4 (Nox4 -/- ). We found that SS rats fed a 4% NaCl diet have significantly elevated ENaC activity even 3 days post diet change. ENaC activity (NP o ) was 0.57±0.08, 1.32±0.3 and 1.69±0.06 before, 3 days and 3 weeks after high salt diet, respectively. In contrast, ENaC activity was not significantly different in SS Nox4-/- animals after high salt diet. To study the role of Nox4 in hyperglycemic conditions, diabetes was induced in 6 weeks old male wild type or Nox4 -/- rats with a single i.p. injection of STZ. We found that ENaC activity in the animals that were hyperglycemic for 11 weeks was elevated compared to control rats (0.71±0.10 and 1.27±0.2; p<0.05) and this effect was mediated via changes in channel open probability. Nox4 deficiency blunts the effect of hyperglycemia on ENaC activity (in STZ-treated animals open probability significantly increased from 0.43±0.06 to 0.86±0.07 in WT rats, but in the Nox4 -/- group did not change: P o was 0.51±0.06 and 0.51±0.07, respectively) that delineates the importance of Nox4-mediated ROS production for regulation of ENaC open probability. Taken together, our data indicate that ENaC activity in Dahl SS rats is elevated following a change of salt diet (3 days and 3 weeks at high salt) and after the development of type 1 diabetes. Furthermore, Nox4 plays a crucial role in these effects of high salt and hyperglycemia on ENaC activity.

Abstract 105: Therapeutic Suppression of Mtorc2 Signaling Reduces Salt-induced Hypertension and Kidney Injury in SS Rats

The present study explored the protective effect of mTORC2 inhibition in salt-induced hypertension and kidney injury. We have previously reported that enhanced blood pressure salt-sensitivity with renal chronic interstitial infusion of H 2 O 2 (347 nmol/kg/min) in normotensive Sprague Dawley (SD) rats. In the present study, in vivo experiment was performed in which H 2 O 2 (347 nmol/Kg/min) was chronically infused for 3 days into renal interstitium of unilaterally nephrectomized SD rats. A significant increase of mTORC2 activity (pAKT/AKT) was observed in the renal cortex of SD rats infused with H 2 O 2 compared to saline infused rats. We have recently shown that excess production of H 2 O 2 in the SS rat kidneys is the hallmark of salt-induced hypertension. We hypothesized that mTORC2 in the kidney contributes to the development of salt-induced hypertension in SS rats. Rats were treated with PP242 which is an ATP-competitor inhibitor which inhibits the activities of both mTORC1 and mTORC2 whereas rapamycin specifically inhibits mTORC1. PP242 was administrated daily (i.p., 15 mg/Kg/day) for 21 days to SS rats fed a 4.0% NaCl diet. Remarkably, salt-induced hypertension was significantly reduced in SS rats which averaged 119 ± 2 mmHg in PP242 treated rats (n=7) compared to 168 ± 3 mmHg in vehicle treated rats (n=7). Albuminuria was greatly reduced with urine albumin excretion (mg/day) averaging 32.8 ± 3 in PP242 treated rats compared to 256 ± 37 in vehicle treated rats. PP242 treatment notably resulted in reduced infiltration of T lymphocytes in the kidneys of SS rats fed a 4.0% NaCl diet. CD3 + cells/mm 2 averaging 157.0 ± 40.0 compared to 36.0 ± 11.0 in the renal cortex and 218.0 ± 24.0 compared to 24.0 ± 9.0 in the outer medulla in PP242 versus vehicle treated rats. These data show that mTORC2 is required for the intiation of salt-induced hypertension and therapeutic suppression of this pathway virtually abolished salt-sensitivity blood pressure and kidney injury.

Intravital imaging of the kidney in a rat model of salt-sensitive hypertension.

Hypertension is one of the most prevalent diseases worldwide and a major risk factor for renal failure and cardiovascular disease. The role of albuminuria, a common feature of hypertension and robust predictor of cardiorenal disorders, remains incompletely understood. The goal of this study was to investigate the mechanisms leading to albuminuria in the kidney of a rat model of hypertension, the Dahl salt-sensitive (SS) rat. To determine the relative contributions of the glomerulus and proximal tubule (PT) to albuminuria, we applied intravital two-photon-based imaging to investigate the complex renal physiological changes that occur during salt-induced hypertension. Following a high-salt diet, SS rats exhibited elevated blood pressure, increased glomerular sieving of albumin (GSCalb = 0.0686), relative permeability to albumin (+Δ16%), and impaired volume hemodynamics (-Δ14%). Serum albumin but not serum globulins or creatinine concentration was decreased (-0.54 g/dl), which was concomitant with increased filtration of albumin (3.7 vs. 0.8 g/day normal diet). Pathologically, hypertensive animals had significant tubular damage, as indicated by increased prevalence of granular casts, expansion and necrosis of PT epithelial cells (+Δ2.20 score/image), progressive augmentation of red blood cell velocity (+Δ269 µm/s) and micro vessel diameter (+Δ4.3 µm), and increased vascular injury (+Δ0.61 leakage/image). Therefore, development of salt-induced hypertension can be triggered by fast and progressive pathogenic remodeling of PT epithelia, which can be associated with changes in albumin handling. Collectively, these results indicate that both the glomerulus and the PT contribute to albuminuria, and dual treatment of glomerular filtration and albumin reabsorption may represent an effective treatment of salt-sensitive hypertension.

