Female Salt-sensitive Rats Research Articles

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.

Protective Effects of Female Gut Microbiota Transplantation on Hypertension and Renal Damage in Male Dahl Salt-Sensitive Rats

We have previously demonstrated through microbiota transplantation studies that the gut microbiota importantly contributes to the development of hypertension, renal damage, and renal inflammation in the Dahl Salt-Sensitive (SS) rat fed a 4.0% NaCl high salt diet (HS). We also know that female SS rats are significantly protected from salt-induced hypertension, renal injury, and renal immune cell infiltration compared to males. One potential explanation for this sex difference is the substantial divergence we have observed in gut microbiota composition between male and female SS rats. Therefore, we hypothesized that gut microbiota transplantation between males and females would modulate the observed sex differences in salt-sensitivity. At 8 weeks of age, we first suppressed the native gut microbiota of male SS rat recipients via broad-spectrum antibiotics treatment (bacitracin and streptomycin, 2 g each/L, p.o.) for 3 days, followed by inoculation with either male or female donor cecal contents (oral gavage, 1 mL) for 7 consecutive days. Thus, our experimental groups are 1) male recipient rats repopulated with male donor microbiota (M+M, n=4) and 2) male recipient rats repopulated with female donor microbiota (M+F, n=3). Donor rats were maintained on the 0.4% NaCl low salt diet (LS) to more closely mimic the intrinsic microbiota of each sex. Telemeters were implanted at 10 weeks to allow for the continuous monitoring of blood pressure, followed by 3 weeks of HS challenge. At baseline, there were no differences in mean arterial pressure (MAP) between groups (123.4±1.3 vs 124.5±3.6 mmHg, M+M vs M+F on HS day 0, p=0.765). However, during the last week of HS challenge, males receiving female cecal microbiota transplantation had significantly reduced MAP compared to controls (153.6±3.4 vs 144.1±4.8 mmHg, M+M versus M+F on HS day 19, p=0.013). At the conclusion of the study, this reduction in blood pressure also coincided with a protection from renal damage, indicated by reduced urinary albumin excretion (209.0±37.1 vs 143.7±19.2 mg/day, M+M vs M+F, p=0.035), and an improvement in renal function, indicated by an increase in creatinine clearance (0.36±0.02 vs 0.48±0.01 ml/min/g kidney, M+M vs M+F, p=0.002). However, there appeared to be no effects on renal inflammation, with no changes to either the circulating or renal immune cell profile between rats receiving male or female microbiota. Furthermore, in female SS rat recipients that were subsequently transplanted with either male or female donor cecal contents, we observed no significant effects related to sex of donor microbiota in terms of blood pressure, renal damage, or renal inflammation. Together, these data demonstrate the anti-hypertensive and renoprotective effects of the female gut microbiota to recipient male SS rats. Moreover, there are additional factors within the female host that appear to be protective against the male microbiota. 19CDA34660184, HL161231 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.

Abstract P225: Dahl Salt-sensitive And Salt-resistant Rats Exhibit Highly Disparate Blood Pressure Responses To Lifetime High Fat Diet Feeding

In an effort to understand obesity associated hypertension, we and others are using Dahl salt-sensitive (SS) rats fed a high fat diet as an experimental model. One reason for choosing this rat strain is that it is well-known that SS rats exhibit accelerated hypertension development not just in response to high salt intake but to numerous other stimuli as well, when compared to Dahl salt resistant (SR) rats. However, it is not known whether this general dichotomous sensitivity to hypertension development in SS and SR rats also extends to the impact of high fat feeding. Therefore, in the present study we tested the hypothesis that high fat feeding would cause a smaller increase in blood pressure in SR rats versus SS rats in both females and males (n=8 each). To explore possible strain differences in the mechanisms known to contribute to obesity associated hypertension, we also compared the activity of the renin angiotensin and sympathetic nervous systems in SS and SR rats. Rats were fed a high fat diet (60% kcal fat) from weaning. Blood pressure was measured by telemetry starting at 12 weeks of age. At 12 weeks, mean arterial pressures (MAP, mmHg) were 109±8 and 158±6 in male SR and SS rats, and 108±1 and 150±5 in female SR and SS rats, respectively. By 18 weeks MAP were 109±7 and 175±4 in male SR and SS rats, and 108±1 and 180±4 in female SR and SS rats, respectively. In males, plasma renin was lower, and aldosterone the same, in SS rats than SR rats. However, SS rats showed larger falls in blood pressure during week-long ACE inhibitor treatment and to acute sympathoinhibition with hexamethonium than did SR rats. In females, plasma renin and aldosterone both were higher in SS rats than SR rats. Female SS rats also showed larger falls in blood pressure to chronic ACE inhibition and acute ganglion blockade than did SR rats. These results confirm that both male and female SR rats are highly resistant to obesity associated hypertension, and that less robust activation by high fat diet of the renin angiotensin and sympathetic nervous systems may be a causative factor.

