Dahl Salt-sensitive Genetic Background Research Articles

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.

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.

Abstract 008: Sex-dependency Of T Cell-induced Salt-sensitive Hypertension And Kidney Damage

We have previously demonstrated that female Dahl Salt-Sensitive (SS) rats are protected from salt-induced hypertension and renal injury compared to males. There is an immunological component to this sex difference, since genetic deletion of T cells via mutation of the CD247 gene on the Dahl SS genetic background (CD247-/-) normalizes sex differences in blood pressure, though females are still protected from salt-sensitive renal damage compared to males. The current study hypothesized that sex of the T cell is an important factor in determining the severity of salt-sensitivity. We tested the hypothesis via a sex-specific splenocyte transfer by freshly isolating 10 7 male or female splenocytes and injecting them i.p. on postnatal day 5 into either male or female CD247-/- recipients. At 9 weeks of age, rats underwent a 3 week high salt challenge (HS, 4.0% NaCl, AIN-76A), with blood pressure measured continuously by telemetry and overnight urine collected weekly to assess renal damage. All data presented are from HS day 21. Transfer of either male or female donor splenocytes into CD247-/- recipient males exacerbated salt-induced hypertension compared to PBS vehicle controls (+PBS: 143.7±2.9, +Male: 152.8±5.2, +Female: 151.8±3.9 mmHg, n=11-14, p<0.05). Similarly reflected in proteinuria, splenocyte transfer, regardless of sex, also worsened kidney damage in male CD247-/- recipients (+PBS: 191.9±26.7, +Male: 304.6±51.3, +Female: 283.9±37.1 mg/day, n=12-16, p<0.05). In CD247-/- female recipients, there was an exacerbation in blood pressure with the transfer of male splenocytes (+PBS: 144.8±6.1, +Male: 151.5±3.9 mmHg, n=6-13, p<0.05), but no significant change with the transfer of female splenocytes (+Female: 145.3±2.6 mmHg). There was a trend for male splenocytes to worsen proteinuria in CD247-/- female recipients (+PBS: 103.9±11.1, +Male: 157.7±21.2 mg/day, n=6-14, p=0.056), but there was a significant reduction in the transfer of female versus male splenocytes (+Female: 113.2±15.7, p<0.05). Through flow cytometry, we confirmed similar extent of T cell reconstitution in both male and female CD247-/- recipients. These data demonstrate that salt-sensitive hypertension and renal damage are amplified if the sex of the T cell or the recipient are male.

Blood Pressure, Vascular Reactivity, and SOD Expression/Activity in Mas1 Receptor Knockout Rats in the Dahl Salt‐Sensitive Genetic Background

Dahl salt‐sensitive (SS) rats are the most widely used rodent genetic model of human salt‐sensitive hypertension. Earlier studies have established the importance of impaired regulation of the renin gene in contributing to endothelial dysfunction in SS rats and the protective effect of low dose angiotensin II (ANG II) infusion in restoring endothelial function via the ANG II type1 receptor (AT1R) in the SS genetic background. Activation of the Mas1 receptor (Mas1R) for angiotensin (1‐7) [ANG (1‐7)] generally opposes ANG II‐mediated effects of AT1R activation. In Sprague‐Dawley rats fed high salt diet, binding of ANG (1‐7) to the Mas1R can activate NRF2 defenses and restore impaired endothelial function. The goal of the present study was to provide initial phenotyping of a Mas1R knockout (Mas1R KO) rat in the Dahl SS genetic background. Mas1R KO rats and wild type (WT) controls (12–15 weeks old) were maintained on a low salt (LS; 0.4% NaCl) or switched to a high salt (HS; 4% NaCl) diet for 3 days or 10 days. Arterial pressure measured via telemetry was similar in Mas1R KO rats (121±7 mmHg, n=3) and WT rats fed LS diet (124 mmHg, n=2) and increased in WT (138 mmHg, n=2) and Mas1R KO rats (132±5 mmHg, n=3) fed HS diet for 10 days. In rats fed LS diet, Mas1R KO eliminated vasodilator responses to ANG (1‐7) and AVE0991 (Mas receptor agonist) in middle cerebral arteries (MCA) and mesenteric resistance arteries (MRA) and reduced acetylcholine (ACh) reactivity in MCA. Because superoxide dismutase (SOD) enzymes contribute to antioxidant defense by catalyzing the dismutation of superoxide radicals and help to prevent diseases related to oxidative stress and endothelial dysfunction, we measured superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2) activity in the liver of WT and Mas1R KO rats after 3 days of HS diet. High salt diet increased SOD1 activity (WT‐LS 1.04±0.16 U/ml vs WT‐HS 1.65±0.12 U/ml) and SOD2 activity (WT‐LS 0.76±0.03 U/ml vs WT‐HS 1.06±0.11 U/ml) in WT rats, but not in Mas1R KO rats. SOD1 and SOD2 mRNA expression (evaluated via RT‐PCR) tended to increase in WT rats fed HS diet vs. LS diet (1.3 – 1.8 fold change, p > 0.05). SOD1 mRNA expression was significantly higher in HS‐fed Mas1R KO rats vs. Mas1R KO rats fed LS diet (2.8 fold change, p = 0.01) and SOD2 mRNA tended to increase as well (1.7 fold change; p = 0.1). SOD1 and SOD2 protein expression measured by Western blot analysis was similar in WT and Mas1R KO rats fed either LS or HS diet. The present study using the Mas1 receptor knockout in the Dahl SS rat genetic background suggests that Mas1R activation by its agonists (AVE 0991) and ANG (1‐7) improves endothelial function and may provide novel therapeutic strategies for the treatment of low‐renin salt‐sensitive hypertension.Support or Funding InformationNIH #HL‐128242 and NIH #R21‐OD024781

