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Background: Renal T cells contribute importantly to hypertension, but the underlying mechanism is incompletely understood. We reported that CD8Ts directly stimulate distal convoluted tubule cells (DCTs) to increase NCC (sodium chloride co-transporter) expression and salt reabsorption. However, the mechanistic basis of this pathogenic pathway that promotes hypertension remains to be elucidated. Methods: We used mouse models of DOCA+salt (DOCA) treatment and adoptive transfer of CD8 + T cells (CD8T) from hypertensive animals to normotensive animals in in vivo studies. Co-culture of mouse DCTs and CD8Ts was used as in vitro model to test the effect of CD8T activation in promoting NCC-mediated sodium retention and to identify critical molecular players contributing to the CD8T-DCT interaction. Interferon (IFNγ)-KO mice and mice receiving renal tubule-specific knockdown of PDL1 were used to verify in vitro findings. Blood pressure was continuously monitored via radio-biotelemetry, and kidney samples were saved at experimental end points for analysis. Results: We identified critical molecular players and demonstrated their roles in augmenting the CD8T-DCT interaction leading to salt-sensitive hypertension. We found that activated CD8Ts exhibit enhanced interaction with DCTs via IFN-γ-induced upregulation of MHC-I and PDL1 in DCTs, thereby stimulating higher expression of NCC in DCTs to cause excessive salt retention and progressive elevation of blood pressure. Eliminating IFN-γ or renal tubule-specific knockdown of PDL1 prevented T cell homing into the kidney, thereby attenuating hypertension in 2 different mouse models. Conclusions: Our results identified the role of activated CD8Ts in contributing to increased sodium retention in DCTS through the IFNγ-PDL1 pathway. These findings provide a new mechanism for T cell involvement in the pathogenesis of hypertension and reveal novel therapeutic targets.

Impaired salt handling by the kidneys acts as one source of hypertension. However, the exact identity of this kidney defect remains unknown. As established recently, the adaptive immunity, T cells in particular, acts as an important contributor to the pathogenesis of hypertension. In this regard, our laboratory has reported that CD8+ T cells (CD8Ts), when adoptively transferred from salt-sensitive hypertensive mice to normotensive mice, stimulate Sodium Chloride Cotransporter (NCC). Through this stimulation, sodium retention occurs, and salt-sensitive hypertension develops. However, whether other sodium transporters within the nephron are affected by hypertensive CD8Ts and contribute to this pathogenesis of hypertension remains uncharacterized. We hypothesize hypertensive CD8Ts will also have an impact on other sodium channels. Here, we demonstrate the impact of hypertensive CD8Ts on sodium-potassium-co-transporter (NKCC) and epithelial sodium channel (ENaCs) in the kidney and the potential of these regulations contributing to salt-sensitive hypertension.Through in vivo studies, we employed a CD8T-adoptive transfer model as described in our previous publications. The kidneys from mice receiving adoptive transfer of hypertensive CD8Ts were harvested and protein levels of NKCC, αENaC, βENaC, and γENaC were analyzed by western blot. With in vitro studies, we co-cultured mouse CD8Ts with Collecting Duct epithelial cells (CDs, M1 cells) to examine the direct interaction between CD8Ts and CDs in media containing various levels of corticosteroids. Similar western blots were performed after this co-culture, as well as measurement of sodium retention through an intracellular sodium indicator and flow cytometry, as reported previously.In the results of in vivo studies, we found that the kidneys of mice receiving adoptive transfer of hypertensive CD8Ts demonstrated higher expression of NKCC, βENaC and γENaC, but no change in αENaC. In the results from in vitro studies, we further found that in charcoal-stripped co-culture conditions, pre-activated CD8Ts stimulate higher expression of ENaC subunits and ENaC-mediated sodium retention in M1 cells. However, these effects are masked in culture conditions with physiological hormone levels or with additional corticosteroids.Overall, our results suggest that, as a potential kidney defect, hypertensive CD8Ts strongly stimulate NCC and NKCC in the kidney to mediate excessive salt retention. Although CD8Ts themselves can increase ENaC, this effect might be masked in physiologic conditions. NIH R01-HL146713 (Mu), AHA 15BGIA25730047 (Mu), and SPaT T32-GM10699 (Deck) 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.

