BACKGROUND:Pig-to-human kidney xenotransplantation offers a potential solution to the organ shortage. However, the ability of the transplanted pig kidney to regulate blood pressure and fluid balance remains uncertain. The renin-angiotensin-aldosterone system (RAAS) plays a crucial role in these functions, but species differences may impair its effectiveness in xenotransplantation. METHODS:Gene-edited pig kidneys were transplanted into six immunosuppressed baboons. Group A (n = 2) underwent bilateral native nephrectomy, while Group B (n = 4) had unilateral nephrectomy with one native kidney remaining in situ (with its ureter ligated) to avoid it contributing to salt and volume regulation. Plasma creatinine, potassium, renin, angiotensinogen, angiotensin I, and aldosterone levels were measured. RESULTS:Group A, but not Group B, exhibited increases in plasma creatinine and potassium levels, indicating hypovolemia that could be corrected by frequent fluid administration. Pig-specific renin was undetectable at all post-transplant time points in both groups. Baboon renin concentration and activity were measurable only in Group B, indicating that the native kidney contributed to renin production. Aldosterone levels remained unchanged in both groups. CONCLUSIONS:The absence of detectable pig renin highlights a potential physiological challenge in xenotransplantation. However, the retention of a native kidney may help maintain RAAS function and mitigate fluid and electrolyte imbalances. In clinical pig-to-human kidney transplantation, both native kidneys are usually retained, thus minimizing the development of hypovolemia.

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.

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 [...]

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. Despite the importance of these central circuits, the cellular properties of pre-sympathetic liver-related neurons remain to be determined. Here, we tested the hypotheses that the activity of pre-sympathetic liver-related neurons in the PVN and VLM/VMM is altered in diet induced obese (DIO) mice, as well as their sensitivity to insulin. Whole-cell patch-clamp recordings were conducted from liver-related neurons in male DIO (15–19-week-old) and control mice. During a step protocol, current steps (0-30pA, duration 1s) were applied to reveal the firing activity of neurons. Our data showed increased excitability of liver-related PVN neurons in DIO mice (n=9) compared to Control mice (n=10) (simple linear regression, different elevation, p=0.04). Moreover, in DIO mice, insulin decreased the frequency of action potentials (basal: 0.83±0.28 Hz vs insulin: 0.47±0.22 Hz, n=10; Wilcoxon test, p=0.01), while it had no effect on the firing in control mice (basal: 0.79±0.37 Hz vs insulin: 0.51±0.26 Hz, n=8, Wilcoxon test, p=0.11). Similarly, in DIO mice, insulin decreased the firing of liver-related PVN neurons after current injection (simple linear regression, different elevation, p=0.01, n=9). Next, liver-related PVN neurons were identified based on their projection to the VLM/VMM, and we found that the excitability of pre-sympathetic liver-related PVN neurons (n=7) was increased compared to liver-related PVN neurons (n=10) (simple linear regression, different elevation, p=0.00). Intriguingly, insulin did not alter the excitability of pre-sympathetic liver-related PVN neurons neither in control (n=8) or DIO mice (n=7) (simple linear regression, different elevation (Control: p=0.27; DIO: p=0.21). Since insulin sensitivities of liver-related PVN and pre-sympathetic liver-related PVN neurons were different, recordings were conducted from liver-related neurons in the VLM/VMM, which is well-known for its control over the sympathetic tone. In control mice, during the step protocol insulin had no effect on the excitability (simple linear regression, different elevation, p=0.37 n=7) whereas it decreased the firing rate of liver-related VLM/VMM neurons in DIO mice (nonlinear fit, different curve, p=0.03, n=4). Additionally, insulin receptor expression was confirmed by single cell digital droplet PCR in a subset of liver-related neurons by collecting mRNA from the cytoplasm of the recorded neurons. In summary, these results demonstrate that the firing activity of liver-related neurons are altered in DIO mice as well as their sensitivity to insulin. These data provide further evidence for cellular changes involving neurons regulating the sympathetic output to the liver. This work was supported by the NIH (DK-122842 to AZs and AVD), and Marko Spark Innovation Research Fund to AZs and AVD. We also thank the Tulane Brain Institute Cell and Tissue Imaging Core and the NIH Center for Neuroanatomy and Neurotropic viruses for the PRVs (P40 OD010996). 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.

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.

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.

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.