Abstract 105: Maternal Diet Leads to Nephron Count Differences After Postnatal Day 14 and Time-dependent Differences in Apoptosis and Unfolded Protein Response in Dahl Salt-sensitive Rats

We have shown that maternal exposure to Casein-based AIN-76A diet [SS/JrHsdMcwi (SS/Mcw) rats] compared to grain-based 5L2F diet [SS/JrHsdMcwiCrl (SS/Crl) rats] during the gestational-lactational period exacerbates the development of salt-induced hypertension and renal injury in adult SS rats. We hypothesized that SS/Mcw rats will have significantly fewer glomeruli than SS/Crl rats, and determined its potential mechanisms. At different time points during development [embryonic day 20 (E 20), day of life (DOL) 14, 21, 56]: glomerular counts were determined by maceration method and by glomerular density (total glomerular number/renal cortex area); apoptosis was measured by TUNEL assay; RT-PCR, Western Blot and immunostaining were used to examine the expression level of genes involved in the apoptotic pathway and unfolded protein response (UPR). We found lower glomerular counts in SS/Mcw compared to SS/Crl rats at DOL 21 [26048±1423 per kidney vs. 34766±1821 (p<0.001] and DOL 56 [15717±2052 vs. 27999±3362 (p <0.001)], and lower glomerular densities [DOL 21, 10.8±1.0/mm2 vs. 13.2±1.6 (p<0.001); DOL 56, 2.4±0.1 vs. 3.6±0.2 (p < 0.001)]. Glomerular density was not different between SS/Mcw and SS/Crl at DOL 14 when nephrogenesis is complete. We found higher glomerular apoptosis in SS/Mcw compared to SS/Crl rats at DOL 14 [9.5±1.4 per 100 glomeruli vs. 5.1±1.8 (p<0.01)] and DOL 21 [5.2±0.7 vs. 3.6±0.5 (p <0.01)]. mRNA levels of several genes involved in intrinsic apoptotic pathway and UPR were significantly upregulated in SS/Mcw rats compared to SS/Crl rats at different time points. Significantly lower UPR-related protein expression in SS/Mcw compared to SS/Crl rats was detected for JNK and GRP78 (DOL 14 and 56) and CHOP (DOL 56). Significantly higher UPR-related protein expression in SS/Mcw compared to SS/Crl rats were detected for GRP78 (E 20), JNK (DOL 21) and Caspase 12 (DOL 14, 21 and 56). Caspase 12 was located in the glomeruli in DOL 21 and 56 kidneys. Our results show marked decrease of glomerular counts with age in SS/Mcw compared to SS/Crl after DOL 14. The time-dependent differences in the expression of apoptosis and UPR-related genes leading to glomerular apoptosis may be a potential mechanism for the nephron count differences between SS/Mcw and SS/Crl.

Abstract 533: Effect of Dipeptidyl Peptidase-4 Inhibition on Blood Pressure and Its Dipping Pattern in Dahl Salt-Sensitive Rats

Several studies have indicated that treatment with dipeptidyl peptidase-4 (DPP-4) inhibitors decreases blood pressure. We aimed to examine the effects of vildagliptin, a specific DPP-4 inhibitor, on blood pressure and its dipping pattern in Dahl salt-sensitive (DSS) rats. Male DSS rats were treated with a high salt (8% NaCl) diet and vehicle (0.5% carboxymethylcellulose) or vildagliptin (3 or 10 mg/kg, twice daily, p.o.) for 7 days (n = 7 per group). Mean arterial pressure (MAP) was measured by telemetry. High salt diet for 7 days significantly increased MAP with an extreme dipping pattern of blood pressure in DSS rats. Vildagliptin dose-dependently inhibited DPP-4 enzymatic activity and increased plasma GLP-1 levels. Furthermore, vildagliptin dose-dependently attenuated the development of salt-induced hypertension. Interestingly, vildagliptin significantly increased urine sodium excretion and normalized the dipping pattern. In anesthetized high salt-diet DSS rats, acute intra-cerebroventricular infusion of vildagliptin (50, 500 or 2500 μg in 10 μl solution) did not alter MAP or heart rate (n = 4). These data suggest that treatment with the DPP-4 inhibitor, vildagliptin, attenuates the extreme dipping pattern of blood pressure and development of salt sensitive hypertension by increasing urinary sodium excretion. These effects of vildagliptin may not be mediated through the central nervous system.

