Physiologic homeostasis after pig-to-human kidney xenotransplantation.

Demand for kidney grafts outpaces supply, limiting kidney transplantation as a treatment for kidney failure. Xenotransplantation has the potential to make kidney transplantation available to many more patients with kidney failure, but the ability of xenografts to support human physiologic homeostasis has not been established. A brain-dead adult decedent underwent bilateral native nephrectomies followed by 10 gene-edited (four gene knockouts, six human transgenes) pig-to-human xenotransplantation. Physiologic parameters and laboratory values were measured for seven days in a critical care setting. Data collection aimed to assess homeostasis by measuring components of the renin-angiotensin-aldosterone system, parathyroid hormone signaling, glomerular filtration rate, and markers of salt and water balance. Mean arterial blood pressure was maintained above 60 mmHg throughout. Pig kidneys secreted renin (post-operative day three to seven mean and standard deviation: 47.3 ± 9 pg/mL). Aldosterone and angiotensin II levels were present (post-operative day three to seven, 57.0 ± 8 pg/mL and 5.4 ± 4.3 pg/mL, respectively) despite plasma renin activity under 0.6 ng/mL/hr. Parathyroid hormone levels followed ionized calcium. Urine output down trended from 37 L to 6 L per day with 4.5 L of electrolyte free water loss on post-operative day six. Aquaporin 2 channels were detected in the apical surface of principal cells, supporting pig kidney response to human vasopressin. Serum creatinine down trended to 0.9 mg/dL by day seven. Glomerular filtration rate ranged 90-240 mL/min by creatinine clearance and single-dose inulin clearance. Thus, in a human decedent model, xenotransplantation of 10 gene-edited pig kidneys provided physiologic balance for seven days. Hence, our in-human study paves the way for future clinical study of pig-to-human kidney xenotransplantation in living persons.

Renin-angiotensin-aldosterone system function in the pig-to-baboon kidney xenotransplantation model.

After pig-to-baboon kidney transplantation, episodes of hypovolemia and hypotension from an unexplained mechanism have been reported. This study evaluated the renin-angiotensin-aldosterone system post-kidney xenotransplantation. Kidneys from genetically-engineered pigs were transplanted into 5 immunosuppressed baboons after the excision of the native kidneys. Immunosuppressive therapy was based on the blockade of the CD40/CD154 costimulation pathway. Plasma renin, angiotensinogen (AGT), angiotensin II (Ang II), aldosterone levels, and urine osmolality and electrolytes were measured in healthy pigs, healthy nonimmunosuppressed baboons, and immunosuppressed baboons with life-supporting pig kidney grafts. After pig kidney transplantation, plasma renin and Ang II levels were not significantly different, although Ang II trended lower, even though plasma AGT and potassium were increased. Plasma aldosterone levels were unchanged. Urine osmolality and sodium concentration were decreased. Even in the presence of increasing AGT and potassium levels, lower plasma Ang II concentrations may be because of reduced, albeit not absent, the reactivity of pig renin to cleave baboon AGT, suggesting an impaired response of the renin-angiotensin-aldosterone system to hypovolemic and hypotensive episodes. The maintenance of aldosterone may be protective. The reduced urine osmolality and sodium concentration reflect the decreased ability of the pig kidney to concentrate urine. These considerations should not prohibit successful clinical pig kidney xenotransplantation.

The Renin-Angiotensin-Aldosterone System in Metabolic Diseases and Other Pathologies.

It has been our pleasure to have been able to develop two special issues within the International Journal of Molecular Sciences: (1) Renin-Angiotensin-Aldosterone System in Pathologies and (2) Renin-Angiotensin-Aldosterone System in Metabolism & Disease [...].

Immunosuppression by Mycophenolate Mofetil Mitigates Intrarenal Angiotensinogen Augmentation in Angiotensin II-Dependent Hypertension.