Abstract P174: Sex Differences In The Pro-hypertensive Effect Of Carnitine, The Precursor Of Gut-derived Metabolite TMAO

Recent evidence reports sexually divergent mechanisms that differentially drive the severity of hypertension. Our data show that female Dahl Salt-Sensitive (SS) rats are significantly protected from salt-induced hypertension and renal injury and have stark differences in gut microbiota composition compared to males. Gut-derived metabolites are increasingly being recognized as mechanistic links between the gut microbiota and hypertension. One such metabolite is trimethylamine N-oxide (TMAO), which is derived from the bacterial metabolism of carnitine and is gaining notoriety for its role in cardiovascular disease. Metabolomics analysis in high salt-fed SS rats revealed a trend for increased TMAO (1.3-fold, p=0.11) in the serum of males compared to females (n=6). TMAO appears to be specifically derived from gut bacteria since oral antibiotic treatment nearly eliminated circulating TMAO levels in both males and females (99.3% and 88.9% reduction, respectively; p<0.001). Interestingly, antibiotic treatment reduced salt-sensitive hypertension in males but not females. There was also a corresponding increase in the TMAO precursor carnitine (1.9-fold, p<0.01) in the serum of males versus females. Thus, we hypothesized that administration of carnitine (400 mg/kg/day) in the drinking water would exacerbate salt-sensitive hypertension, renal damage, and gut inflammation in male and female SS rats challenged with high salt (4% NaCl). There was a trend for carnitine treatment to exacerbate mean arterial pressure in both males (160±9 vs 146±2 mmHg, n=4-6, p=0.22) and females (155±6 vs 139±2 mmHg, n=2, p=0.14) compared to vehicle. Despite elevated pressure in both sexes, carnitine-treated males exhibited greater increases in albuminuria (340±136 vs 194±29 mg/day, carnitine vs vehicle, p=0.28) than females (55±33 vs 26±5 mg/day). Carnitine treatment also significantly increased the number of CD3+ T cells in the colonic lamina propria (24.3±6.0 vs 2.4±0.5 x 10 6 cells/g tissue, n=5, p<0.05) of male rats compared to vehicle. Together, these data identify gut microbiota-mediated carnitine/TMAO metabolism as a potentially detrimental pathway that promotes greater salt-sensitivity, renal damage, and gut inflammation in males versus females.

Sex‐Specific Influence of Broad‐Spectrum Antibiotic Treatment on Dahl Salt‐Sensitive Hypertension