SPAK (Stk39) is Involved in NKCC2 Phosphorylation and Salt‐sensitive Hypertension in Dahl Salt‐sensitive Rats

In the thick ascending limb (TAL), NaCl reabsorption is mediated by the Na/K/2Cl cotransporter NKCC2, which plays a crucial role in the regulation of blood pressure. NKCC2 activity is partly controlled by phosphorylation via STE20/SPS1‐related proline‐alanine‐rich kinase (SPAK, gene name Stk39) at Thr96 and Thr101 residues in the NH2‐terminus of NKCC2. Previously, we found that NKCC2 phosphorylation was 5‐fold higher in TALs from Dahl salt‐sensitive (DSS) rats, suggesting that this pathway may be related to the salt‐sensitivity in this model. However, the kinase responsible for NKCC2 hyperphosphorylation is unknown. In addition, the role of SPAK in the development of salt‐sensitive hypertension remains unclear. We hypothesized that SPAK is involved in salt‐sensitive hypertension in part by increasing NKCC2 phosphorylation during normal or high salt diet. To test this, we generated SPAK knockout (KO) rats in a DSS genetic background via Crispr/Cas9 targeting of exon 5 in collaboration with the Gene Editing Rat Resource Center (Medical College of Wisconsin). Gene deletion was confirmed by sequencing and RT‐PCR. We used radiotelemetry to continuously measure mean blood pressure (MBP) in these rats on normal Na (0.22% Na chow) or high salt diet (4% Na chow). At baseline, SPAK KO rats had lower MBP than wild‐type DSS (WT) rats (SPAK KO: 117±1 mmHg; WT: 127±2 mmHg; p<0.05, n=7 each). After 4 weeks on high salt diet, MBP rose in all rats reaching 137±3 in SPAK KO and 154±3 mmHg in WT rats (p<0.05). The increase in MBP after switching rats from normal to high salt was lower in SPAK KO than WT rats; at 7 days (8±1 vs 13±1 mmHg, p<0.05), 14 days (12±2 vs 17±2 mmHg, p<0.05), and 28 days (20±3 vs 27±2 mmHg, p<0.05). We then explored total and phosphorylated‐NKCC2 (Thr96 and Thr101) expression in isolated TALs by Western blot. In rats fed normal salt (0.22% Na), phosphorylated‐NKCC2 (corrected over total NKCC2 expression) was lower in TALs isolated from SPAK KO (WT: 100; SPAK KO: 39±25%; p<0.05, n=4) whereas total NKCC2 expression (corrected by GAPDH) was not different between both groups (WT: 100; SPAK KO: 103±17%; n=5). After 4 weeks on high‐salt (4% Na), phosphorylated‐NKCC2 was also lower in TALs isolated from SPAK KO (WT: 100; SPAK KO: 38±13%; p<0.05, n=4) whereas total NKCC2 expression (corrected by GAPDH) was not different between both groups (WT: 100; SPAK KO: 113±22%; n=4). We conclude that SPAK KO rats exhibit a lower blood pressure at baseline and decreased salt sensitivity. This may be in part caused by lower NKCC2 phosphorylation in TALs of SPAK KO DSS rats. Our findings support an important role for SPAK in NKCC2 hyperphosphorylation and salt sensitive hypertension in DSS rats.Support or Funding InformationNIH and NIDDK R01, Grant Number: 5R01DK107263‐02.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 103: Decreased Ability to Excrete a Na Load and Hypertension in the ALMS1 (Alstrom Syndrome 1) Knockout Rat

