Dahl Salt-sensitive Research Articles

Genetically conditioned interaction among microRNA-155, alpha-klotho, and intra-renal RAS in male rats: Link to CKD progression.

Incident chronic kidney disease (CKD) varies in populations with hypertension of similar severity. Proteinuria promotes CKD progression in part due to activation of plasminogen to plasmin in the podocytes, resulting in oxidative stress-mediated injury. Additional mechanisms include deficiency of renal alpha-klotho, that inhibits Wnt/beta-catenin, an up regulator of intra-renal renin angiotensin system (RAS) genes. Alpha-klotho deficiency therefore results in upregulation of the intra-renal RAS via Wnt/beta-catenin. In hypertensive, Dahl salt sensitive (DS) and spontaneously hypertensive rats (SHR), we investigated renal and vascular injury, miR-155, AT1R, alpha-klotho, and TNF-α. Hypertensive high salt DS (DS-HS), but not SHR developed proteinuria, plasminuria, and glomerulosclerosis. Compared to DS low salt (DS-LS), in hypertensive DS-HS alpha-klotho decreased 5-fold in serum and 2.6-fold in kidney, whereas serum mir-155 decreased 3.3-fold and AT1R increased 52% in kidney and 77% in aorta. AT1R, alpha-klotho, and miR-155 remained unchanged in prehypertensive and hypertensive SHR. TNF-α increased by 3-fold in serum and urine of DS-HS rats. These studies unveiled in salt sensitive DS-HS, but not in SHR, a genetically conditioned dysfunction of the intermolecular network integrated by alpha-klotho, RAS, miR-155, and TNF-α that is at the helm of their end-organ susceptibility while plasminuria may participate as a second hit.

Quercetin Prevents Hypertension in Dahl Salt-sensitive Rats F ed a High-salt Diet Through Balancing Endothelial Nitric Oxide Synthase and Sirtuin 1.

A high-salt diet is a leading dietary risk factor for elevated blood pressure and cardiovascular disease. Quercetin reportedly exhibits cardioprotective and antihypertensive therapeutic effects. The objective of this study is to examine the effect of quercetin on high-salt dietinduced elevated blood pressure in Dahl salt-sensitive (SS) rats and determine the underlying molecular mechanism. Rats of the Dahl SS and control SS-13 BN strains were separated into five groups, SS-13 BN rats fed a low-salt diet (BL group), SS-13 BN rats fed a high-salt diet (BH group), Dahl SS rats fed a low-salt diet (SL group), Dahl SS rats fed a high-salt diet (SH group), and SH rats treated with quercetin (SHQ group). Blood pressure was checked three weeks into the course of treatment, and biochemical markers in the urine and serum were examined. Additionally, western blot was done to evaluate the sirtuin 1 (SIRT1) and endothelial nitric oxide synthase (eNOS) expression levels. Immunohistochemical analysis was performed to verify SIRT1 levels. We demonstrated that a high-salt diet elevated blood pressure in both SS-13 BN and Dahl SS rats, and quercetin supplementation alleviated the altered blood pressure. Compared with the SH group, quercetin significantly elevated the protein expression of SIRT1 and eNOS. Immunohistochemistry results further confirmed that quercetin could improve the protein expression of SIRT1. Quercetin reduced blood pressure by enhancing the expression of SIRT1 and eNOS in Dahl SS rats fed a high-salt diet.

Intact NOX2 in T Cells Mediates Pregnancy-Induced Renal Damage in Dahl SS Rats.

Hypertensive disorders of pregnancy are associated with increased risk for cardiovascular disease, renal disease, and mortality. While the exact mechanisms remain unclear, T cells and reactive oxygen species have been implicated in its pathogenesis. We utilized Dahl salt-sensitive (SS), SSCD247-/- (Dahl SS CD247 knockout rat; lacking T cells), and SSp67phox-/- (Dahl SS p67phox [NOX2 (NADPH [nitcotinamide adenine dinucleotide phosphate] oxidase 2)] knockout rat; lacking NOX2) rats to investigate these mechanisms in primigravida and multigravida states. We assessed blood pressure and renal damage phenotypes in SS, SSCD247-/-, and SSp67phox-/- rats during primigravida and multigravida states. To investigate the contribution of NOX2 in T cells, we performed adoptive transfers of splenocytes or cluster of differentiation (CD)4+ T cells from either SS or SSp67phox-/- donors into SSCD247-/- recipients to determine pregnancy-specific alterations in phenotype. Multigravida SS rats developed significant pregnancy-induced renal damage and renal functional impairment associated with elevated maternal mortality rates, whereas deletion of T cells or NOX2 garnered protection. During primigravida states, this attenuation in renal damage was observed, with the greatest protection in the SSp67phox-/- rat. To demonstrate that NOX2 in T cells contributes to adverse pregnancy phenotypes, adoptive transfer of SS splenocytes into SSCD247-/- rats resulted in significant pregnancy-induced renal damage, whereas transfer of SSp67phox-/- splenocytes garnered protection. Specifically, the transfer of SS CD4+ T cells resulted in pregnancy-induced proteinuria and increases in uterine artery resistance index, an effect not seen with the transfer of SSp67phox-/- CD4+ T cells. T cells and NOX2-derived reactive oxygen species, thus, contribute to end-organ damage in both primigravida and multigravida pregnancies in the SS rat leading to increases in maternal mortality.