Our laboratory has previously demonstrated through fecal microbiota transfer studies that the gut microbiota importantly contributes to the development of hypertension, renal damage, and renal inflammation in the Dahl Salt-Sensitive (SS) rat fed a 4.0% high salt diet (HS). We have furthermore provided evidence that female SS rats are significantly protected from salt-induced hypertension, renal injury, and renal immune cell infiltration compared to males. One potential explanation for this sex difference is the substantial divergence we have observed in gut microbiota composition between male and female SS rats. Given the established role of the gut microbiota, we hypothesized that gut microbiota ablation via broad-spectrum antibiotic treatment would modulate the hypertensive response to high salt in both male and female SS rats. SS rats were treated with a broad-spectrum antibiotic cocktail of bacitracin and streptomycin (Bac/Strep, 2 g/L in drinking water) throughout the 3-week duration of HS challenge. SS males treated with Bac/Strep resulted in a significant reduction in mean arterial pressure (170.8±5.1 vs 156.0±4.1 mmHg, Vehicle vs Bac/Strep, n=12-13/group, p=0.001), proteinuria (365.0±39.7 vs 254.1±23.2 mg/day, p=0.002), and albuminuria (207.6±24.4 vs 124.3±10.3 mg/day, p<0.001) compared to male vehicle-treated rats after 3 weeks of HS. Due to the immunological nature of Dahl SS hypertension, we analyzed the infiltrating immune cell profile in the kidneys of Bac/Strep-treated males and found a specific decrease in B cells (40.7% reduction, p=0.012) as compared to vehicle-treated male SS kidneys, with no change in the number of T cells or monocytes/macrophages. These data altogether indicate that Bac/Strep treatment eliminated gut bacteria responsible for driving the hypertensive and renal injury phenotype in male Dahl SS rats. However, these protective effects of Bac/Strep were not observed in female SS rats. There was no significant difference in mean arterial pressure (134.6±3.1 vs 134.7±2.7 mmHg, Vehicle vs Bac/Strep, n=7-9/group, p= 0.974), proteinuria (39.5±3.8 vs 45.6±8.5 mg/day, p=0.408), or albuminuria (16.7±2.3 vs 18.7±3.7 mg/day, p=0.572) between vehicle- and Bac/Strep-treated female SS rats after 3 weeks of HS. Interestingly, despite the lack of an effect on the salt-induced hypertensive phenotype in females, there was still a significant decrease in renal B cells (38.5% reduction, p=0.05) in Bac/Strep-treated female SS rats compared to vehicle, perhaps indicating a specific effect of antibiotic treatment as opposed to a consequence of reduced blood pressure and renal damage. In conclusion, broad-spectrum antibiotic treatment reduced hypertension, renal damage, and renal inflammation in male Dahl SS rats, providing further evidence for the contribution of the gut bacteria in the response to HS. However, it appears that salt-induced elevations in blood pressure in female rats occur independent of Bac/Strep-sensitive gut bacteria.

Abstract MP42: T Cell Deletion Normalizes Sex Differences Of Blood Pressure But Not Renal Damage In Dahl Salt-sensitive Rats

The immune system has a clear role in the development of hypertension and renal damage in male Dahl salt-sensitive (SS) rats, but far less is known about this phenotype in females, especially in regard to the contribution of immune-related mechanisms. The current study examined the hypertensive and kidney injury phenotype in response to a 3 week high salt challenge (HS, 4.0% NaCl, AIN-76A) in female versus male SS rats (n&gt;10/group). Measured via radiotelemetry, there was no difference in low salt (LS, 0.4% NaCl) mean arterial pressure (MAP) between females and males (129±2 vs 124±2 mmHg, respectively). However, after 3 weeks of HS, females had a significant attenuation in the development of hypertension compared to males (161±3 vs 177±7 mmHg). This coincided with significantly less renal damage, evident by markedly reduced albuminuria (105±16 vs 183±16 mg/day) and medullary protein cast formation (7.0±0.7 vs 14.6±1.1%) in the females. Assessed via flow cytometry, there were fewer CD45+ total leukocytes (48% reduction), CD3+ T cells (51%), CD45R+ B cells (73%), and CD11b/c+ monocytes/macrophages (47%) in female versus male kidneys. To interrogate the contribution of adaptive immunity, specifically T cells, to the development of this sex difference, the same parameters were investigated in female and male SS CD247-/- rats which lack CD3+ T cells. The absence of functional T cells significantly reduced blood pressure in both females (147±6 vs 161±3 mmHg; SS CD247-/- vs SS) and males (157±8 vs 177±7 mmHg) after 3 weeks of HS, with no statistical difference between female and male SS CD247-/- rats. While a reduction in albuminuria (females: 37±12 vs 105±16 mg/day; males: 88±13 vs 183±16 mg/day, SS CD247-/- vs SS) and medullary protein cast formation (females: 1.8±0.4 vs 7.0±0.7%; males: 9.1±0.7 vs 14.6±1.1%) was observed in SS CD247-/- versus SS rats regardless of sex, there was greater protection from these measures of kidney damage in SS CD247-/- female versus SS CD247-/- males. Together, our data indicate that female SS rats are significantly protected from high salt-induced hypertension and renal disease compared to males rats. The deletion of T cells normalized high salt blood pressure between male and female rats but sex differences in renal damage still persisted.

