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.

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.

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 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 [...].

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.

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.

Alterations in the estrogen receptor profile of cardiovascular tissues during aging.

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.

STAT1 regulates interferon-γ-induced angiotensinogen and MCP-1 expression in a bidirectional manner in primary cultured mesangial cells.

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.

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.

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.

Chymase inhibition retards albuminuria in type 2 diabetes.

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.

Measuring Respiration in Isolated Murine Brain Mitochondria: Implications for Mechanistic Stroke Studies.

Measuring mitochondrial respiration in brain tissue is very critical in understanding the physiology and pathology of the central nervous system. Particularly, measurement of respiration in isolated mitochondria provides the advantage over the whole cells or tissues as the changes in respiratory function are intrinsic to mitochondrial structures rather than the cellular signaling that regulates mitochondria. Moreover, a high-throughput technique for measuring mitochondrial respiration minimizes the experimental time and the sample-to-sample variation. Here, we provide a detailed protocol for measuring respiration in isolated brain non-synaptosomal mitochondria using Agilent Seahorse XFe24 Analyzer. We optimized the protocol for the amount of mitochondria and concentrations of ADP, oligomycin, and trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) for measuring respiratory parameters for complex I-mediated respiration. In addition, we measured complex II-mediated respiratory parameters. We observed that 10 µg of mitochondrial protein per well, ADP concentrations ranging between 2.5 and 10 mmol/L along with 5 µmol/L of oligomycin, and 5 µmol/L of FCCP are ideal for measuring the complex I-mediated respiration in isolated mouse brain mitochondria. Furthermore, we determined that 2.5 µg of mitochondrial protein per well is ideal for measuring complex II-mediated respiration. Notably, we provide a discussion of logical analysis of data and how the assay could be utilized to design mechanistic studies for experimental stroke. In conclusion, we provide detailed experimental design for measurement of various respiratory parameters in isolated brain mitochondria utilizing a novel high-throughput technique along with interpretation and analysis of data.

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.

Estrogen receptor profiles across tissues from male and female Rattus norvegicus.

BACKGROUND: Estrogen is formed by the enzyme aromatase (CYP19A1) and signals via three identified receptors ERα (ESR1), ERß (ESR2), and the G protein-coupled estrogen receptor (GPER). Understanding the relative contribution of each receptor to estrogenic signaling may elucidate the disparate effects of this sex hormone across tissues, and recent developments in PCR technology allow absolute quantification and direct comparison of multiple targets. We hypothesized that this approach would reveal tissue- and sex-specific differences in estrogen receptor mRNA. METHODS: ESR1, ESR2, GPER, and CYP19A1 were measured in four cardiovascular tissues (heart, aorta, kidney, and adrenal gland), three brain areas (somatosensory cortex, hippocampus, and prefrontal cortex), and reproductive tissues (ovaries, mammary gland, uterus, testes) from six male and six female adult Sprague-Dawley rats. RESULTS: GPER mRNA expression was relatively stable across all tissues in both sexes, ranging from 5.49 to 113 copies/ng RNA, a 21-fold difference. In contrast, ESR1/ESR2 were variable across tissues although similar within an organ system. ESR1 ranged from 4.46 to 614 copies/ng RNA (138-fold difference) while ESR2 ranged from 0.154 to 83.1 copies/ng RNA (540-fold). Significant sex differences were broadly absent except for renal ESR1 (female 206 vs. male 614 copies/ng RNA, P < 0.0001) and GPER (62.0 vs. 30.2 copies/ng RNA, P < 0.05) as well as gonadal GPER (5.49 vs. 47.5 copies/ng RNA, P < 0.01), ESR2 (83.1 vs. 0.299 copies/ng RNA, P < 0.01), and CYP19A1 (322 vs. 7.18 copies/ng RNA, P < 0.01). Cardiovascular tissues showed a predominance of ESR1, followed by GPER. In contrast, GPER was the predominant transcript in the brain with similarly low levels of ESR1 and ESR2. CYP19A1 was detected at very low levels except for reproductive tissues and the hippocampus. CONCLUSION: While the data indicates a lack of sex differences in most tissues, significant differences were found in the range of receptor gene expression across tissues as well as in the receptor profile between organ systems. The data provide a guide for future studies by establishing estrogen receptor expression across multiple tissues using absolute PCR quantification. This knowledge on tissue-specific estrogen receptor profiles will aid the development of hormonal therapies that elicit beneficial effects in specific tissues.

Advanced Glycation End Products Stimulate Angiotensinogen Production in Renal Proximal Tubular Cells.

BACKGROUND: Elevated advanced glycation end products (AGE) in diabetes mellitus (DM) are implicated in the progression of DM-associated tissue injury, including diabetic nephropathy. The intrarenal renin-angiotensin system, in particular augmentation of angiotensinogen (AGT) in proximal tubular cells (PTC), plays a crucial role in the development of diabetic nephropathy. This study investigated hypothesis that AGE stimulates AGT production in PTC. MATERIALS AND METHODS: Urinary AGT and AGE levels in streptozotocin-induced DM mice were measured by enzyme-linked immunosorbent assays. AGT expression and secretion were evaluated in cultured rat PTC receiving 0-200 µg/ml AGE-BSA treatments for 24 hours. Furthermore, intracellular signaling pathways activated by AGE were elucidated. RESULTS: DM mice exhibited greater urinary AGT and AGE levels compared to control mice (AGT: 21.6 ± 5.5 ng/day vs. 190.1 ± 57.8 ng/day, AGE: 139.1 ± 21.6 µg/day vs. 332.8 ± 102.7 µg/day). In cultured PTC, treatment with AGE-BSA enhanced AGT mRNA expression (3.43 ± 0.11-fold compared to control), intracellular AGT protein levels (3.60 ± 0.38-fold), and secreted AGT levels (2.11 ± 0.18-fold). On the other hand, AGT levels were not altered in PTC receiving nonglycated BSA. Recombinant soluble AGE receptor, which competes with endogenous AGE receptor, diminished the AGE-induced AGT upregulation, suggesting that AGE-BSA stimulates AGT expression via activation of the AGE receptor. Enhanced phosphorylation of ERK1/2 and c-Jun, but not p38 MAP kinase, were observed in AGE-BSA-treated PTC. AGE-induced AGT augmentation was attenuated by an ERK inhibitor. CONCLUSIONS: The findings indicate that AGE enhances proximal tubular AGT expression via ERK1/2, which can exacerbate the development of diabetic related kidney injury.

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.

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.