Few genes involved in obesity are known to be involved in hypertension. The Alstrom Syndrome 1 protein (ALMS1) is involved in obesity in humans. We found it is expressed in the thick ascending limb (TAL) where it interacts with the Na/K/2Cl cotransporter (NKCC2). ALMS1 is also expressed in other nephron segments such as the proximal tubule and collecting ducts. We hypothesized that ALMS1 deletion leads to hypertension which is likely due to a decrease ability to excrete a salt load secondary to enhanced NaCl transport by the TAL and other nephron segments. To study the role of ALMS1 in renal function we generated ALMS1 knockout (KO) rats in a Dahl-salt sensitive genetic background via zinc-finger nuclease targeting by MCW gene targeting core. Using radio-telemetry we found the KO rats to have a higher baseline systolic blood pressure on normal Na chow compared to the Wild-type salt sensitive (WT) rats (KO:146±2 and WT:136±1 mmHg, *p=0.0012). To explore whether hypertension is related to increase renal Na reabsorption in KO rats we performed metabolic cage protocols to measure the acute excretion of a Na load (1% of body weight) in conscious rats. Our data show that it took longer for KO rats to excrete the Na load; cumulative or at individual time points (6, 9, 24 h) (cumulative Na, KO:4251±590 μmols/24h and WT:8788±994 μmols/24h, *p=0.0028). To test the role of different nephron segments in higher Na reabsorption we used a single dose of diuretics and measured urine Na and volume excretion. The NKCC2 inhibitor bumetanide (5mg/kg), induced a higher natriuretic response in KO rats (KO:485.2±37.1 μmols/8h, WT:221.8±32 μmols/8h, *p<0.05). The NCC inhibitor hydrochlorothiazide (HCTZ) 200mg/kg, caused a similar natriuretic effect in KO rats (KO:206.8±16 μmols/8h and WT:155.6±20 μmols/8h, p=0.0717). The ENaC inhibitor benzamil (10mg/kg), caused a similar natriuretic response in KO rats (KO:214.5±33 μmols/8h and WT:253.4±22 μmols/8h, p=0.3477). We conclude that ALMS1 KO have a decreased ability to excrete a salt load and this is primarily mediated by enhanced TAL-mediated Na reabsorption. Higher TAL Na absorption is likely involved in hypertension in ALMS1 KO rats. These data show that ALMS1 is important for blood pressure control and renal function.

Abstract 006: Mutation of SH2B3, a GWAS Candidate Gene for Hypertension, Attenuates Dahl SS Pathology

The immune system mediates hypertension in many experimental models, though the mechanisms remain unclear. In the Dahl salt-sensitive (SS) rat, infiltration of T lymphocytes and macrophages in the kidneys parallels the onset of hypertension and renal disease in response to high salt diet (4% NaCl). Immunosuppressive treatment blunts the infiltration of immune cells in the kidneys and attenuates the hypertensive pathology. SH2B3, a gene associated with human hypertension in many GWAS, encodes an intracellular adaptor protein shown to play an important role in inflammatory signaling. Mutation of SH2B3 via zinc-finger nucleases on the Dahl SS genetic background reduces immune cell infiltration in the kidneys and significantly attenuates the hypertensive pathology. Bone marrow transplant studies (n=5-6/group; below) show that Dahl SS or SH2B3 mutant rats receiving the SH2B3 mutant bone marrow (open circles, light bars) have significantly attenuated hypertension and albuminuria compared to rats receiving the Dahl SS bone marrow (closed circles, dark bars; * indicates P<0.05 vs. final day of 0.4% NaCl, † indicates P<0.05 vs. Dahl SS on the same day). These studies indicate a role for the immune system in the altered phenotype in the SH2B3 mutant rat. One possible immune mechanism involved is interleukin 6 (IL-6) signaling, a proinflammatory pathway in which SH2B3 functions. After 21 days on 4% NaCl diet (n=5-6/group), SH2B3 mutants have significantly lower mRNA expression of IL-6 (expressed as fold change of Dahl SS) in the renal cortex (0.54, p<0.05) and renal medulla (0.45, p<0.05). Further studies of IL-6 signaling will elucidate its role in Dahl SS hypertension.