Augmentation of intrarenal angiotensinogen (AGT) leads to further formation of intrarenal angiotensin II (Ang II) and the development of hypertensive kidney injury. Recent studies demonstrated that macrophages and the enhanced production of pro-inflammatory cytokines can be crucial mediators of renal AGT augmentation in hypertension. Accordingly, this study investigated the effects of immunosuppression by mycophenolate mofetil (MMF) on intrarenal AGT augmentation. Ang II (80 ng/min) was infused with or without daily administration of MMF (50 mg/kg) to Sprague-Dawley rats for 2 weeks. Mean arterial pressure (MAP) in Ang II infused rats was slightly higher (169.7 ± 6.1 mmHg) than the Ang II + MMF group (154.7 ± 2.0 mmHg), but was not statistically different from the Ang II + MMF group. MMF treatment suppressed Ang II-induced renal macrophages and IL-6 elevation. Augmentation of urinary AGT by Ang II infusion was attenuated by MMF treatment (control: 89.3 ± 25.2, Ang II: 1194 ± 305.1, and Ang II + MMF: 389 ± 192.0 ng/day). The augmentation of urinary AGT by Ang II infusion was observed before the onset of proteinuria. Elevated intrarenal AGT mRNA and protein levels in Ang II infused rats were also normalized by the MMF treatment (AGT mRNA, Ang II: 2.5 ± 0.2 and Ang II + MMF: 1.5 ± 0.1, ratio to control). Ang II-induced proteinuria, mesangial expansion and renal tubulointerstitial fibrosis were attenuated by MMF. Furthermore, MMF treatment attenuated the augmentation of intrarenal NLRP3 mRNA, a component of inflammasome. These results indicate that stimulated cytokine production in macrophages contributes to intrarenal AGT augmentation in Ang II-dependent hypertension, which leads to the development of kidney injury.

Angiotensin II biphasically regulates cell differentiation in human iPSC-derived kidney organoids.

Human kidney organoid technology holds promise for novel kidney disease treatment strategies and utility in pharmacological and basic science. Given the crucial roles of the intrarenal renin-angiotensin system (RAS) and angiotensin II (ANG II) in the progression of kidney development and injury, we investigated the expression of RAS components and effects of ANG II on cell differentiation in human kidney organoids. Human induced pluripotent stem cell-derived kidney organoids were induced using a modified 18-day Takasato protocol. Gene expression analysis by digital PCR and immunostaining demonstrated the formation of renal compartments and expression of RAS components. The ANG II type 1 receptor (AT1R) was strongly expressed in the early phase of organoid development (around day 0), whereas ANG II type 2 receptor (AT2R) expression levels peaked on day 5. Thus, the organoids were treated with 100 nM ANG II in the early phase on days 0-5 (ANG II-E) or during the middle phase on days 5-10 (ANG II-M). ANG II-E was observed to decrease levels of marker genes for renal tubules and proximal tubules, and the downregulation of renal tubules was inhibited by an AT1R antagonist. In contrast, ANG II-M increased levels of markers for podocytes, the ureteric tip, and the nephrogenic mesenchyme, and an AT2R blocker attenuated the ANG II-M-induced augmentation of podocyte formation. These findings demonstrate RAS expression and ANG II exertion of biphasic effects on cell differentiation through distinct mediatory roles of AT1R and AT2R, providing a novel strategy to establish and further characterize the developmental potential of human induced pluripotent stem cell-derived kidney organoids.NEW & NOTEWORTHY This study demonstrates angiotensin II exertion of biphasic effects on cell differentiation through distinct mediatory roles of angiotensin II type 1 receptor and type 2 receptor in human induced pluripotent stem cell-derived kidney organoids, providing a novel strategy to establish and further characterize the developmental potential of the human kidney organoids.

Elevated cerebrospinal fluid sodium in hypertensive human subjects with a family history of Alzheimer's disease.