Irisin mitigates salt-sensitive hypertension via regulating renal AMPK-Rac1 pathway

ABSTRACT Background Irisin, as a myokine, plays a protective role against cardiovascular disease, including myocardial infarction, atherosclerosis and hypertension. However, whether irisin attenuates salt-sensitive hypertension and the related underlying mechanisms is unknown. Methods Male Dahl salt-resistant (DSR) and Dahl salt-sensitive (DSS) (12 weeks) rats were fed a high salt diet (8% NaCl) with or without irisin treatment by intraperitoneal injection for 8 weeks. Results Compared with DSR rats, DSS rats showed higher systolic blood pressure (SBP), impaired natriuresis and diuresis and renal dysfunction. In addition, it was accompanied by downregulation of renal p-AMPKα and upregulation of renal RAC1 and nuclear mineralocorticoid receptor (MR). Irisin intervention could significantly up-regulated renal p-AMPKα level and down-regulated renal RAC1-MR signal, thereby improving renal sodium excretion and renal function, and ultimately reducing blood pressure in DSS rats. Ex vivo treatment with irisin reduced the expression of RAC1 and nuclear MR in primary renal distal convoluted tubule cells from DSS rats and the effects of irisin were abolished by cotreatment of compound C (AMPK inhibitor), indicating that the regulation of RAC1-MR signals by irisin depended on the activation of AMPK. Conclusions Irisin administration lowered salt-sensitive hypertension through regulating RAC1-MR signaling via activation of AMPK, which may be a promising therapeutic approach for salt-sensitive hypertension.

Abstract 33: CD4+ T Cell-Induced Renal Damage and Salt-Sensitive Hypertension is Not Modified by CD8+ T Cells

Salt-Sensitive (SS) hypertension and renal damage in the Dahl SS rat mirrors human disease with T cell infiltration in the kidney accompanying the development of SS hypertension and renal damage. Deletion of T cells on the Dahl SS genetic background (SS CD247-/- ) attenuates hypertension and renal damage, and subsequent replacement of CD4+ T cells alone restores the disease phenotype while the transfer of only CD8+ T cells had no significant effect on the disease phenotype. The present study tested the hypothesis that CD4+ and CD8+ T cells will synergistically act to enhance salt-sensitive hypertension and renal damage in the Dahl SS compared to the injection of only CD4+ T cells. This hypothesis was tested by adoptive transferring purified CD4+ T cells from Dahl SS (~5 million, CD4+ SS→), a combination of ~5 million CD4+ T cells with ~5 million CD8+ T cells from Dahl SS (CD4+CD8+ SS→) or PBS vehicle (PBS→) into the SS CD247-/- rat on postnatal day 5. Animals were instrumented with radiotelemeters at 8 weeks of age. After a week of recovery, baseline measurements of blood pressure and albumin excretion were obtained while rats were fed a low salt (LS, 0.4% NaCl) diet. Rats were then switched to a high salt (HS, 4.0% NaCl) diet for 21 days, with continuous blood pressure measurements and urine collections every 7 days. On day 21, the rats were euthanized, and their kidneys were perfused and collected for immune cell analysis by flow cytometry. During the LS baseline period there was no significant difference observed in mean arterial pressure (MAP) between the CD4+ SS→, CD4+CD8+ SS→, or PBS→ groups (121±2, 122±2, 119±1 mmHg; n=9, 10, 6, respectively). After three weeks of HS, both CD4+ SS→ and CD4+CD8+ SS→ rats demonstrated an equivalent amplification of SS hypertension (176±6 and 179±5 mmHg) and albuminuria (261±53 and 285±37 mg/day, respectively) which was significantly greater than the MAP (159±5 mmHg) and albuminuria (169±29 mg/day) in PBS→. Finally, a flow cytometric analysis at the end of the experiment confirmed the absence of CD3+ T cells in the PBS→ and documented the reconstitution of equal numbers of CD4+ cells in the blood and kidney of the rats receiving CD4+ cells or the combined CD4+ CD8+ cell transfer. We also confirmed the CD4+CD8+ was the only group that had reconstituted CD8+ T cells. These studies demonstrated that CD4+ T cells amplify salt-sensitive hypertension and renal damage in the Dahl SS independently from CD8+ T cells.

Abstract 04: Renal- and TAL-Specific Inhibition of Traf2 and NCK Interacting Kinase (TNIK) Decreases NKKC2 Phosphorylation and Blunts Salt-Sensitive Hypertension in Dahl Salt-Sensitive Rats

Up to 50% of hypertensive patients are salt-sensitive. Abnormally enhanced NaCl reabsorption by the thick ascending limb of the loop of Henle (TAL) in the kidney contributes to the development of salt-sensitivity. NaCl reabsorption in the TAL is entirely mediated by the apical Na/K/2Cl cotransporter NKCC2. We showed that NKCC2 phosphorylation at Thr-96,101 is increased in TALs from Dahl salt-sensitive (DSS) rats. We discovered a new kinase, TNIK (Traf2-and-NCK-interacting kinase), that binds the amino-terminus of NKCC2 and phosphorylates NKCC2 at Thr-96,101. TNIK is abundantly expressed in TALs and other nephron segments. We hypothesized that TNIK contributes to salt-sensitive hypertension by increasing NKCC2 phosphorylation in TALs from DSS rats. First, we measured TNIK expression in isolated TALs. TNIK expression was 8±4-fold higher in TALs from DSS compared to SD rats (n=5, p<0.05). Then, we used a pharmacological specific inhibitor of TNIK, NCB-0846. We implanted renal medullary catheters connected to minipumps to directly infuse NCB-0846 or vehicle into the left kidney of uninephrectomized DSS rats. After recovery rats were fed 4% NaCl diet and measured systolic blood pressure (SBP) via tail-cuff. NCB-0846 decreased SBP by 21±6 mmHg within four days compared to the vehicle-infused group, and BP remained lower for 7 days (n=5, p<0.03 vs vehicle). To specifically decrease TNIK gene expression in medullary TALs, we used Cas9-mediated gene editing. We injected the renal medulla of the left kidney with AAV-gRNA-TNIK simultaneously with AV-NKCC2promoter-Cas9 (drives Cas9 expression in TALs) or AV-NKCC2promoter-Cas9 alone (AV-control) in DSS rats. After 10 days, rats were fed a 4% NaCl diet and SBP was measured. Rats transduced with AAV-gRNA-TNIK had a 18±5 mmHg lower BP compared to AV-control group (AV-control:147 ± 4.7 vs AAV-gRNA-TNIK:129 ± 5.6 mmHg n=5, p<0.03 vs control) which remained lower after 14 days (AV-control:163 ± 4.8 vs AAV-gRNA-TNIK:140 ± 4.9 mmHg, n=5, p<0.01). After 14 days on high salt, TNIK expression was decreased by 30 ± 4% (n=4, p<0.05), and pThr-96,101-NKCC2 decreased by 43 ± 11% in AAV-gRNA-TNIK transduced TALs compared to AV-control transduced TALs (n=4, p<0.02). We conclude that hypertension and enhanced NKCC2 phosphorylation in the DSS rat is partly due to higher TNIK expression in TALs and that inhibition of this novel kinase could be a target for salt-sensitive hypertension.