Heart failure is the top killer in the U.S and worldwide, often caused by hypertension, aging, or cardiac toxicity. Diastolic Dysfunction (DD), the often the first step of heart failure, occurs when the chambers of the heart are not able to expand during the filling phase. In addition, hypertension is the leading cause of DD. Though this is known, the progression of hypertension into DD remains poorly understood. Of note is the ability of M2 macrophages to differentiate into a myofibroblast-like phenotype, known as macrophage to myofibroblast transition (MMT). This transition has been observed in interstitial fibrosis in chronic renal allograft injury. The researchers noted that MMT account for 50% of the myofibroblast population. To our knowledge, the extent of fibrotic remodeling due to MMT in the heart has not been ardently investigated. Therefore, we aim to answer whether MMT plays a causal role in the progression of hypertension to DD and the pathogenesis of heart failure.Utilizing the uninephrectomized, DOCA-salt model, mice were subjected to this treatment and hypertension was induced. As a negative control, another group of mice received a uninephrectomy and the same salt in their drinking water, however, DOCA was not administered (Sham group). MMT prevalence, identified as CD68+ α-SMA+ expressions, was observed inside the hearts of DOCA and Sham mice. The hearts were isolated and homogenized via gentleMACS tissue Dissociator, the monocytes were purified via Percoll isolation, and flow cytometry was used to identify the population of CD68+ α-SMA+ expressing cells.As soon as 10 days post DOCA implantation, the DOCA group exhibited worsening diastolic function as it took longer for the pressure to change in the hearts of DOCA mice than in Sham (-dp/dt). These data were gathered via left ventricular (LV) catheter insertion. At a later time, utilizing a different cohort of mice preliminary data show an increase of monocyte (CD68+) and fibroblast (α-SMA+) double-positive expressing cells in the hearts of hypertensive mice.MMT has been observed in the hearts of hypertensive mice. This transition may exacerbate Diastolic Dysfunction. More research must be performed to investigate the wide variety of functions of macrophages (i.e., M1 to M2 ratio and transition, macrophage residence or infiltration, and macrophage stimulation of myofibroblasts), as well as the specific interactions between macrophages and cardiomyocytes, or other immune cells, that might mediate this transition. These data provide a preliminary, but interesting glimpse, into the role of macrophages in exacerbating Diastolic Dysfunction and mediating Heart Failure. NIH RO1-HL146713 and AHA 15BGIA25730047 (Shengyu Mu) 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.

Background and Hypothesis: T cells play a critical role in hypertension; however, the molecules driving this interaction were unclear. We recently showed that CD8Ts are activated during the initial phase of blood pressure elevation and directly interact with the distal convoluted tubule (DCT) through the interferon gamma (IFNγ)-PDL1 pathway. Here we identified that these CD8Ts are activated during this initial phase via pro-inflammatory extracellular ATP stimulating their P2X7 receptors, leading to enhanced IFNγ production and increased sodium retention in tubular cells that interacted with these CD8Ts. We predict the CD8T P2X7 receptor is contributing to hypertension and abrogating this receptor on CD8Ts will block CD8T-tubular interaction and lower blood pressure. Methods: To determine whether CD8Ts activation in hypertension requires T cell receptor (TCR) stimulation, we used transgenic OT1 mice and induced hypertension via the DOCA + salt (DOCA) model, measuring blood pressure and confirming enhanced CD8T activation despite no exposure to ovalbumin. In in-vitro studies, we co-cultured DCTs with pre-stimulated CD8Ts and measured PDL1 expression and sodium retention in DCTs via flow cytometry. CD8Ts were obtained from WT or P2X7 KO mice and stimulated with BZATP or ATP using calcium influx (intracellular calcium indicator Fluo-4), IFNγ transcription, and glycolytic activity (Seahorse) as activation endpoints. To confirm in-vivo, the activation profiles of CD8Ts obtained from sham or DOCA treated WT and P2X7 KO mice were measured using rt-qPCR and flow cytometry. To confirm the role of P2X7 receptors on CD8Ts, we performed adoptive transfer of DOCA CD8Ts (WT or P2X7 KO) to the alternative model and transferred naive WT CD8Ts to P2X7 KO mice receiving DOCA treatment. All blood pressure was measured via radio-biotelemetry. Results: We found OT1 CD8Ts become activated to the DOCA presser, produce more IFNγ, and contribute to salt-sensitive hypertension without antigen-mediated TCR stimulation. P2X7 receptor agonists BZATP and ATP drove CD8T calcium influx, increased IFNγ transcription, and increased glycolytic activity in CD8Ts – indicative of T cell activation; as such, CD8Ts pre-stimulated with the P2X7 agonist BZATP increased tubular PDL1 expression and sodium retention more than naive CD8Ts in our co-culture model. DOCA treated P2X7 KO CD8Ts were unable to increase blood pressure in our adoptive transfer model; however, transferring sham WT CD8Ts to DOCA P2X7 KO mice restored blood pressure response to the presser to WT levels. Conclusion: Stimulation of the P2X7 receptor contributes to CD8T activation in the pathogenesis of hypertension, resulting in enhanced sodium retention. Deficiency of this receptor on CD8Ts prevents activation by ATP and results in blunted blood pressure elevation in two models of hypertension; as such, the CD8T P2X7 receptor may be a promising target for improving blood pressure management. NIH R01-HL146713, AHA 15BGIA25730047, SPaT 5T32GM106999-09 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.