High salt (sodium) intake leads to the development of hypertension despite the fact that plasma sodium concentration ([Na+]) is usually normal in hypertensive human patients. Increased cerebrospinal fluid (CSF) sodium contributes to elevated sympathetic activity and high blood pressure (BP) in rodent models of hypertension. However, whether there is an increased accumulation of sodium in the CSF of humans with chronic hypertension is not well defined. Here, we investigated CSF [Na+] from hypertensive and normotensive human subjects with family histories of Alzheimer's disease in samples collected in a clinical trial, as spinal tap is not a routine clinical procedure for hypertensive patients. The [Na+] and osmolality in plasma and CSF were measured by flame photometry. Daytime ambulatory BP was monitored while individuals were awake. Participants were deidentified and data were analyzed in conjunction with a retrospective analysis of patient history and diagnosis. We found that CSF [Na+] was significantly higher in participants with high BP compared with normotensive participants; there was no difference in plasma [Na+], or plasma and CSF osmolality between groups. Subsequent multiple linear regression analyses controlling for age, sex, race, and body mass index revealed a significant positive correlation between CSF [Na+] and BP but showed no correlation between plasma [Na+] and BP. In sum, CSF [Na+] was higher in chronic hypertensive individuals and may play a key role in the pathogenesis of human hypertension. Collectively, our findings provide evidence for the clinical significance of CSF [Na+] in chronic hypertension in humans.

Blockade of sodium-glucose cotransporter 2 suppresses high glucose-induced angiotensinogen augmentation in renal proximal tubular cells.

Renal proximal tubular angiotensinogen (AGT) is increased by hyperglycemia (HG) in diabetes mellitus, which augments intrarenal angiotensin II formation, contributing to the development of hypertension and kidney injury. Sodium-glucose cotransporter 2 (SGLT2) is abundantly expressed in proximal tubular cells (PTCs). The present study investigated the effects of canagliflozin (CANA), a SGLT2 inhibitor, on HG-induced AGT elevation in cultured PTCs. Mouse PTCs were treated with 5-25 mM glucose. CANA (0-10 µM) was applied 1 h before glucose treatment. Glucose (10 mM) increased AGT mRNA and protein levels at 12 h (3.06 ± 0.48-fold in protein), and 1 and 10 µM CANA as well as SGLT2 shRNA attenuated the AGT augmentation. CANA did not suppress the elevated AGT levels induced by 25 mM glucose. Increased AGT expression induced by treatment with pyruvate, a glucose metabolite that does not require SGLT2 for uptake, was not attenuated by CANA. In HG-treated PTCs, intracellular reactive oxygen species levels were elevated compared with baseline (4.24 ± 0.23-fold), and these were also inhibited by CANA. Furthermore, tempol, an antioxidant, attenuated AGT upregulation in HG-treated PTCs. HG-induced AGT upregulation was not inhibited by an angiotensin II receptor antagonist, indicating that HG stimulates AGT expression in an angiotensin II-independent manner. These results indicate that enhanced glucose entry via SGLT2 into PTCs elevates intracellular reactive oxygen species generation by stimulation of glycolysis and consequent AGT augmentation. SGLT2 blockade limits HG-induced AGT stimulation, thus reducing the development of kidney injury in diabetes mellitus.

Nitric oxide synthase inhibitors negatively regulate respiration in isolated rodent cardiac and brain mitochondria.

Nitric oxide (NO) is known to exert inhibitory control on mitochondrial respiration in the heart and brain. Evidence supports the presence of NO synthase (NOS) in the mitochondria (mtNOS) of cells; however, the functional role of mtNOS in the regulation of mitochondrial respiration is unclear. Our objective was to examine the effect of NOS inhibitors on mitochondrial respiration and protein S-nitrosylation. Freshly isolated cardiac and brain nonsynaptosomal mitochondria were incubated with selective inhibitors of neuronal (nNOS; ARL-17477, 1 µmol/L) or endothelial [eNOS; N5-(1-iminoethyl)-l-ornithine, NIO, 1 µmol/L] NOS isoforms. Mitochondrial respiratory parameters were calculated from the oxygen consumption rates measured using Agilent Seahorse XFe24 analyzer. Expression of NOS isoforms in the mitochondria was confirmed by immunoprecipitation and Western blot analysis. In addition, we determined the protein S-nitrosylation by biotin-switch method followed by immunoblotting. nNOS inhibitor decreased the state IIIu respiration in cardiac mitochondria and both state III and state IIIu respiration in brain mitochondria. In contrast, eNOS inhibitor had no effect on the respiration in the mitochondria from both heart and brain. Interestingly, NOS inhibitors reduced the levels of protein S-nitrosylation only in brain mitochondria, but nNOS and eNOS immunoreactivity was observed in the cardiac and brain mitochondrial lysates. Thus, the effects of NOS inhibitors on S-nitrosylation of mitochondrial proteins and mitochondrial respiration confirm the existence of functionally active NOS isoforms in the mitochondria. Notably, our study presents first evidence of the positive regulation of mitochondrial respiration by mitochondrial nNOS contrary to the current dogma representing the inhibitory role attributed to NOS isoforms.NEW & NOTEWORTHY Existence and the role of nitric oxide synthases in the mitochondria are controversial. We report for the first time that mitochondrial nNOS positively regulates respiration in isolated heart and brain mitochondria, thus challenging the existing dogma that NO is inhibitory to mitochondrial respiration. We have also demonstrated reduced protein S-nitrosylation by NOS inhibition in isolated mitochondria, supporting the presence of functional mitochondrial NOS.

