Infiltrating T cells in the kidney amplify salt-sensitive (SS) hypertension and renal damage, but the mechanisms are not known. Genetic deletion of T cells (SS CD247-/- ) or the p67phox subunit of NOX2 (SS p67phox-/- ) attenuates salt-sensitive hypertension in the Dahl SS rat. We hypothesized that reactive oxygen species (ROS) produced by NOX2 in T cells drive the SS phenotype and renal damage. T cells were reconstituted by adoptively transferring ~10 million splenocytes from the Dahl SS (SS→CD247, n=14), the SS p67phox-/- (p67phox→CD247, n=19), or PBS only (PBS→CD247, n=8), into the peritoneum of SS CD247-/- rats on postnatal day 5. Animals were instrumented with radiotelemeters at 8 weeks of age. After a week of recovery, blood pressure and albumin excretion were measured 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 22, the rats were euthanized, and their kidneys were perfused and collected for histology and renal immune cell characterization by flow cytometry. At baseline there was no detectable difference in mean arterial pressure (MAP) between groups (124±2 vs 123±2 vs 116±2 mmHg; SS→CD247 vs p67phox→CD247 vs PBS→CD247, respectively). After 21 days of HS, MAP was significantly greater in the SS→CD247 compared to the p67phox→CD247 rats (155±3 vs 144±2 mmHg; p<0.001) and the PBS→CD247 rats (155±3 vs 141±3 mmHg; p<0.001). Indices of renal damage identified no difference in albuminuria between SS→CD247 and p67phox→CD247 on LS (25.8±4.7 vs 23.2±3.2 mg/day). After 21 days of HS, albuminuria was significantly greater in the SS→CD247 (242.8±30.3 mg/day) compared to the p67phox→CD247 (175.5 ± 14.3 mg/day, p<0.03) and PBS→CD247 (135.9±27.8 mg/day, p<0.005). There was no difference between p67phox→CD247 and PBS→CD247 in albuminuria or MAP during LS or HS. The lack of CD3+ cells in the PBS→CD247 but presence of CD3+ cells in rats receiving the T cell transfer demonstrated the effectiveness of the adoptive transfer. These results indicate that ROS production by T cells participates in the amplification of salt-sensitive hypertension and renal damage.
Histamine is involved in the immune response, vasodilation, neurotransmission, and gastric acid secretion. Although elevated histamine levels and increased expression of histamine metabolizing enzymes have been reported in renal disease, there is gap in knowledge regarding the mechanisms of histamine-related pathways in the kidney. We report here that all four histamine receptors, as well as enzymes responsible for metabolism of histamine are expressed in human and rat kidney tissues. In this study, we hypothesized that the histaminergic system plays a role in salt-induced kidney damage in Dahl Salt-Sensitive (DSS) rat, a model characterized with inflammation-driven renal lesions. To induce renal damage related to salt-sensitivity, DSS rats were challenged with a 21 days of high salt diet (HS, 4% NaCl); normal salt diet (NS, 0.4% NaCl) fed rats were used as a control. We observed lower histamine decarboxylase (HDC), and higher histamine N-methyltransferase (HNMT) levels in HS diet-fed rats, indicative of a shift in the histaminergic tone; metabolomics showed higher histamine and histidine levels in the kidneys HS diet-fed rats, while plasma levels for both compounds were lower. Acute systemic inhibition of histamine receptor 2 (HR2) in the DSS rat revealed that it lowered vasopressin receptor 2 and AQP2 abundance in the kidney. In summary, we established here the existence of the local histaminergic system, revealed a shift in the renal histamine balance during salt-induced kidney damage, and provided evidence that blockage of HR2 in the DSS rat affects water balance and urine concentrating mechanisms.