Background & Hypothesis: The immune system plays a critical role in the development of hypertension. We recently found that CD8 + T cells (CD8Ts) infiltrate the kidneys and stimulate distal convoluted tubular cells (DCTs), upregulating sodium-chloride co-transporter (NCC) activity, leading to excessive sodium retention. However, the precise molecules driving this direct interaction between CD8Ts and DCTs have not yet been identified. Here we hypothesize that activated CD8Ts release gamma interferon (IFNγ) that primes the tubular cell to express PDL1 which functions as a co-stimulatory ligand promoting interactions between activated CD8Ts and DCTs contributing to the development of salt-sensitive hypertension. We predict blocking this molecular pathway will reduce CD8T-homing into the kidney, lowering blood pressure. Results: We found that CD8Ts isolated from DOCA-salt treated hypertensive mice exhibit higher activity compared to those from sham normotensive mice. Pre-activated CD8Ts demonstrated augmented ability to interact with DCTs compared to naïve CD8Ts, and as a consequence, DCTs that had been co-cultured with pre-activated CD8Ts demonstrated increased expression of NCC (7 fold) and increased PDL1 and high sodium retention (47% vs. 0-3%). These effects were abolished by neutralizing IFNγ or knockdown of PDL1 in DCTs. In-vivo results verified the in-vitro studies. We found that IFNγ-KO mice demonstrated reduced blood pressure elevation and T cell infiltration within their kidneys to DOCA-salt compared to WT mice receiving the same treatment (last day systolic blood pressure average 155.2 mmHg compared to 189 mmHg from WT). Blood pressure elevation was blunted in DOCA-salt treated mice with renal tubule-specific knockdown of PDL1 (last day systolic blood pressure average 124 mmHg for mice with siPDL1 nanoparticle and 144 mmHg for mice with scrambled siRNA); NCC elevation was also blunted in this model. Conclusion: Activated CD8Ts demonstrate enhanced ability to interact with DCTs leading to increased expression of NCC and sodium retention through a IFNγ-PDL1 mediated mechanism. Blocking the IFNγ-PDL1 pathway prevents CD8T-DCT interaction both in vitro and in vivo , thereby ameliorating salt-sensitive hypertension.

Aims Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease and lacks for safe and effective drug to therapy completely. Ginsenoside-Rg1 is one of the main components of ginseng and has been proved to counteract a variety of diseases. However, there is currently a lack of sufficient evidence to support the efficacy of ginsenoside-Rg1 in the treatment of NAFLD. Our aim was to investigate whether Ginsenoside-Rg1 is a potential drug for NAFLD. Main methods NAFLD model in rats was established by giving a high-fat diet (HFD), ginsenoside-Rg1 was intragastrically administered 100 mg/kg/d for 8 weeks in NAFLD rat. Serum biochemical indices were measured. Liver tissues were stained with hematoxylin and eosin (HE) and oil red O. Total RNA was extracted from liver and was used for high throughput sequencing to identify the changes of transcriptome. The relevant hub genes were verified by quantitative real-time PCR and western blot. Key findings Serum biochemical analysis indicated that ginsenoside-Rg1 improved liver function. Additionally, the staining of HE and oil red O indicated ginsenoside-Rg1 could remit pathology process of NAFLD. The transcriptome changes also support this result and reveals Atf3 and Acox2 were key genes. Significance Taken together, these results suggest that the efficiency of ginsenoside-Rg1 against NAFLD and confirmed that ginsenoside-Rg1 is a potential effective drug in treatment of NAFLD.