Abstract MP31: The effect of dual SGLT1/2 inhibition on the progression of salt-sensitive hypertension

The dual inhibition of sodium-glucose co-transporter 1 and 2 (SGLT1/SGLT2) presents a novel therapeutic approach to cardiovascular diseases. The FDA recently approved the dual SGLT1/2 inhibitor sotagliflozin (Sota) for treating heart failure regardless of ejection fraction. However, our understanding of the effects and mechanisms of SGLT2 and dual SGLT1/2 inhibitors in salt-sensitive hypertension (SS HTN) remains limited. Thus, additional studies are needed to reveal the role and mechanism(s) of SGLTi in SS hypertension. In our previous studies, we demonstrated the beneficial effect of SGLT2i dapagliflozin (Dapa) on SS HTN in male and female Dahl SS rats on blood pressure (MAP after 3 weeks on high salt diet were: Dapa vs. vehicle: 136 ± 3 vs. 156 ± 6 mmHg in males and 140 ± 5 vs. 160±9 mmHg in females). This study aims to define the effects and potential mechanisms of dual SGLT1/2 inhibition in managing SS HTN. Dual SGLT1/2 inhibition with Sota (30 mg/kg/day) prevented the development of SS HTN in Dahl SS in both sexes without changes in heart rate. By the 21 st day of the high-salt challenge, MAP was lower more than 30 mmHg in treated groups (Sota vs. vehicle: male rats 126 ± 2 vs 157 ± 5 mmHg (N=7, p=0.0002); female rats 126 ± 3 vs 167 ± 10 mmHg (N=6, p=0.0095). Under Sota treatment, rats had significantly increased diuresis (Sota vs. vehicle: males 69 ± 3 vs 35 ± 2 ml and females 48 ± 5 vs 31 ± 2 ml on day 21), glucose and sodium excretion. In both sexes, Sota treatment resulted in a lower heart-to-body weight ratio, and kidney weight was not changed. Also, Sota attenuated kidney fibrosis and protein cast formation (Sota vs. vehicle: cortex fibrosis in males 2.3 ± 0.4 vs 4.6 ± 0.5 % and females 2.0 ± 0.2 vs 4.0 ± 0.7 %, outer medullary protein casts in males 2.5 ± 0.2 vs 16.0 ± 2.6 % and 1.9 ± 0.3 vs 7.5 ± 0.9 % in females) without changes in albuminuria in both sexes. However, no changes in glomerular filtration rate or creatine clearance were detected. Based on our data, it can be inferred that dual SGLT1/2 inhibition prevents the development of SS HTN and kidney damage. Potential mechanisms underlying those beneficial effects of SGLT1/2 inhibition will be further explored.

Abstract 13: Vascular Chemerin, Particularly from PVAT, contributes to Norepinephrine and Serotonin-Induced Vasoconstriction and Vascular Stiffness in a Sex-Dependent Manner

The adipokine chemerin has been implicated in healthy blood pressure maintenance and adiposity-associated hypertension, evidenced by falls in mean arterial pressure after administration of an antisense oligonucleotide against chemerin in Dahl SS rats. Circulating levels of chemerin are positively associated, in humans, with hypertension and aortic stiffening—a blood pressure-independent risk factor of cardiovascular disease. While chemerin’s role in blood pressure is observed in whole animals, mechanisms of chemerin’s influence on vascular health and disease remain unknown. Chemerin causes direct vasoconstriction through its main receptor Chemerin1 and indirect vasoconstriction by amplifying electrical field stimulation-induced contraction. We identified chemerin production at the vasculature—the blood vessel and its perivascular adipose tissue (PVAT)—and are working to discover the mechanistic role of vascular chemerin in blood vessel function and disease. Using RNAScope®, QPCR, isometric contractility, western blot, and in vivo imaging, we test the hypothesis that vascular chemerin increases arterial contractility through Chemerin1 and plays a role in aortic stiffening. RNAScope® probes detected CMKLR1 expression in the tunica media and PVAT of the thoracic aorta. QPCR revealed Rarres2 expression was higher in the PVAT, and CMKLR1 expression was higher in the media. Chemerin1 antagonism via the selective inhibitor CCX832 (1μM) significantly reduced maximal contraction to both norepinephrine (NE) and serotonin (5-HT) (see figure), but not angiotensin II, in the male rat +PVAT groups. This supports that PVAT-derived chemerin plays a role in sympathetic stimulation of the vasculature. Interestingly, in females, this change in contractile response to NE and 5-HT was not observed. Western blot showed that females express significantly lower levels of chemerin protein in PVAT, suggesting PVAT as the primary source of active chemerin. Finally, male, but not female, genetic chemerin knockout Dahl SS rats have lower aortic arch pulse wave velocity than wild types (M WT: 3.7 ± .7 M KO: 1.4 ± .3 F WT: 2.6 ± .5, F KO: 1.5 ± .3 m/s, N=4-6/group), highlighting a role of chemerin in aortic stiffening. Thus, chemerin, sourced by the PVAT, plays a significant role in vascular contractility and aortic stiffening in males. Future work will determine if chemerin’s influence is amplified in adiposity-associated hypertension, where chemerin levels are elevated in both sexes.