Interaction between TRPV1-expressing neurons in the hypothalamus.

Transient receptor potential vanilloid type 1 (TRPV1) is a ligand-gated ion channel expressed in the peripheral and central nervous systems. TRPV1-dependent mechanisms take part in a wide range of physiological and pathophysiological pathways including the regulation of homeostatic functions. TRPV1 expression in the hypothalamus has been described as well as evidence that TRPV1-dependent excitatory inputs to hypothalamic preautonomic neurons are diminished in diabetic conditions. Here we aimed to determine the functional expression of TRPV1 in two hypothalamic nuclei known to be involved in the central control of metabolism and to test the hypothesis that TRPV1-expressing neurons receive TRPV1-expressing inputs. A mouse model (TRPV1Cre/tdTom) was generated to identify TRPV1-expressing cells and determine the cellular properties of TRPV1-expressing neurons in adult mice. Our study demonstrated the functional expression of TRPV1 in the dorsomedial hypothalamic nucleus and paraventricular nucleus in adult mice. Our findings revealed that a subset of TRPV1Cre/tdTom neurons receive TRPV1-expressing excitatory inputs, indicating direct interaction between TRPV1-expressing neurons. In addition, astrocytes likely play a role in the modulation of TRPV1-expressing neurons. In summary, this study identified specific hypothalamic regions where TRPV1 is expressed and functional in adult mice and the existence of direct connections between TRPV1Cre/tdTom neurons. NEW & NOTEWORTHY Transient receptor potential vanilloid type 1 (TRPV1) is expressed in the hypothalamus, and TRPV1-dependent regulation of preautonomic neurons is decreased in hyperglycemic conditions. Our study demonstrated functional expression of TRPV1 in two hypothalamic nuclei involved in the control of energy homeostasis. Our results also revealed that a subset of TRPV1-expressing neurons receive TRPV1-expressing excitatory inputs. These findings suggest direct interaction between TRPV1-expressing neurons.

Canagliflozin Prevents Intrarenal Angiotensinogen Augmentation and Mitigates Kidney Injury and Hypertension in Mouse Model of Type 2 Diabetes Mellitus.

BACKGROUND: Hypertension and renal injury are common complications of type 2 diabetes mellitus (T2DM). Hyperglycemia stimulates renal proximal tubular angiotensinogen (AGT) expression via elevated oxidative stress contributing to the development of high blood pressure and diabetic nephropathy. The sodium glucose cotransporter 2 (SGLT2) in proximal tubules is responsible for the majority of glucose reabsorption by renal tubules. We tested the hypothesis that SGLT2 inhibition with canagliflozin (CANA) prevents intrarenal AGT augmentation and ameliorates kidney injury and hypertension in T2DM. METHODS: We induced T2DM in New Zealand obese mice with a high fat diet (DM, 30% fat) with control mice receiving regular fat diet (ND, 4% fat). When DM mice exhibited > 350 mg/dL blood glucose levels, both DM- and ND-fed mice were treated with 10 mg/kg/day CANA or vehicle by oral gavage for 6 weeks. We evaluated intrarenal AGT, blood pressure, and the development of kidney injury. RESULTS: Systolic blood pressure in DM mice (133.9 ± 2.0 mm Hg) was normalized by CANA (113.9 ± 4.0 mm Hg). CANA treatment ameliorated hyperglycemia-associated augmentation of renal AGT mRNA (148 ± 21 copies/ng RNA in DM, and 90 ± 16 copies/ng RNA in DM + CANA) and protein levels as well as elevation of urinary 8-isoprostane levels. Tubular fibrosis in DM mice (3.4 ± 0.9-fold, fibrotic score, ratio to ND) was suppressed by CANA (0.9 ± 0.3-fold). Furthermore, CANA attenuated DM associated increased macrophage infiltration and cell proliferation in kidneys of DM mice. CONCLUSIONS: CANA prevents intrarenal AGT upregulation and oxidative stress and which may mitigate high blood pressure, renal tubular fibrosis, and renal inflammation in T2DM.