Regulation of systemic and local angiotensinogen (AGT) levels is a key determinant of tissue angiotensin II (Ang II) levels and inappropriate AGT augmentation promotes the development of hypertension and tissue injury. Kidney and urinary AGT levels are increased in Ang II-mediated hypertension. Recent studies have demonstrated that circulating hepatocyte-derived AGT (hAGT) enters kidneys sustaining kidney and urinary AGT levels. However, roles of hAGT in blood pressure elevation and kidney injury in Ang II-mediated hypertension have not been delineated. This study tested if hAGT contributes to the development of the pathophysiological events in Ang II-infused mice. A low dose of Ang II (400 ng/kg/min) was infused to male wild type (WT) and hAGT gene knockout (KO) mice (N=9 and 13) for 4 weeks. The control group in each genotype received vehicle (Veh) infusion (N=5 and 6). Western blot confirmed non-detectable levels of hAGT in KO mice. hAGT KO markedly decreased plasma AGT levels (WT+Veh:12.2±0.6 vs. hAGT KO+Veh: 0.8±0.1 μg/ml). Ang II infusion did not elevate plasma AGT levels in either WT and hAGT KO mice. Although hAGT KO mice exhibited a lower baseline of systolic blood pressure (SBP) than WT mice, Ang II-mediated increases in SBP was not attenuated in hAGT KO mice (ΔSBP in WT+Ang II: 30.1±4.4 vs. hAGT KO+Ang II: 26.0±4.2 mmHg). Kidney AGT mRNA levels were increased by Ang II infusion to the same extent in both WT and hAGT mice (WT+Ang II: 1.30±0.04 vs. hAGT KO+Ang II: 1.34±0.06, ratio to control). Likewise, Ang II infusion increased IL-6 mRNA to the same magnitude in both WT and hAGT KO mice. Urinary AGT was sustained in hAGT KO+Veh mice (66±9%) compared to WT+Veh mice. Ang II infusion did not alter urinary AGT levels in both groups. Glomerular mesangial expansion and fibrosis by Ang II infusion were not observed. Ang II infusion developed tubulointerstitial fibrosis in renal cortex and medulla. hAGT KO prevented the fibrosis only in the medulla. These outcomes demonstrate that elevation of SBP, augmentation of intrarenal AGT and IL-6 expression, and the development of renal cortical fibrosis in Ang II-mediated hypertension do not require hAGT. In contrast, hAGT contributes to renal medullary fibrosis which may be due to the lower absolute levels of blood pressure.

Estrogen exerts protective effects on the cardiovascular system via three known estrogen receptors: alpha (ERα), beta (ERß), and the G protein-coupled estrogen receptor (GPER). Our laboratory has previously showed the importance of GPER in the beneficial cardiovascular effects of estrogen. Since clinical studies indicate that the protective effects of exogenous estrogen on cardiovascular function are attenuated or reversed 10 years post-menopause, the hypothesis was that GPER expression may be reduced during aging. Vascular reactivity and GPER protein expression were assessed in female mice of varying ages. Physiological parameters, blood pressure, and estrogen receptor transcripts via droplet digital PCR (ddPCR) were assessed in the heart, kidney, and aorta of adult, middle-aged, and aged male and female C57BL/6 mice. Vasodilation to estrogen (E2) and the GPER agonist G-1 were reduced in aging female mice and were accompanied by downregulation of GPER protein. However, ERα and GPER were the predominant receptors in all tissues, whereas ERß was detectable only in the kidney. Female sex was associated with higher mRNA for both ERα and GPER in both the aorta and the heart. Aging impacted receptor transcript in a tissue-dependent manner. ERα transcript decreased in the heart with aging, while GPER expression increased in the heart. These data indicate that aging impacts estrogen receptor expression in the cardiovascular system in a tissue- and sex-specific manner. Understanding the impact of aging on estrogen receptor expression is critical for developing selective hormone therapies that protect from cardiovascular damage.

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.