Preeclampsia (PE) is a pregnancy-specific disorder that is characterized by hypertension and proteinuria (maternal syndrome). Our group has demonstrated that Dahl salt-sensitive rats maintained on a low salt casein-based diet (SSC) develop maternal syndrome, while Dahl SS rats maintained on a low salt wheat gluten-based diet (SSWG) are protected. Population-based data demonstrated woman that experience a preeclamptic event are at an increased risk for developing hypertension and kidney disease after the pregnancy relative to woman who have a normotensive pregnancy. Since the SSC are susceptible to developing maternal syndrome, we tested the hypothesis that following a single pregnancy SSC rats would exhibit greater salt sensitive hypertension and renal damage relative to SSWG rats in response to a postpartum high salt (HS) challenge. To test this hypothesis SSC and SSWG rats were exposed to a single pregnancy, allowed to deliver naturally and then pups were weaned at 3 weeks. Following a postpartum urinalysis, dams recovered for one week before a two week HS diet challenge. Blood pressure and proteinuria were tracked throughout the study and immune cell infiltration was assessed at the end of the HS challenge. Despite exhibiting comparable baseline MAP measured via telemetry (135±6 vs 126±4mmHg, p>0.05) and urinary protein excretion levels (42.3±5 vs 26.7.1±7 mg/day, p>0.05, n=4-5/group), the SSC rats exhibit significantly elevated postpartum MAP (147±7 vs 131±2 mmHg, p<0.05) and proteinuria (230±40 vs 75±11 mg/day, p<0.05) relative to the SSWG mated rats. Additionally, when these animals were challenged with a postpartum HS diet, the SSC mated rats exhibited an exacerbated response in both MAP (ΔMAP: 22±5 vs 6±3, p<0.05) and proteinuria (300±25 vs 122±20 mg/day, p<0.05) that is accompanied with a significant reduction in creatinine clearance (CC: 0.31±0.08 vs 0.63±0.02 mL/min/g, p<0.05) relative to the SSWG mated rats. These findings are specific to pregnancy in the SSC rats since diet-matched virgin controls only exhibited a slight ΔMAP (SSC Virgin: 10±4mmHg, SSWG Virgin: 9±1mmHg) in response to a HS diet and comparable CC to the SSWG mated rats (SSC Virgin: 0.48±0.07, SSWG Virgin: 0.63±0.06 mL/min/g, p>0.05). These renal damage phenotypes observed in the mated SSC are associated with a two-fold increase in infiltrating immune cells including monocytes/macrophages, T and B cells relative to all other groups. These data suggest the renal demand of an adverse pregnancy that occurs in maternal syndrome is exacerbated with a secondary insult such as a high salt diet. Future studies will investigate the mechanisms behind these pregnancy-induced renal damage phenotypes and how an adverse pregnancy can lead to worsened health outcomes for both mother and offspring. HL157549, 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.
Infiltration of T cells into the kidney of the Dahl Salt Sensitive (SS), a 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 the hypertension and renal damage, and subsequent replacement of T cells by splenocyte transfer restores the disease phenotype. These studies demonstrate a role for T cells to amplify salt-sensitive hypertension, but the T cell subtype and the mechanism of action are not understood. A potential mechanism of T cell action is free radical production since the deletion of the p67phox subunit of NADPH oxidase 2 (NOX2) in adoptively transferred splenocytes attenuates Dahl SS hypertension and kidney damage. The present study was therefore performed to test the hypothesis that NOX2 in CD4+ T cells amplifies the SS phenotype and associated renal damage in the Dahl SS. We tested this hypothesis by adoptively transferring CD4+ T cells (~5 million) from splenocytes isolated from the Dahl SS (SS→CD247), from SS rats lacking p67phox (SSp67phox-/-), a critical subunit of NOX2 (p67phox→CD247), or PBS (PBS→CD247) into the SSCD247-/- rat on postnatal day 5. Animals were instrumented with radiotelemeters at 7 weeks of age. After a week of recovery, baseline measurements of blood pressure and albumin excretion were obtained while they were maintained on 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 22, the rats were euthanized, and their kidneys were perfused and collected for histology and renal immune cell characterization through flow cytometry. During the baseline period there was no significant difference in mean arterial pressure (MAP) between the SS→CD247, p67phox→CD247, and PBS→CD247 groups (112±1, 108±2, 109±2 mmHg; n=8,7,7, respectively). After 20 days on a high salt challenge there was a significant difference in MAP between the SS→CD247 (149±4 mmHg, n=8) and the p67phox→CD247 (133±2 mmHg, n=7, p<0.05) but no significant difference between the p67phox→CD247 MAP and the PBS→CD247 group (132±5 mmHg, n=7). Albuminuria trended higher in the SS→CD247 (172±22 mg/day) when compared to p67phox→CD247 (153±10 mg/day) and PBS→CD247 (143±44 mg/day) but did not reach significance. Data collected from the flow analysis confirmed the absence of CD3+ T cells in the PBS→CD247 and demonstrated no difference in the number of CD4+ T cells that infiltrated the kidneys between the SS→CD247 and the p67phox→CD247. These results indicate that ROS production from CD4+ T cells significantly amplifies the salt sensitive hypertension and renal HL161231, Georgia Research Alliance 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.