Inflammation as a Regulator of the Renin-Angiotensin System and Blood Pressure.

PURPOSE OF REVIEW: Mechanisms facilitating progression of hypertension via cross stimulation of the renin-angiotensin system (RAS) and inflammation have been proposed. Accordingly, we review and update evidence for regulation of RAS components by pro-inflammatory factors. RECENT FINDINGS: Angiotensin II (Ang II), which is produced by RAS, induces vasoconstriction and consequent blood pressure elevation. In addition to this direct action, chronically elevated Ang II stimulates several pathophysiological mechanisms including generation of oxidative stress, stimulation of the nervous system, alterations in renal hemodynamics, and activation of the immune system. In particular, an activated immune system has been shown to contribute to the development of hypertension. Recent studies have demonstrated that immune cell-derived pro-inflammatory cytokines regulate RAS components, further accelerating systemic and local Ang II formation. Specifically, regulation of angiotensinogen (AGT) production by pro-inflammatory cytokines in the liver and kidney is proposed as a key mechanism underlying the progression of Ang II-dependent hypertension.

Regulation of Nephron Progenitor Cell Self-Renewal by Intermediary Metabolism.

Nephron progenitor cells (NPCs) show an age-dependent capacity to balance self-renewal with differentiation. Older NPCs (postnatal day 0) exit the progenitor niche at a higher rate than younger (embryonic day 13.5) NPCs do. This behavior is reflected in the transcript profiles of young and old NPCs. Bioenergetic pathways have emerged as important regulators of stem cell fate. Here, we investigated the mechanisms underlying this regulation in murine NPCs. Upon isolation and culture in NPC renewal medium, younger NPCs displayed a higher glycolysis rate than older NPCs. Inhibition of glycolysis enhanced nephrogenesis in cultured embryonic kidneys, without increasing ureteric tree branching, and promoted mesenchymal-to-epithelial transition in cultured isolated metanephric mesenchyme. Cotreatment with a canonical Wnt signaling inhibitor attenuated but did not entirely block the increase in nephrogenesis observed after glycolysis inhibition. Furthermore, inhibition of the phosphatidylinositol 3-kinase/Akt self-renewal signaling pathway or stimulation of differentiation pathways in the NPC decreased glycolytic flux. Our findings suggest that glycolysis is a pivotal, cell-intrinsic determinant of NPC fate, with a high glycolytic flux supporting self-renewal and inhibition of glycolysis stimulating differentiation.

Macrophage-derived IL-6 contributes to ANG II-mediated angiotensinogen stimulation in renal proximal tubular cells.