Abstract 06: Role of the Atrial Natriuretic Peptide in Mitochondria-mediated Proximal Tubule Epithelial Oxidative Stress in Type 1 Diabetic Kidney Disease

Introduction: Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease. Most studies of DKD are glomerulo-centric. The recent progress in the control of renal glucose handling by SGLT2i makes the proximal tubule (PT) a prime candidate. Mitochondrial (mito) dysfunction and mito-induced oxidative stress in DKD is well-studied and is known to accelerate tubular injury. Some studies have suggested the role of the Atrial Natriuretic Peptide (ANP) in DKD. ANP is a hormone that regulates renal salt/water homeostasis and renal hemodynamics. We hypothesize that in DKD, ANP is a beneficial factor that improves proximal tubule mitochondrial function via protection from oxidative stress-induced ferroptosis. Methods: Using male ANP knock-out (SS NPPA-/- ) and wild-type (SS WT ) rats on the Dahl SS background, T1DKD was induced by streptozotocin (STZ; 75mg/kg) at 9-10 weeks of age. Baseline, midpoint, and endpoint blood glucose, urine, and body weights were collected. Western blotting was done on isolated cortical tissue and tissue fibrosis was measured in PicroSirius Red staining. ANOVA through Origin 2019b was used for comparisons. Results: STZ- SS WT and STZ-SS NPPA-/- groups exhibited similar body weight (231.7 ± 14.8 vs. 230.9 ± 8.4 g, respectively), two-kidney weight to body weight ratio (31.8 ± 0.9 vs. 28.4 ± 1.5 mg/g), and endpoint hyperglycemia (with blood glucose levels >650 mg/dL). Both, STZ- SS WT and STZ-SS NPPA-/- groups had high diuresis (59.9 ± 3.1 vs. 52.7 ± 3.2 mL/24hr/100gTBW). Histological analysis revealed elevated cortical fibrosis in the STZ-SS NPPA-/- compared to STZ- SS WT (4.6 ± 0.2 vs. 3.1 ± 0.2 % area, respectively, p<0.05). Moreover, in the kidney cortex, STZ-SS NPPA-/- rats compared to STZ- SS WT rats exhibit a significant increase in OXPHOS complexes IV and V (p<0.05) and a decrease in glutathione peroxidase 4 (GPX4; a core regulator of ferroptosis) abundance (1.5 ± 0.2 vs. 1.9 ± 0.1 a.u., respectively, p<0.05). Conclusion: Data showed that lack of ANP may promote DKD-related tubulointerstitial fibrosis and suggested a relationship with renal cortical mitochondrial function. Future studies will provide mechanistic insight into the role of ANP in mito-related oxidative stress and further assess the role of ANP in PT ferroptosis.

Abstract P452: Cell Populations and Transcriptomes Differ Across Blood Pressure and Sex in Perivascular Adipose Tissue: A Single-Nuclei RNA Sequencing Study

The perivascular adipose tissue (PVAT), fat that surrounds many blood vessels, is capable of modulating vascular tone through multiple modalities. Despite increasing research interest, PVAT remains less understood compared to other vascular layers. We hypothesized that the transcriptome and cell population of thoracic aorta PVAT (taPVAT) differ between hypertensive and normotensive rats. To test this hypothesis, we performed single-nucleus RNA-sequencing (snRNA-seq) on twenty-five taPVAT samples from male and female Dahl SS rats, fed either control (Con) chow or a high-fat diet (HFD) for 8 or 24 weeks. Mean arterial pressure (MAP) measurements showed elevated values in female rats fed a HFD for 24 weeks with a trend towards increasing blood pressure in male rats (Fig. 1a). Our analysis included at least three taPVAT samples per treatment group, resulting in 71,813 high-quality sequenced nuclei, representing eight major cell types (Fig. 1b) and twenty-eight cell sub-types. Within each cell type, the differences in population and gene expression across diet, diet duration, and sex were examined. The most substantial population proportion differences were observed across diet duration, with fewer differences noted across diet and sex (Fig. 1c). The total number of differentially expressed genes (DEGs) (adjusted p-value ≤ 0.05, |log 2 (fold change)| ≥ 1) across diet, diet duration, and sex were 191, 424, and 119, respectively. Interestingly, in the taPVAT from HFD fed rats, the gene Csmd1 (CUB and Sushi multiple domains 1) was consistently upregulated across nearly all cell types. Whereas Scd (stearoyl-CoA desaturase), which is involved in fatty acid biosynthesis, was frequently the most downregulated gene. Pathway enrichment analysis revealed that the DEGs related to the HFD were associated with a decrease in lipid and fatty acid biosynthetic processes. DEGs related to rats fed a HFD for twenty-four weeks versus eight weeks were linked to an enrichment in inflammatory response and extracellular matrix organization pathways, and in HFD fed male rats compared to females, there was enrichment for leukocyte aggregation and peptidyl-cysteine modification pathways. In conclusion, our findings help paint a more detailed picture of the molecular and cellular changes in taPVAT associated with hypertension.

Abstract P220: Abdominal Aortic Perivascular Adipose Tissue Lipolysis Is Activated In Hypertensive Dahl SS Rats Fed a High-Fat Diet

Perivascular adipose tissues (PVAT) possess anti-contractile functions that are lost during hypertension (HTN). PVAT stores fatty acids (FA) and releases them through lipolysis upon adrenergic or inflammatory stimuli. Visceral AT lipolysis in humans with high abdominal adiposity and hypertension elevates FA release, promoting HTN via lipotoxicity and increased oxylipin synthesis. We hypothesize that lipolytic activity in abdominal aortic PVAT (aaPVAT) is increased during HTN. Plasma and aaPVAT were harvested from Dahl SS rats fed a high-fat (HF) or a control (CON) diet for 8 (♂=20, ♀=20), 16 (♂=10, ♀=10) and 24 (♂=14, ♀=14) wks. Mean arterial pressure (MAP±SEM; mmHg) was measured with sphyngomanometry. Free FA (FFA), β-hydroxybutyrate (BHB), glucose, and triglycerides were quantified with the CataChemWell-T analyzer, and individual FA using HPLC–MS/MS (reported as ng/dL). Adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and phosphorylated pHSL(Ser563) were quantified in the Abby Western Blot. HF increased MAP compared to CON by 8 (131.5 vs 119.51±2.52), 16 (139.34 vs 118.26±5.7), and 24 wks (168.56 vs 121.94±3.93). HF at 24 wks had higher MAP compared to 8 and 16 wks. HF increased FFA and BHB at all time points and glucose at 24 wks. Plasma triglycerides were increased in HF vs. CON, and ♂ had higher levels than ♀ at all time points. ATGL, basal lipolysis rate-limiting enzyme, increased in aaPVAT from HF ♂ by 2.2, 2.53, and 1.82-fold at 8, 16, and 24 wks, respectively, compared to CON. In HF ♀, ATGL increased (1.95-fold change) at 16 wks vs. 8 wks. HSL content increased in HF vs. CON at 16 wks (2.1-fold change) but not at 24 wks. In contrast, pHSL abundance and the pHSL:HSL ratio were not affected by diet at 16 wks; however, both were reduced by 35.2% at 24 wks in HF ♂ and ♀. Lipolytic activity in HF coincided with higher plasma arachidonic (AA) and linoleic acid (LA) at 8 (AA HF=19.93, CON= 16.13±1.3; LA HF=29.22, CON=21.74±2.7) and 16 wks (AA HF=14.83, CON=11.61±1.34; LA HF=22.84, CON=11.66±2.9). Sex had no effect on AA or LA concentrations. Our results provide evidence that hypertensive Dahl SS rats fed HF exhibit higher aaPVAT basal lipolysis that may be driven by ATGL. In contrast, demand lipolysis, controlled by HSL activity, is reduced. Dysregulated basal lipolysis may lead to alterations in PVAT FA trafficking, enhance AA availability, and promote the biosynthesis of inflammatory oxylipins, thus impairing its vasoactive functions.