Introgression of the Brown Norway Renin Gene onto the Dahl Salt Sensitive Genetic Background Restores Endothelium‐Dependent Vascular Relaxation by Reducing Oxidative Stress in the Cerebral Vasculature

Dahl salt sensitive (SS) rats fed low salt (LS) diet are normotensive but have severe endothelial dysfunction, chronically low plasma renin activity and low plasma angiotensin II levels. This study assessed endothelial function in isolated middle cerebral arteries (MCA) from male SS rats and congenic rats receiving a 2.0 Mbp portion of Brown Norway (BN) chromosome 13 containing the BN renin allele (Ren1‐BN). All rats were fed LS diet (0.4% NaCl) since weaning. MCAs from Ren1‐BN congenic rats exhibited dose‐dependent dilation to acetylcholine (10−10 – 10−5 M) whereas MCAs from the SS rats did not. Vascular superoxide (dihydroethidium fluorescence) and hydrogen peroxide levels (Amplex Red) were significantly elevated in cerebral arteries of SS rats compared to Ren1‐BN congenic rats, and chronic antioxidant treatment with tempol (15 mg/kg/day) restored vascular relaxation in SS rats. Western blot analysis of multiple antioxidant enzymes showed significantly greater expression of Cu/Zn superoxide dismutase in cerebral arteries of the Ren1‐BN congenic rats. These results suggest that restoring normal regulation of the renin‐angiotensin system by introgressing the BN renin allele onto the Dahl SS genetic background rescues endothelial function by increasing antioxidant enzyme expression and reducing oxidative stress. (NIH #HL65289, #HL72920, #HL92026).

Blood pressure and proteinuria effects of multiple quantitative trait loci on rat chromosome 9 that differentiate the spontaneously hypertensive rat from the Dahl salt-sensitive rat

A blood pressure (BP) quantitative trait locus (QTL) was previously located within 117 kb on rat chromosome 9 (RNO9) using hypertensive Dahl salt-sensitive and normotensive Dahl salt-resistant rats. An independent study between two hypertensive rat strains, the Dahl salt-sensitive rat and the spontaneously hypertensive rat (SHR), also detected a QTL encompassing this 117 kb region. Dahl salt-sensitive alleles in both of these studies were associated with increased BP. To map SHR alleles that decrease BP in the Dahl salt-sensitive rat, a panel of eight congenic strains introgressing SHR alleles onto the Dahl salt-sensitive genetic background were constructed and characterized. S.SHR(9)x3B, S.SHR(9)x3A and S.SHR(9)x2B, the congenic regions of which span a portion or all of the 1 logarithm of odds (LOD) interval identified by linkage analysis, did not significantly alter BP. However, S.SHR(9), S.SHR(9)x4A, S.SHR(9)x7A, S.SHR(9)x8A and S.SHR(9)x10A, the introgressed segments of which extend distal to the 1 LOD interval, significantly reduced BP. The shortest genomic segment, BP QTL1, to which this BP-lowering effect can be traced is the differential segment of S.SHR(9)x4A and S.SHR(9)x2B, to which an urinary protein excretion QTL also maps. However, the introgressed segment of S.SHR(9)x10A, located outside of this QTL1 region, represented a second BP QTL (BP QTL2) having no detectable effects on urinary protein excretion. In summary, the data suggest that there are multiple RNO9 alleles of the SHR that lower BP of the Dahl salt-sensitive rat with or without detectable effects on urinary protein excretion and that only one of these BP QTLs, QTL1, overlaps with the 117 kb BP QTL region identified using Dahl salt-sensitive and Dahl salt-resistant rats.

Elevated BSC-1 and ROMK Expression in Dahl Salt-Sensitive Rat Kidneys

This study compared the expression of enzymes and transport and channel proteins involved in the regulation of sodium reabsorption in the kidney of Dahl salt-sensitive (DS) and salt-resistant Brown-Norway (BN) and consomic rats (SS.BN13), in which chromosome 13 from the BN rat has been introgressed into the DS genetic background. The expression of the Na+/K+/2Cl- (BSC-1) cotransporter, Na+/H+ exchanger (NHE3), and Na+-K+-ATPase proteins were similar in the renal cortex of DS, BN, and SS.BN13 rats fed either a low-salt (0.1% NaCl) or a high-salt (8% NaCl) diet. The expression of the BSC-1 and the renal outer medullary K+ channel (ROMK) were higher, whereas the expression of the cytochrome P4504A proteins responsible for the formation of 20-hydroxyeicosatetraenoic (20-HETE) was lower in the outer medulla of the kidney of DS than in BN or SS.BN13 rats fed either a low-salt or a high-salt diet. In addition, the renal formation and excretion of 20-HETE was lower in DS than in BN and SS.BN13 rats. These results suggest that overexpression of ROMK and BSC-1 in the thick ascending limb combined with a deficiency in renal formation of 20-HETE may predispose Dahl S rats fed a high-salt diet to Na+ retention and hypertension.