Contribution of T‐lymphocytes to the Sex Differences Observed in Dahl Salt‐sensitive Hypertension and Renal Damage

Previous studies in our laboratory have established a clear role for the immune system in the development of hypertension and renal damage in male Dahl salt‐sensitive (SS) rats. Far less is known about the salt‐sensitive phenotype in female Dahl SS rats and how it compares to males, especially in regard to the contribution of immune‐related mechanisms. Using radiotelemetry to measure blood pressure, urine and renal histological analysis to assess kidney damage, and flow cytometry to assess renal immune cell infiltration, the current study examined the hypertensive and kidney injury phenotype in response to a 3 week high salt challenge (HS, 4.0% NaCl, AIN‐76A) in female versus male Dahl SS rats (n&gt;10/group). During the low‐salt (LS, 0.4% NaCl) period, there was no difference in baseline mean arterial pressure (MAP) between females and males (129.4±2.0 vs 123.7±1.9 mmHg, respectively). However, after 3 weeks of HS, females were significantly protected from developing the same extent of hypertension observed in the males (161.5±3.1 vs 176.7±6.7 mmHg). This protection in the females coincided with significantly less renal damage, evident by markedly reduced albuminuria (105.0±15.7 vs 183.2±15.6 mg/day) and medullary protein cast formation (6.96±0.72 vs 14.62±1.14%) compared to the males. There was also a significant 48.5% reduction in the number of CD45+ total leukocytes in the female versus male kidneys (3.3±.3 vs 6.3±0.5 ×106 cells/kidney), which was true across all immune subsets – CD3+ T‐lymphocytes (50.8% reduction), CD45R+ B‐lymphocytes (73.5% reduction), and CD11b/c+ monocytes/macrophages (46.7% reduction). To interrogate the potential contribution of adaptive immunity, specifically T‐lymphocytes, to the development of this sex difference, the same parameters were investigated in both female and male SSCD247−/− rats. The lack of functional CD3+ T‐lymphocytes in SSCD247−/− rats significantly reduced blood pressure in both females (147.1±5.9 vs 161.5±3.1 mmHg; SSCD247−/− vs SS) and males (157.2±8.1 vs 176.7±6.7 mmHg) after a 3 week salt challenge, where there was no statistical difference between female and male SSCD247−/− rats. There was a corresponding decrease in kidney damage, with reduced albuminuria in both female (37.2±12.1 vs 105.0±15.7 mg/day) and male (88.4±12.7 vs 183.2±15.6 mg/day) SSCD247−/− rats when compared to SS rats with the full complement of immune cells. Flow cytometric analysis confirmed the lack of T‐lymphocytes in all SSCD247−/− rats, and additionally, there was significantly less medullary protein cast formation in the SSCD247−/− rats compared to the SS, regardless of sex (females: 1.82±0.37 vs 6.96±0.72%; males: 9.10±0.75 vs 14.62±1.14%; SSCD247−/− vs SS). Together, our data indicate that although female SS rats are significantly protected from progressing to the same severity of disease compared to male SS rats, female SS rats do still develop salt‐induced hypertension and renal damage that is amplified by T‐lymphocytes.Support or Funding Information19CDA34660184, HL116264, HL137748

Chronic intermittent hypoxia orchestrates cardiorespiratory cacophony--adapting melody to malady.

Chronic intermittent hypoxia orchestrates cardiorespiratory cacophony--adapting melody to malady.