The development of ANG II-dependent hypertension involves increased infiltration of macrophages (MΦ) and T cells into the kidney and the consequent elevation of intrarenal cytokines including IL-6, which facilitates the progression of hypertension and associated kidney injury. Intrarenal renin-angiotensin system (RAS) activation, including proximal tubular angiotensinogen (AGT) stimulation, has also been regarded as a cardinal mechanism contributing to these diseases. However, the interaction between immune cells and intrarenal RAS activation has not been fully delineated. Therefore, the present study investigated whether ANG II-treated MΦ induce AGT upregulation in renal proximal tubular cells (PTCs). MΦ were treated with 0-10(-6) M ANG II for up to 48 h. PTCs were incubated with the collected medium from MΦ. In ANG II-treated MΦ, IL-6 mRNA and protein levels were increased (1.86 ± 0.14, protein level, ratio to control); moreover, IL-6 levels were higher than TNF-α and IL-1ß in culture medium isolated from ANG II-treated MΦ. Elevated AGT expression (1.69 ± 0.04, ratio to control) accompanied by phosphorylated STAT3 were observed in PTCs that received culture medium from ANG II-treated MΦ. The addition of a neutralizing IL-6 antibody to the collected medium attenuated phosphorylation of STAT3 and AGT augmentation in PTCs. Furthermore, a JAK2 inhibitor also suppressed STAT3 phosphorylation and AGT augmentation in PTCs. These results demonstrate that ANG II-induced IL-6 elevation in MΦ enhances activation of the JAK-STAT pathway and consequent AGT upregulation in PTCs, suggesting involvement of an immune response in driving intrarenal RAS activity.

Quantification of intact plasma AGT consisting of oxidized and reduced conformations using a modified ELISA.

The pleiotropic actions of the renin-angiotensin system (RAS) depend on the availability of angiotensinogen (AGT) which generates angiotensin I (ANG I) when cleaved by renin. Thus, quantification of the intact AGT (iAGT) concentrations is important to evaluate the actual renin substrate available. The iAGT conformation exists as oxidized AGT (oxi-AGT) and reduced AGT (red-AGT) in a disulfide bond, and oxi-AGT has a higher affinity for renin, which may exacerbate RAS-associated diseases. Accordingly, we determined iAGT, oxi-AGT, and red-AGT levels in plasma from rats and mice. Blood samples were obtained by cardiac puncture and then immediately mixed with an inhibitor solution containing a renin inhibitor. Total AGT (tAGT) levels were measured by tAGT ELISA which detects both cleaved and iAGT. iAGT levels were determined by iAGT ELISA which was found to only detect red-AGT. Thus, it was necessary to treat samples with dithiothreitol, a reducing agent, to quantify total iAGT concentration. tAGT levels in rat and mouse plasma were 1,839 ± 139 and 1,082 ± 77 ng/ml, respectively. iAGT levels were 53% of tAGT in rat plasma but only 22% in mouse plasma, probably reflecting the greater plasma renin activity in mice. The ratios of oxi-AGT and red-AGT were ∼4:1 (rat) and 16:1 (mouse). Plasma iAGT consists of oxi-AGT and red-AGT, suggesting that oxidative stress can influence ANG I generation by the AGT conformation switch. Furthermore, the lower availability of plasma iAGT in mice suggests that it may serve as a limiting factor in ANG I formation in this species.

Increased angiotensinogen expression, urinary angiotensinogen excretion, and tissue injury in nonclipped kidneys of two-kidney, one-clip hypertensive rats.

In angiotensin II (ANG II)-dependent hypertension, there is an angiotensin type 1 receptor-dependent amplification mechanism enhancing intrarenal angiotensinogen (AGT) formation and secretion in the tubular fluid. To evaluate the role of increased arterial pressure, AGT mRNA, protein expression, and urinary AGT (uAGT) excretion and tissue injury were assessed in both kidneys of two-kidney, one-clip Sprague-Dawley hypertensive rats subjected to left renal arterial clipping (0.25-mm gap). By 18-21 days, systolic arterial pressure increased to 180 ± 3 mmHg, and uAGT increased. Water intake, body weights, 24-h urine volumes, and sodium excretion were similar. In separate measurements of renal function in anesthetized rats, renal plasma flow and glomerular filtration rate were similar in clipped and nonclipped kidneys and not different from those in sham rats, indicating that the perfusion pressure to the clipped kidneys remained within the autoregulatory range. The nonclipped kidneys exhibited increased urine flow and sodium excretion. The uAGT excretion was significantly greater in nonclipped kidneys compared with clipped and sham kidneys. AGT mRNA was 2.15-fold greater in the nonclipped kidneys compared with sham (1.0 ± 0.1) or clipped (0.98 ± 0.15) kidneys. AGT protein levels were also greater in the nonclipped kidneys. The nonclipped kidneys exhibited greater glomerular expansion and immune cell infiltration, medullary fibrosis, and cellular proliferation than the clipped kidneys. Because both kidneys have elevated ANG II levels, the greater tissue injury in the nonclipped kidneys indicates that an increased arterial pressure synergizes with increased intrarenal ANG II to stimulate AGT production and exert greater renal injury.