Oxidative/nitrative stress plays an important role in myocardial ischemia/reperfusion (MI/R) injury. Notch1 participates in the regulation of cardiogenesis and cardiac response to hypertrophic stress, but the function of Notch1 signaling in MI/R has not been explored. This study aims to determine the role of Notch1 in MI/R, and investigate whether Notch1 confers cardioprotection. Notch1 specific small interfering RNA (siRNA, 20 μg) or Jagged1 (a Notch ligand, 12 μg) was delivered through intramyocardial injection. 48 h after injection, mice were subjected to 30 min of myocardial ischemia followed by 3 h (for cell apoptosis and oxidative/nitrative stress), 24 h (for infarct size and cardiac function), or 2 weeks (for cardiac fibrosis and function) of reperfusion. Cardiac-specific Notch1 knockdown resulted in significantly aggravated I/R injury, as evidenced by enlarged infarct size, depressed cardiac function, increased myocardial apoptosis and cardiac fibrosis. Downregulation of Notch1 increased expression of inducible NO synthase (iNOS) and gp(91phox), enhanced the production of NO metabolites and superoxide, as well as their cytotoxic reaction product peroxynitrite. Moreover, Notch1 blockade also reduced phosphorylation of endothelial NO synthase (eNOS) and Akt, and increased expression of PTEN, a key phosphatase involved in the regulation of Akt phosphorylation. In addition, activation of Notch1 by Jagged1 or administration of peroxynitrite scavenger reduced production of peroxynitrite and attenuated MI/R injury. These data indicate that Notch1 signaling protects against MI/R injury partly though PTEN/Akt mediated anti-oxidative and anti-nitrative effects.

Impaired mitochondrial biogenesis causes skeletal muscle damage in diabetes. However, whether and how mitochondrial biogenesis is impaired in the diabetic heart remains largely unknown. Whether adiponectin (APN), a potent cardioprotective molecule, regulates cardiac mitochondrial function has also not been previously investigated. In this study, electron microscopy revealed significant mitochondrial disorders in ob/ob cardiomyocytes, including mitochondrial swelling and cristae disorientation and breakage. Moreover, mitochondrial biogenesis of ob/ob cardiomyocytes is significantly impaired, as evidenced by reduced Ppargc-1a/Nrf-1/Tfam mRNA levels, mitochondrial DNA content, ATP content, citrate synthase activity, complexes I/III/V activity, AMPK phosphorylation, and increased PGC-1α acetylation. Since APN is an upstream activator of AMPK and APN plasma levels are significantly reduced in ob/ob mice, we further tested the hypothesis that reduced APN in ob/ob mice is causatively related to mitochondrial biogenesis impairment. One week of APN treatment of ob/ob mice activated AMPK, reduced PGC-1α acetylation, increased mitochondrial biogenesis, and attenuated mitochondrial disorders. In contrast, knocking out APN inhibited AMPK-PGC-1α signaling and impaired both mitochondrial biogenesis and function. The ob/ob mice exhibited lower survival rates and exacerbated myocardial injury after MI, when compared to controls. APN supplementation improved mitochondrial biogenesis and attenuated MI injury, an effect that was almost completely abrogated by the AMPK inhibitor compound C. In high glucose/high fat treated neonatal rat ventricular myocytes, siRNA-mediated knockdown of PGC-1α blocked gAd-enhanced mitochondrial biogenesis and function and attenuated protection against hypoxia/reoxygenation injury. In conclusion, hypoadiponectinemia impaired AMPK-PGC-1α signaling, resulting in dysfunctional mitochondrial biogenesis that constitutes a novel mechanism for rendering diabetic hearts more vulnerable to enhanced MI injury.

Objectives Evidence exists that the increase of TNFα is associated with reperfusion injury in patients with myocardial infarction after PCI. Adiponectin (APN) is anti-inflammatory and cardioprotective protein suppressed by TNFα, and is significantly reduced during MI/R. Whether neutralising TNFα protects against MI/R injury through upregulation of APN level has not been previously investigated. Methods and Results Adult male C57 mice were subjected to 30 min MI followed by 3h or 24h reperfusion or sham MI/R for this study. Etanercept, a TNFα neutralising drug for treating rheumatoid arthritis in clinic, was intraperitoneally injected 10 min before reperfusion. Etanercept administration ameliorated MI/R injury evidenced by increased cardiac function (1 day, 7 day, 14 day p Conclusions Overall, we have demonstrated for the first time that upregulating of adiponectin is involved in the cardioprotective effect of Etanercept, suggesting that using a single administration of Etanercept during PCI might improve the outcome of myocardial infarction patients.