Abstract P455: Integrins play a role in stress relaxation of perivascular adipose tissue

Perivascular adipose tissue (PVAT) is well known for being anti-contractile (reducing potency, maximum) in healthy tissues. We discovered a new function of PVAT, the ability to stress relax in response to a stretch. This is of note because stress relaxation has been attributed largely to smooth muscle, of which PVAT has none that is organized in a functional layer. Here we test the hypothesis that stress relaxation depends on the interactions of the cell membrane delimited heterodimer integrins with collagen. Our model is the thoracic aorta of the male Dahl SS rat on a normal diet. The PVAT and aorta were physically separated for most assays. Results from snRNA seq in PVAT, histochemistry and isometric contractility were used. Collagen was evident in PVAT as stained by Masson Trichrome. snRNA seq of whole PVAT determined expression of many collagen isoforms and integrins, with mRNA for both the α1 and β1 integrin being most highly expressed. Isolated aortic rings and separated PVAT were challenged with 2 or 4 grams of passive tension. Pharmacological inhibition of integrin/collagen interaction was effected by the specific α1β1 distintegrin obtustatin (5 uM) or general inhibitor RGD peptide (1 mM). RGD peptide but not obtustatin reduced the ability of the isolated PVAT ring to maintain applied tone at both the 2 and 4 gram challenge (~15% gain in relaxation/loss of tone, figure ). By contrast, stress relaxation in the aorta itself was not reduced by either inhibitor. In addition to a1b1 and collagen interaction, cell-cell communication inference identified integrins av and a5, two major RGD motif containing isoforms, as potential partners of collagens with fibroblast-like cells most likely involved. Collectively, these findings validate that stress relaxation can occur in a non-smooth muscle tissue, doing so at least in part through integrin-collagen interactions that may not include α1β1 heterodimers. The importance of this lies in considering PVAT as a vascular layer that possesses mechanical functions.

Abstract P203: Nicotine Exposure: A Catalyst for Nitrosative and Oxidative Stress in Glomerular Podocytes and Renal Impairment

Introduction: Nicotine consumption through vaping, cigarettes, or nicotine replacement therapy poses major risks for renal health, especially in individuals with hypertension or kidney disease. Nicotine induces oxidative stress and nitric oxide synthase (NOS) remodeling in endothelial cells, but a gap in knowledge persists regarding its impact on glomerular epithelial cells, particularly podocytes. We hypothesize that nicotine promotes nitrosative stress in podocytes, generating peroxynitrite (ONOO - ), causing mitochondrial damage, and glomerular impairment. Methods: We performed confocal imaging on human cultured podocytes to detect ONOO - , Ca 2+ , and nitric oxide (NO) in response to nicotine. We used Seahorse assay (Agilent XFe24) to test mitochondrial respiration. Nicotine was then chronically infused in Dahl SS rats (0.2 mg/kg/day s.c., 0.4% NaCl diet) for 21 days, and recorded blood pressure with telemetry. Glomerular damage was evaluated, and electron microscopy was employed to assess mitochondrial ultrastructure. Electron paramagnetic resonance (EPR) spectroscopy was used to access the NO levels in cultured podocytes and in vivo . OriginPro was used for statistical analysis. Results: Acute application of nicotine promoted intracellular Ca 2+ and ONOO - transients in podocytes. The ONOO - response was blocked in the presence of SOD, indicating that ONOO - production requires superoxide. The application of specific α7, α4β2, α2β4, α4β4, and α3β4 nicotinic acetylcholine receptor (nAChR) agonists elicited Ca 2+ transients but did not produce ONOO - , suggesting that nitrosative stress occurs independently from nAChR activation. Incubation with nicotine (12 hrs) resulted in a decrease in podocytes’ mitochondrial respiration (two-sample t-test, p<0.05). In vivo , nicotine infusion did not affect blood pressure but promoted mitochondrial damage in podocytes, which exhibited swelling, loss of cristae, and mitophagy (t-test, p<0.05). Histopathological assessment showed higher glomerular damage in nicotine-exposed rats (t-test, p<0.05). Both EPR and confocal approaches demonstrated nicotine-mediated changes in NO bioavailability and an increase in NOS2 activity (t-test, p<0.05). Conclusion: Nicotine products promote NOS uncoupling in podocytes, leading to ONOO - formation, redox stress, mitochondrial impairment, and glomerular damage. Understanding nicotine's impact on podocytes is crucial for preventing or developing therapies against smoking and vaping-related pathologies.