Objective: Activated inflammasomes enhance maturation of pro-inflammatory cytokines, which facilitates the development of kidney injury. NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), one of major subunits in the inflammasome complex, plays a crucial role in innate immunity and inflammation. NLRP3 inflammasome is activated by ATP-P2X7 axis and reactive oxygen species. Expression of pro-NLRP3 is promoted by activated NF-κB by cytokines or PAMPs/DAMPs. In angiotensin II (Ang II)-dependent hypertension, there is augmentation of renal immune cells leading to enhanced production of cytokines and consequent activation of NF-κB. Therefore, we hypothesized that intrarenal NLRP3 levels are increased in Ang II-dependent hypertension in association with the kidney injury, and treatment with mycophenolate mofetil (MMF), an immunosuppressant, prevents the augmentation of NLRP3 and attenuates the consequent progression of kidney injury. Design and method: Ang II (80 ng/min) was infused with/without daily MMF administration (50 ng/kg) to male rats for 2 weeks. mRNA levels of intrarenal NLRP3 and AIM2, which forms another type of inflammasome complex by viral or bacterial infections, were measured by droplet digital PCR. Furthermore, levels of intrarenal angiotensinogen (AGT), a critical contributor to activation of intrarenal renin-angiotensin system leading to the development of hypertension and associated kidney injury, were evaluated. Histological analyses of kidney injury were also performed. Results: MMF prevented increases in intrarenal macrophage/monocyte and IL-6 levels induced by Ang II infusion, confirming immunosuppression by the drug. Ang II infusion significantly increased intrarenal NLRP3 levels (control group: 4.12 ± 1.1 copies/ng RNA vs. Ang II-infused group: 9.96 ± 1.8 copies). The elevated NLRP3 expression was attenuated by MMF (6.24 ± 1.4 copies). In contrast, intrarenal AIM2 levels were not altered by Ang II infusion or MMF. Urinary protein and AGT levels were elevated in Ang II-infused rats, which were prevented by MMF. Histological analyses showed mesangial expansion and tubulointerstitial fibrosis observed in Ang II-infused group, but these injury markers were in normal ranges in the group receiving Ang II + MMF. Conclusions: These results demonstrate activation of the NLRP3 inflammasome in Ang II dependent hypertension and indicate that immunosuppression by MMF mitigates the inflammasome activation, which contributes to attenuation of the kidney injury.

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.

Arterial stiffness is associated with cardiovascular risk independent of blood pressure. Previously, we showed that pulse pressure, a measure of arterial stiffness, is higher in female mice with global deletion of G protein‐coupled estrogen receptor (GPER) despite similar blood pressure. Additionally, we found that the GPER agonist, G1, provides protection from arterial remodeling. Therefore, the current study assessed the impact of GPER, sex, and age on arterial stiffness and aortic expression of four profibrotic genes: decorin (DCN), endothelin 1 (EDN1), type III collagen α1 (COL3A1), and connective tissue growth factor (CTGF). We hypothesized that global GPER knockout (KO) would increase fibrotic gene expression in females more than males, would be exacerbated with age, and would correlate with increased arterial stiffness.MethodsPulse wave velocity (PWV) was obtained in the carotid artery of 20 week‐old and 52 week‐old male and female wildtype and GPER KO mice (n = 3/group) using a Vevo 1100 high resolution ultrasound. RNA was isolated from aortas using a Qiagen RNeasy Mini Kit, and gene expression was quantified using droplet digital polymerase chain reaction (ddPCR). Statistical analysis was performed using three‐way ANOVA and Pearson r Correlation.ResultsIn females, GPER deletion increased expression of all four profibrotic genes in 20 week‐old mice, but this relationship was reversed in 52 week‐old mice. In males, GPER deletion was associated with lower expression of fibrotic genes in both age groups. PWV significantly correlated with increased expression of CTGF (r = 0.66, p = 0.019) and EDN1 (r = 0.61, p = 0.036). In addition, the expression of CTGF, COL3A1, EDN1, and DCN were positively correlated (p < 0.005). Interestingly, expression of both ERα and GPER were increased with aging in female mice (p < 0.05).ConclusionIncreased PWV was accompanied by upregulation of four profibrotic genes in the aorta. Adult female GPER KO mice had increased stiffness and higher expression of fibrotic genes. However, GPER deletion was associated with lower PWV and fibrotic gene expression in middle‐aged mice. Therefore, the protective effect of GPER in the vasculature was reversed with aging, despite the fact that aging upregulated estrogen receptor expression. Future studies will determine the mechanism by which GPER influences the expression of fibrotic genes and arterial stiffness.Support or Funding InformationNIH HL133619

Activated inflammasomes enhance maturation of pro-inflammatory cytokines, which facilitates the development of kidney injury. NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), one of major subunits in the inflammasome complex, plays a crucial role in innate immunity and inflammation. Although NLRP3 inflammasome is activated by ATP-P2Y7 axis and reactive oxygen species, the expression of pro-NLRP3 is promoted by NF-κB activated by cytokines or PAMPs/DAMPs. Thus, we hypothesized that mycophenolate mofetil (MMF), an immunosuppressant, attenuates augmentation of intrarenal NLRP3 and consequent progression of kidney injury in angiotensin II (Ang II)-dependent hypertension. Ang II (80 ng/min) was infused with/without daily MMF administration (50 ng/kg) to Sprague-Dawley rats for 2 weeks. mRNA levels of intrarenal NLRP3 and AIM2, which forms another type of inflammasome complex by viral or bacterial infections, were measured by droplet digital PCR. Furthermore, kidney injury was evaluated. MMF treatment mitigated Ang II-induced macrophage infiltration into kidneys, suggesting immunosuppression by the drug. Ang II infusion significantly increased intrarenal NLRP3 mRNA levels (normotensive control group: 4.12±1.1 copies/ng RNA vs. Ang II-infused group: 9.96±1.8 copies, N=5). The elevated NLRP3 expression in kidneys of Ang II-infused rats was attenuated by MMF treatment (6.24±1.4 copies). In contrast, intrarenal AIM2 levels were lower than NLRP3 in the control group and the levels were not altered by Ang II infusion or MMF treatment (normotensive control group: 0.42±0.1 copies, Ang II-infused group: 0.35±0.06 copies and Ang II+MMF group: 0.35±0.08 copies). Urinary protein and angiotensinogen levels were elevated in Ang II-infused rats and MMF treatment suppressed the augmentations. Histological analyses also showed the development of kidney injury including mesangial expansion and tubulointerstitial fibrosis observed in the hypertensive rats, but these injury markers were mitigated by MMF. These results demonstrate activation of the NLRP3 inflammasome in Ang II dependent hypertension and indicate that immunosuppression by MMF mitigates the inflammasome activation, which contributes to attenuation of the kidney injury.