Abstract 479: Shortening of Mitochondria and Dilation of Endoplasmic Reticulum in the Medullary Thick Ascending Limb of Dahl Salt-Sensitive Rats

Functional abnormalities in the kidney appear to precede or coincide with the initiation of salt-induced hypertension in the Dahl salt-sensitive SS rat. However, the structural basis or mechanism of early renal abnormalities, especially in the renal tubules, is poorly understood. We performed electron microscopy analysis in 7 week old SS rats and salt-insensitive consomic SS.13 BN rats and Sprague-Dawley (SD) rats fed a 4% NaCl diet for 7 days. Long mitochondria (>2 μm), an indication of healthy mitochondria, accounted for a significantly smaller fraction of all mitochondria in medullary thick ascending limbs in SS rats (4% ± 1%) than in SS.13 BN rats (8% ± 1%, P<0.05 vs. SS) and SD rats (9% ± 1%, P<0.01 vs. SS). This is consistent with our previous findings of mitochondrial insufficiency in the medulla of SS rats. Long mitochondria in proximal tubules, however, were more abundant in SS rats than in SS.13 BN and SD rats. The width of endoplasmic reticulum, an index of endoplasmic reticulum stress, was significantly greater in medullary thick ascending limbs of SS rats (107 ± 1 nm) than SS.13 BN rats (95 ± 2 nm, P<0.001 vs. SS) and SD rats (74 ± 3 nm, P<0.01 vs. SS or SS.13 BN ). The rat strain differences in ultrastructure were observed even though the tubules were indistinguishable by light microscopy and less than 5% of the kidney area was affected by inflammation, interstitial fibrosis, or tubular casts. These data indicate that ultrastructural abnormalities in the mitochondria and endoplasmic reticulum in the medullary thick ascending limbs occur in the early stage of, and could contribute to, the development of salt-induced hypertension.

Mineralocorticoid receptors/epithelial Na+ channels in the choroid plexus are involved in hypertensive mechanisms in stroke-prone spontaneously hypertensive rats

Increase in cerebrospinal fluid (CSF) Na(+) concentration ([Na(+)]) precedes hypertension and is a key step in the development of salt-induced hypertension. In the choroid plexus (CP), epithelial Na(+) channels (ENaCs) have an important role in Na(+) transport from the blood into the CSF. However, it remains unknown whether the mineralocorticoid receptors (MR)/ENaCs pathway in the CP of stroke-prone spontaneously hypertensive rats (SHRSP) is involved in neural mechanisms of hypertension. Therefore, we examined the role of the MR/ENaCs pathway in the CP in the development of hypertension in SHRSP associated with an increase in CSF [Na(+)]. As a marker of MR activation, serum/glucocorticoid-inducible kinase 1 (Sgk1) expression levels in the CP were measured and found to be greater in SHRSP than in Wistar-Kyoto (WKY) rats. CSF [Na(+)] levels were also higher in SHRSP than in WKY rats. In SHRSP, high-salt intake (8%) increased blood pressure and urinary norepinephrine excretion compared with those in animals fed a regular salt diet (0.5%) for 2 weeks. Furthermore, the expression levels of MR, Sgk1 and ENaCs in the CP and the increase in CSF [Na(+)] were greater in SHRSP fed a high-salt diet than in those fed a regular salt diet. These alterations were attenuated by intracerebroventricular infusion of eplerenone (10 μg kg(-1) per day), except for α-ENaC and β-ENaC. We conclude that activation of the MR/ENaCs pathway in the CP contributes to hypertension via an increase in CSF [Na(+)], thereby exaggerating salt-induced hypertension with sympathetic hyperactivation in SHRSP.

Regulation of pendrin by cAMP: possible involvement in β-adrenergic-dependent NaCl retention

The sodium-independent anion exchanger pendrin is expressed in several tissues including the kidney cortical collecting duct (CCD), where it acts as a chloride/bicarbonate exchanger and has been shown to participate in the regulation of acid-base homeostasis and blood pressure. The renal sympathetic nervous system is known to play a key role in the development of salt-induced hypertension. This study aimed to determine whether pendrin may partly mediate the effects of β adrenergic receptors (β-AR) on renal salt handling. We investigated the regulation of pendrin activity by the cAMP/protein kinase A (PKA) signaling pathway, both in vitro in opossum kidney proximal (OKP) cells stably transfected with pendrin cDNA and ex vivo in isolated microperfused CCDs stimulated by isoproterenol, a β-AR agonist. We found that stimulation of the cAMP/PKA pathway in OKP cells increased the amount of pendrin at the cell surface as well as its transport activity. These effects stemmed from increased exocytosis of pendrin and were associated with its phosphorylation. Furthermore, cAMP effects on the membrane expression and activity of pendrin were abolished by mutating the serine 49 located in the intracellular N-terminal domain of pendrin. Finally, we showed that isoproterenol increases pendrin trafficking to the apical membrane as well as the reabsorption of both Cl(-) and Na(+) in microperfused CCDs. All together, our results strongly suggest that pendrin activation by the cAMP/PKA pathway underlies isoproterenol-induced stimulation of NaCl reabsorption in the kidney collecting duct, a mechanism likely involved in the sodium-retaining effect of β-adrenergic agonists.