ROCK/NF-κB axis-dependent augmentation of angiotensinogen by angiotensin II in primary-cultured preglomerular vascular smooth muscle cells.

In angiotensin II (ANG II)-dependent hypertension, the augmented intrarenal ANG II constricts the renal microvasculature and stimulates Rho kinase (ROCK), which modulates vascular contractile responses. Rho may also stimulate angiotensinogen (AGT) expression in preglomerular vascular smooth muscle cells (VSMCs), but this has not been established. Therefore, the aims of this study were to determine the direct interactions between Rho and ANG II in regulating AGT and other renin-angiotensin system (RAS) components and to elucidate the roles of the ROCK/NF-κB axis in the ANG II-induced AGT augmentation in primary cultures of preglomerular VSMCs. We first demonstrated that these preglomerular VSMCs express renin, AGT, angiotensin-converting enzyme, and ANG II type 1 (AT1) receptors. Furthermore, incubation with ANG II (100 pmol/l for 24 h) increased AGT mRNA (1.42 ± 0.03, ratio to control) and protein (1.68 ± 0.05, ratio to control) expression levels, intracellular ANG II levels, and NF-κB activity. In contrast, the ANG II treatment did not alter AT1a and AT1b mRNA levels in the cells. Treatment with H-1152 (ROCK inhibitor, 10 nmol/l) and ROCK1 small interfering (si) RNA suppressed the ANG II-induced AGT augmentation and the upregulation and translocalization of p65 into nuclei. Functional studies showed that ROCK exerted a greater influence on afferent arteriole responses to ANG II in rats subjected to chronic ANG II infusions. These results indicate that ROCK is involved in NF-κB activation and the ROCK/NF-κB axis contributes to ANG II-induced AGT upregulation, leading to intracellular ANG II augmentation.

Passive antibody transfer from pregnant women to their fetus are maximized after SARS-CoV-2 vaccination irrespective of prior infection.

Background: Pregnancy is associated with a higher risk of adverse symptoms and outcomes for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection for both mother and neonate. Antibodies can provide protection against SARS-CoV-2 infection and are induced in pregnant women after vaccination or infection. Passive transfer of these antibodies from mother to fetus in utero may provide protection to the neonate against infection. However, it is unclear whether the magnitude or quality and kinetics of maternally derived fetal antibodies differs in the context of maternal infection or vaccination. Objective: We aimed to determine whether antibodies transferred from maternal to fetus differed in quality or quantity between infection- or vaccination-induced humoral immune responses. Methods: We evaluated 93 paired maternal and neonatal umbilical cord blood plasma samples collected between October 2020 and February 2022 from a birth cohort of pregnant women from New Orleans, Louisiana, with histories of SARS-CoV-2 infection and/or vaccination. Plasma was profiled for the levels of spike-specific antibodies and induction of antiviral humoral immune functions, including neutralization and Fc-mediated innate immune effector functions. Responses were compared between 4 groups according to maternal infection and vaccination. Results: We found that SARS-CoV-2 vaccination or infection during pregnancy increased the levels of antiviral antibodies compared to naive subjects. Vaccinated mothers and cord samples had the highest anti-spike antibody levels and antiviral function independent of the time of vaccination during pregnancy. Conclusions: These results show that the most effective passive transfer of functional antibodies against SARS-CoV-2 in utero is achieved through vaccination, highlighting the importance of vaccination in pregnant women.

Interferon-γ biphasically regulates angiotensinogen expression via a JAK-STAT pathway and suppressor of cytokine signaling 1 (SOCS1) in renal proximal tubular cells.