Abstract P175: The Effect of TRF on Hypertension Development in Male Dahl Salt-Sensitive Rats

Time restricted feeding (TRF), or food consumption restricted to <10 hrs/day, has been shown to improve blood pressure in various cardiometabolic diseases, yet there is limited data on the effect in SS hypertension. The goal of this study was to examine the effect of TRF on disease development in the Dahl SS rat. We hypothesized that implementing TRF during a critical timepoint in disease progression would reduce mean arterial blood pressure (MAP). To study this, femoral catheters were surgically implanted in male Dahl SS rats at 9±0.5 weeks of age (n=5-7/group), allowing for urine and arterial blood sample collection from conscious rats during continual blood pressure recording. After 1 week of recovery, rats either continued a low salt (LS;0.4% NaCl) diet or were switched to a high salt diet (HS;4% NaCl) with ad libitum (ad lib) access for 1 week. At the start of week 2, a subset of rats began TRF, in which food was provided for 8 hrs (10PM-6AM) during their active period for an additional week. Rats meeting the following criteria were included in analysis: pulse pressure > 10 mmHg, baseline MAP of 115-130 mmHg, and a 10-20 mmHg increase in MAP after 1 week on HS. Differences in MAP were analyzed by 2-way ANOVA with Tukey multiple comparisons and data are reported as mean ± SEM. By the end of week 2, HS TRF rats trended toward lower average MAP compared to HS ad lib rats (141.51±3.41 vs 149.49±1.65 mmHg; p=0.07). However, we found that the HS TRF rats had reduced MAP during their active period (6PM-6AM), compared to the HS ad lib rats (142.60±2.99 vs 151.65±1.862 mmHg; p<0.05), but no difference during the inactive period (6AM-6PM). At the end of the study, average MAP was calculated every 4 hrs to identify the timing at which TRF reduced MAP. We found that the HS TRF rats had lower MAP from 6PM to 10PM (134.61±2.34 vs 146.75±1.21 mmHg; p<0.05) and 10PM to 2AM (144.58±2.79 vs 154.70±2.18 mmHg; p<0.05) when compared to the HS ad lib rats. The LS TRF group also had lower pressure from 6PM-10PM compared to the LS ad lib group (124.93±1.19 vs 130.56±0.73 mmHg; p<0.05), suggesting that this time-specific reduction in MAP may be due to fasting, as there were no other differences in MAP within the LS groups. This suggests that TRF may slow progression of hypertension development in the Dahl SS rat, primarily by affecting MAP during the active period. Future studies will examine how TRF affects Na + handling to further understand these time-dependent effects.

The effects of the atrial natriuretic peptide deficiency on renal cortical mitochondrial bioenergetics in the Dahl SS rat.

Atrial Natriuretic Peptide (ANP) plays an important role in blood pressure regulation. Low levels of ANP correlate with the development of salt-sensitive hypertension (SS-HTN). Our previous studies indicated that ANP deficiency exacerbated renal function decline in SS-HTN. In the heart and fat tissue, ANP was reported to affect lipid peroxidation and mitochondrial bioenergetics but the effects of ANP on mitochondrial function in the kidney are unexplored. We hypothesized that ANP deficiency in SS-HTN causes renal bioenergetic shift, leading to disruption of mitochondrial network and oxidative stress. To address the hypothesis, we placed Dahl SS wild-type (SSWT) and ANP knockout (SSNPPA-/-) rats on 4% NaCl high salt (HS) diet to induce HTN or maintained them on 0.4% NaCl normal salt (NS) diet and assessed mitochondrial bioenergetics and dynamics using spectrofluorimetry, Seahorse assay, electron paramagnetic resonance (EPR) spectroscopy, Western blotting, electron microscopy, PCR and cytokine assays. We report that under high salt conditions, associated with hypertension and renal damage, the SSNPPA-/- rats exhibit a decrease in mitochondrial membrane potential and elevation in mitochondrial ROS levels compared to SSWT. The redox shift is also evident by the presence of more pronounced medullar lipid peroxidation in the SSNPPA-/- strain. We also revealed fragmented, more damaged mitochondria in the SSNPPA-/- rats, accompanied by increased turnover and biogenesis. Overall, our data indicate that ANP deficiency causes disruptions in mitochondrial bioenergetics and dynamics which likely contributes to aggravation of the renal damage and hypertension in the Dahl SS rat; the major pathological effects are evident in the groups subjected to a combined salt and ANP deficiency-induced mitochondrial stress.

Sex differences in dietary sodium evoked NCC regulation and blood pressure in male and female Sprague-Dawley, Dahl salt-resistant, and Dahl salt-sensitive rats.

Hypertension affects approximately one in two United States adults and sex plays an important role in the pathogenesis of hypertension. The Na+-Cl- cotransporter (NCC), regulated by a kinase network including with-no-lysine kinase (WNK)1 and WNK4, STE20/SPS1-related proline alanine-rich kinase (SPAK), and oxidative stress response 1 (OxSR1), is critical to Na+ reabsorption and blood pressure regulation. Dietary salt differentially modulates NCC in salt-sensitive and salt-resistant rats, in part by modulation of WNK/SPAK/OxSR1 signaling. In this study, we tested the hypothesis that sex-dependent differences in NCC regulation contribute to the development of the salt sensitivity of blood pressure using male and female Sprague-Dawley (SD), Dahl salt-resistant (DSR), and Dahl salt-sensitive (DSS) rats. In normotensive salt-resistant SD and DSR rats, a high-salt diet evoked significant decreases in NCC activity, expression, and phosphorylation. In males, these changes were associated with no change in WNK1 expression, a decrease in WNK4 levels, and suppression of SPAK/OxSR1 expression and phosphorylation. In contrast, in females, there was decreased NCC activity associated with suppression of SPAK/OxSR1 expression and phosphorylation. In hypertensive DSS rats, the ability of females to suppress NCC (in opposition to males) via a SPAK/OxSR1 mechanism likely contributes to their lower magnitude of salt-sensitive hypertension. Collectively, our findings support the existence of sex differences in male versus female rats with NCC regulation during dietary salt intake involving suppression of WNK4 expression in male rats only and the involvement of SPAK/OxSR1 signaling in both males and females.NEW & NOTEWORTHY NCC regulation is sex dependent. In normotensive male and female Sprague-Dawley and Dahl salt-resistant rats, which exhibit dietary Na+-evoked NCC suppression, male rats exhibit decreased WNK4 expression and decreased SPAK and OxSR1 levels, whereas female rats only suppress SPAK and OxSR1. In hypertensive Dahl salt-sensitive rats, the ability of females to suppress NCC (in opposition to males) via a SPAK/OxSR1 mechanism likely contributes to their lower magnitude of salt-sensitive hypertension.