Renal inflammation accompanies hypertension and renal damage in Dahl Salt-Sensitive rats fed a high salt diet (HS, 4.0% NaCl). Via a transcriptomics approach, we previously found cluster of differentiation 14 (CD14) and toll-like receptor 4 (TLR4), partner molecules involved in innate immune system activation, to be upregulated in renal medullary tissue of Dahl SS rats after a HS challenge. A subsequent study demonstrated that both CD14 and TLR4 are upregulated in macrophages infiltrating the kidneys of Dahl SS fed HS. Interestingly, CD14 deletion on the Dahl SS background confers an amplification of hypertension and renal damage in females but has no effect in males. Given this finding, we examined sex-specific effects of TLR4 deletion in the pathogenesis of Dahl SS hypertension. The current study tested the hypothesis that TLR4 knockout differentially modulates salt-sensitivity in males versus females. A mutation was generated in the TLR4 gene on the Dahl SS background using CRISPR-Cas9 technology to create a 1-basepair frameshift mutation resulting in a premature stop codon. SSTLR4+/+ and SSTLR4-/- males (n=10) and females (n=6), maintained on a low salt chow (LS, 0.4% NaCl), underwent telemeter implantation at approximately 7 weeks of age for continuous monitoring of blood pressure. Following recovery and baseline blood pressure recordings, animals were switched to a HS chow for 3 weeks. Urine samples were collected once during the LS period and weekly throughout the HS challenge. Kidneys were flushed and harvested for analysis. Mean arterial pressure (MAP) was not different between the groups during the baseline period (117.3 ± 1, 116.9 ± 1, 117.3 ± 1, 116.9 ± 2 mmHg). Throughout the HS challenge, MAP was significantly reduced in the male SSTLR4-/- compared to male SSTLR4+/+ (day 19: 150.8 ± 4 vs. 162.5 ± 5 mmHg, p=0.010), but MAP was unchanged in female SSTLR4-/- compared to SSTLR4+/+ (151.6 ± 4 vs 155.3 ± 4 mmHg, p=0.554). Similarly, urinary albumin excretion was reduced in the male SSTLR4-/- versus SSTLR4+/+ (170.3 ± 27 vs 321.3 ± 63 mg/day, p=0.023), indicative of reduced renal injury in these animals. Albuminuria was not observed to be different between female SSTLR4-/- and SSTLR4+/+ (149.7 ± 24 vs 145.9 ± 14, p=0.448). At the end of the HS challenge, immune cells were isolated from the kidneys of these animals and characterized by flow cytometric analysis. We observed no difference in either the circulating or infiltrating immune cell profile between wildtypes and knockouts. These results indicate a male-specific effect of TLR4 deletion and suggest that TLR4 promotes salt-sensitive hypertension and renal damage in males. This finding contrasts with the effect of TLR4’s partner molecule, CD14, which attenuates SS hypertension in females. Further studies are needed to elucidate the molecular mechanisms of TLR4/CD14 activation and signaling during salt-sensitive hypertension. 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.
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