Objective: Intrarenal interferon-γ significantly contributes to the development of glomerular injury in which angiotensinogen and monocyte chemoattractant protein 1 levels are elevated. However, the exact nature of the role that interferon-γ plays in regulating angiotensinogen and monocyte chemoattractant protein 1 expression has not been fully delineated. Therefore, the aim of this study was to investigate the role that interferon-γ plays in angiotensinogen and monocyte chemoattractant protein 1 expression. Methods: Primary cultured rat mesangial cells were treated with 0–20 ng/mL interferon-γ for 2, 8 or 24 hours. Expression levels of angiotensinogen, monocyte chemoattractant protein 1, suppressors of cytokine signaling 1, an intracellular suppressor of Janus kinase-signal transducers and activators of transcription signaling and activity of the Janus kinase-signal transducers and activators of transcription pathway were evaluated by reverse transcriptase polymerase chain reaction and western blot analysis. Results: Interferon-γ increased angiotensinogen expression in mesangial cells with maximal augmentation observed following 5 ng/mL interferon-γ at 8 hours of treatment (1.87 ± 0.05, mRNA, relative ratio). Further increases were reduced or absent using higher concentrations of interferon-γ. Following treatments, monocyte chemoattractant protein 1 expression was induced in a linear dose-dependent manner (6.85 ± 0.62-fold by 20 ng/mL interferon-γ at 24 hours). In addition, interferon-γ induced STAT1 phosphorylation and suppressors of cytokine signaling 1 expression in a linear dose-dependent manner. The suppression of STAT1 and suppressors of cytokine signaling 1 expression by small interference RNAs facilitated an increase in interferon-γ-induced angiotensinogen expression, indicating that these two factors negatively regulate angiotensinogen expression. In contrast, the increase in interferon-γ-induced monocyte chemoattractant protein 1 expression was attenuated in STAT1-deficient mesangial cells, suggesting that STAT1 positively regulates monocyte chemoattractant protein 1 expression in mesangial cells. Conclusion: These results demonstrate that while interferon-γ increases both angiotensinogen and monocyte chemoattractant protein 1 expression, STAT1 plays an opposing role in the regulation of each factor in mesangial cells.

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.

Chymase released from mast cells produces pro-fibrotic, inflammatory, and vasoconstrictor agents. Studies were performed to test the hypothesis that chronic chymase inhibition provides a renal protective effect in type 2 diabetes. Diabetic (db/db) and control mice (db/m) were chronically infused with a chymase-specific inhibitor or vehicle for 8 weeks. Baseline urinary albumin excretion (UalbV) averaged 42 ± 3 and 442 ± 32 microg/d in control (n = 22) and diabetic mice (n = 27), respectively (p < .05). After administration of chymase inhibitor to diabetic mice, the change in UalbV was significantly lower (459 ± 57 microg/d) than in vehicle-treated diabetic mice (645 ± 108 microg/d). UNGAL V was not different at baseline between diabetic mice that would receive the chymase inhibitor (349 ± 56 ng/d, n = 6) and vehicle (373 ± 99 ng/d, n = 6) infusions, but increased significantly only in the vehicle-treated diabetic mice (p < .05). Glomeruli of diabetic kidneys treated chronically with chymase inhibition demonstrated reduced mesangial matrix expansion compared to glomeruli from untreated diabetic mice. Plasma angiotensin II levels were not altered by chymase inhibitor treatment. In summary, chronic chymase inhibition slowed the progression of urinary albumin excretion in diabetic mice. In conclusion, renal chymase may contribute to the progression of albuminuria in type 2 diabetes renal disease.