Sex-Dependency of T Cell-Induced Salt-Sensitive Hypertension and Kidney Damage.

It is established that the immune system, namely T cells, plays a role in the development of hypertension and renal damage in male Dahl salt-sensitive (SS) rats, but far less is known about this relationship in females. Rats with genetically deleted T cells via CD247 gene mutation on the Dahl SS background (SSCD247-/-) were utilized to interrogate the effect of sex and T cells on salt sensitivity. We assessed the hypertensive and kidney injury phenotypes in male versus female SS and SSCD247-/- rats challenged with 3 weeks of high salt (4.0% NaCl). Differences in T cell activation genes were examined in renal T cells from male and female SS rats, and a sex-specific adoptive transfer was performed by injecting male or female splenocytes into either male or female SSCD247-/- recipients to determine the potential contribution of T cell sex. The lack of functional T cells in SSCD247-/- rats significantly reduced salt-induced hypertension and proteinuria in both sexes, although SSCD247-/- females exhibited greater protection from kidney damage. Adoptive transfer of either Dahl SS male or female splenocytes into SSCD247-/- male recipients exacerbated hypertension and proteinuria compared with controls, while in SSCD247-/- female recipients, exacerbation of disease occurred only upon transfer of male, but not female, SS splenocytes. The absence of T cells in the SSCD247-/- normalized sex differences in blood pressure, though sex differences in renal damage persisted. Splenocyte transfer experiments demonstrated that salt sensitivity is amplified if the sex of the T cell or the recipient is male.

Progesterone Supplementation Improves the Maternal Syndrome of Superimposed Preeclampsia in the Dahl Salt Sensitive Rats

Introduction: Preeclampsia (PE) is characterized by new onset of hypertension after 20 weeks of gestation. It affects 5-7% of all pregnancies in the U.S., and is associated with reduced fetal weight, inflammation and hypertension (HTN). Importantly, 30% of HTN disorders in pregnancy are caused by chronic HTN that is present prior to pregnancy which increases the risk of superimposed PE (SIPE). The mechanisms responsible for the pathogenesis of PE and SIPE are unclear and currently the only treatment is early delivery of the fetus. Progesterone is important for the establishment and maintenance of pregnancy. We have previously shown that progesterone supplementation with 17-hydroxyprogesterone caproate (17-OHPC) improves inflammation, fetal weight and blood pressure in the preclinical RUPP rat model of PE, however the mechanism for this in SIPE is still unknown. Hypothesis: This study was designed to test the hypothesis that 17-OHPC reduces inflammation while improving maternal blood pressure in the preclinical pregnant Dahl Salt Sensitive (DS) rat model of SIPE. Methods: 17-OHPC (3.32mg/kg) or vehicle (Saline) was administrated intraperitoneally on gestation day (GD) 15 to normal pregnant (NP) Sprague-Dawley (SD) and pregnant Dahl Salt Sensitive (DS) rats. On GD 18, Uterine Artery Resistance Index (UARI) was measured by Vevo Doppler Ultrasound and carotid catheters were inserted. On GD 19, mean arterial blood pressure (MAP) and samples were collected. All data are expressed as mean ± standard error means (SEM). Results: MAP was 105±5 mmHg in SD+ Saline rats (n=7) and 105±4 mmHg in SD+17-OHPC rats (n=4), 137±3 mmHg in DS + Saline rats (p<0.05, n=11), which improved to 125±4 mmHg in DS+17-OHPC rats (p<0.05, n=7). Pup and Placenta weights were 2.1± 0.1 g, 0.6 ± 0.1 g in SD + saline rats and significantly reduced to 1.4±0.1 g, 0.5±0.1 g in DS+ Saline rats (p<0.05). Neither placental weight nor pup weight was affected by 17-OHPC. UARI was 0.5 ±0.1 in SD+ Saline rats (n=6) and 0.5 ±0.1 in in SD+17-OHPC (n=4), 0.7±0.1 in DS+Saline rats (n=6, p<0.05), which reduced to 0.5±0.1 in DS+17-OHPC (n=7, p<0.05). TNF-alpha levels were 3.0±1.0 pg/mL in SD+ Saline rats, 11.0±1.2 in DS + Saline rats, which reduced to 4.1±1.6 in DS+17-OHPC (n=4, p<0.05). Importantly, circulating and placental CD4+ T cells were 9.3±3.7 % Gate and 6.4± 1.6 % Gate in SD+ Saline rats (n=4), 38.9 ±2.9 % Gate and 33.1±4.3 % Gate in DS + Saline rats (n=6), which significantly reduced to 26.3 ± 0.1 % Gate and 3.4±1.2 % Gate in DS + 17-OHPC. Conclusion: Collectively, our findings demonstrate that 17-OHPC reduces inflammation and hypertension in the Dahl Salt Sensitive rat model of SIPE. Supported by NIH P20GM121334. 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.