Renal inflammation modulates angiotensinogen (AGT) production in renal proximal tubular cells (RPTCs) via inflammatory cytokines, including interleukin-6, tumor necrosis factor α, and interferon-γ (IFN-γ). Among these, the effects of IFN-γ on AGT regulation in RPTCs are incompletely delineated. This study aimed to elucidate mechanisms by which IFN-γ regulates AGT expression in RPTCs. RPTCs were incubated with or without IFN-γ up to 48 h. AGT expression, STAT1 and STAT3 activities, and SOCS1 expression were evaluated. RNA interference studies against STAT1, SOCS1, and STAT3 were performed to elucidate a signaling cascade. IFN-γ decreased AGT expression at 6 h (0.61±0.05, ratio to control) and 12 h (0.47±0.03). In contrast, longer exposure for 24 and 48 h increased AGT expression (1.76±0.18, EC(50)=3.4 ng/ml, and 1.45±0.08, respectively). IFN-γ treatment for 6 h strongly induced STAT1 phosphorylation and SOCS1 augmentation, and decreased STAT3 activity. However, STAT1 phosphorylation and SOCS1 augmentation waned at 24 h, while STAT3 activity increased. RNA interference studies revealed that activation of STAT1-SOCS1 axis decreased STAT3 activity. Thus, IFN-γ biphasically regulates AGT expression in RPTCs via STAT3 activity modulated by STAT1-SOCS1 axis, suggesting the STAT1-SOCS1 axis is important in IFN-γ-induced activation of the intrarenal renin-angiotensin system.

High-Fat Diet Modulates the Excitability of Neurons within the Brain-Liver Pathway.

Stimulation of hepatic sympathetic nerves increases glucose production and glycogenolysis. Activity of pre-sympathetic neurons in the paraventricular nucleus (PVN) of the hypothalamus and in the ventrolateral and ventromedial medulla (VLM/VMM) largely influence the sympathetic output. Increased activity of the sympathetic nervous system (SNS) plays a role in the development and progression of metabolic diseases; however, despite the importance of the central circuits, the excitability of pre-sympathetic liver-related neurons remains to be determined. Here, we tested the hypothesis that the activity of liver-related neurons in the PVN and VLM/VMM is altered in diet-induced obese mice, as well as their response to insulin. Patch-clamp recordings were conducted from liver-related PVN neurons, VLM-projecting PVN neurons, and pre-sympathetic liver-related neurons in the ventral brainstem. Our data demonstrate that the excitability of liver-related PVN neurons increased in high-fat diet (HFD)-fed mice compared to mice fed with control diet. Insulin receptor expression was detected in a population of liver-related neurons, and insulin suppressed the firing activity of liver-related PVN and pre-sympathetic VLM/VMM neurons in HFD mice; however, it did not affect VLM-projecting liver-related PVN neurons. These findings further suggest that HFD alters the excitability of pre-autonomic neurons as well as their response to insulin.

The sodium-activated sodium channel is expressed in the rat kidney thick ascending limb and collecting duct cells and is upregulated during high salt intake.

Increased dietary salt triggers oxidative stress and kidney injury in salt-sensitive hypertension; however, the mechanism for sensing increased extracellular Na(+) concentration ([Na(+)]) remains unclear. A Na(+)-activated Na(+) channel (Na sensor) described in the brain operates as a sensor of extracellular fluid [Na(+)]; nonetheless, its presence in the kidney has not been established. In the present study, we demonstrated the gene expression of the Na sensor by RT-PCR and Western blotting in the Sprague-Dawley rat kidney. Using immunofluorescence, the Na sensor was localized to the luminal side in tubular epithelial cells of collecting ducts colocalizing with aquaporin-2, a marker of principal cells, and in thick ascending limb, colocalizing with the glycoprotein Tamm-Horsfall. To determine the effect of a high-salt diet (HSD) on Na sensor gene expression, we quantified its transcript and protein levels primarily in renal medullas from control rats and rats subjected to 8% NaCl for 7 days (n = 5). HSD increased Na sensor expression levels (mRNA: from 1.2 ± 0.2 to 5.1 ± 1.3 au; protein: from 0.98 ± 0.15 to 1.74 ± 0.28 au P < 0.05) in the kidney medulla, but not in the cortex. These data indicate that rat kidney epithelial cells of the thick ascending limb and principal cells of the collecting duct possess a Na sensor that is upregulated by HSD, suggesting an important role in monitoring changes in tubular fluid [Na(+)].