Salt-Sensitive Hypertension and Renal Damage are Amplified by Adoptive Transfer of CD4+ and Not CD8+ T Cells: A Role of NADPH Oxidase 2 (NOX2)

Infiltration of T cells into the kidney of the Dahl Salt Sensitive (SS) rat model of human disease accompanies the development of salt-sensitive hypertension and renal damage. Deletion of T cells on the Dahl SS genetic background (SSCD247−/−) attenuates hypertension and renal damage, and subsequent replacement of T cells by splenocyte transfer restores the disease phenotype. The present studies were performed to assess if the T cell subtype (CD4+ vs CD8+) is important in Dahl SS hypertension and interrogate the mechanism of action. In initial experiments, adoptive transfer of purified CD4+ or CD8+ T cells (~5 million) from splenocytes isolated from the Dahl SS (CD4+ SS→CD247 or CD8+ SS→CD247) or PBS vehicle (PBS→CD247) into the SSCD247−/− rat was performed on postnatal day 5. Animals were recovered and instrumented with radiotelemeters at 7 weeks of age. After a week of recovery, baseline measurements of blood pressure and albumin excretion were obtained while rats were fed a low salt (LS, 0.4% NaCl) diet. Rats were then switched to a high salt (HS, 4.0% NaCl) diet for 21 days, with continuous blood pressure measurements and urine collections every 7 days. On day 21, the rats were euthanized, and their kidneys were perfused and collected for immune cell analysis by flow cytometry. During the LS baseline period there was no significant difference observed in mean arterial pressure (MAP) between the CD4+ SS→CD247, CD8+ SS→CD247, or PBS→CD247 groups (113±1, 115±1, 114±1 mmHg; n=21, 10, 24, respectively). After three weeks of HS, rats receiving CD4+ T cells from the SS (CD4+ SS→CD247) demonstrated an amplified salt-sensitive hypertension (154±3 mmHg) and albuminuria (184±22 mg/day) compared to blood pressure (140±3 and 145±3 mmHg) and albuminuria (82±9 and 85±8 mg/day) in the CD8+ SS→CD247 or PBS→CD247 groups, respectively. Since the deletion of the p67phox subunit of NADPH oxidase 2 (NOX2) in adoptively transferred splenocytes attenuated Dahl SS hypertension and kidney damage, parallel studies examined the influence of deletion of p67phox in CD4+ (n=14) and CD8+ T cells (n=8). The adoptive transfer of either CD4+ or CD8+ T cells lacking functional NOX2 did not alter the LS or HS blood pressure or albuminuria when compared to the PBS control group. Finally, a flow cytometric analysis at the end of the experiment confirmed the absence of CD3+ T cells in the PBS→CD247 and documented the reconstitution of equal numbers of CD4+ cells in the blood and kidney of the rats receiving CD4+ cells with intact or deficient NOX2. Interestingly, no difference was observed in CD8+ T cells in the kidney and blood of rats receiving CD8+ cells with intact or deficient NOX2, but a significant number of CD4+ T cells were also observed in the SSCD247−/− rats receiving the purified CD8+ T cells. In summary, these studies demonstrate that CD4+ T cells amplify salt-sensitive hypertension and renal damage in the Dahl SS via a NOX2-dependent mechanism while CD8+ T cells do not alter disease phenotypes when compared to PBS control. GRANTS: HL161231, HL166458, Georgia Research Alliance. 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.

Toll-like Receptor 4 Promotes Salt-Sensitivity in Dahl SS Rats: An Investigation into the Mechanism

Renal inflammation accompanies hypertension and renal damage in Dahl Salt-Sensitive (SS) rats fed a high salt diet (HS, Dyets AIN-76A 4.0% NaCl). Previous studies demonstrated that toll-like receptor 4 (TLR4), a component of innate immune activation, is upregulated in macrophages infiltrating the kidneys of Dahl SS rats fed high salt. Preliminary studies in our laboratory indicated that mean arterial pressure (MAP) (SS: 162.5 ± 4.9 vs. SSTLR4−/−: 147.1 ± 3.5 mmHg, p<0.0001, n=10/group) and albuminuria (321.3 ± 62.7 vs. 170.6 ± 27.1 mg/day, p<0.05, n=10/group) were significantly reduced in male Dahl SS TLR4 knockout rats (SSTLR4−/−) compared to male SS after three weeks of HS. Furthermore, peritoneal macrophages isolated from SSTLR4−/− produce significantly less IL-1β compared to SS macrophages upon stimulation with lipopolysaccharide (LPS) . Since TLR4 is widely expressed in many tissues, we examined the specific effects of TLR4 deletion in immune cells via a total body irradiation/bone marrow transfer approach. The present study tested the hypothesis that TLR4 deletion specifically in hematopoietic cells would attenuate male Dahl SS hypertension and renal damage. At 6 weeks of age, Dahl SS male recipients were irradiated (11Gy) then received either SS or SSTLR4−/− bone marrow transplantation via tail vein injection (~15-20 million cells). Following recovery, rats (maintained on a low salt chow (LS, Dyets AIN-76A 0.4% NaCl)) underwent telemeter implantation at approximately 8 weeks of age for continuous monitoring of blood pressure. Animals were switched to HS following recovery and baseline blood pressure recordings. Urine samples were collected once during the LS period and weekly throughout a 3-week HS challenge. Upon euthanasia, kidneys were flushed and harvested for analysis. MAP was not found to be different between groups during the LS baseline period (SS: 113.4 ± 1.6 vs. SSTLR4−/−: 116.5 ± 2.3 mmHg, n=5-6/group), and our preliminary data also revealed that MAP was not different between groups throughout the high salt challenge (HS day 20: 136.4 ± 10.5 vs 144.8 ± 9.2). Urinary albumin excretion was also not found to be different between rats receiving either SS or TLR4−/− bone marrow transfer (165.2 ± 38.5 vs. 247.9 ± 58.7 mg/day, n=7/group). Furthermore, assessment of BUN at the conclusion of the experiment revealed no differences (33.0 ± 6.1 vs. 29.3 ± 3.2 mg/dL, n=7/group). Immune cells were isolated from the blood and kidneys of these animals and characterized by flow cytometric analysis. There was no difference in the number of circulating leukocytes or lymphocytes (2.1x106 vs. 2.3x106 CD45+ cells per mL blood, n=6-7/group) indicating comparable reconstitution of immune cells in both groups. We also found no difference in renal infiltrating leukocytes or lymphocytes (3.1 x106 vs. 3.2x106 CD45+ cells per kidney, n=7/group). Together, these data contrast the effects of whole-body TLR4 knockout and suggest that TLR4 in hematopoietic cells may not be responsible for promoting Dahl SS hypertension and renal damage in males. Further studies are needed to assess the inflammatory status of immune cells from these animals as well as alternative TLR4-expressing cell types responsible for promoting disease. 23PRE1020121, HL161231, HL166458. 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.