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Cerebral parenchymal arterioles from menopause mice showed increased myogenic tone. We identified an impairment in smooth muscle cell BKCa channel activity, without a reduction in endothelium-dependent dilation or nitric oxide production. Microvascular dysfunction was associated with a reduction in neurovascular responses after somatosensory stimulation. Despite the neurovascular impairment, cognitive abilities were maintained in menopausal mice.

Premenopausal females are protected from angiotensin II (ANG II)-induced hypertension following the adoptive transfer of T cells from normotensive donors. For the present study, we hypothesized that the transfer of hypertensive T cells (HT) or splenocytes (HS) from hypertensive donors would eliminate premenopausal protection from hypertension. Premenopausal recombination-activating gene-1 (Rag-1)(−/−) females received either normotensive (NT) or hypertensive cells 3 wk before ANG II infusion (14 days, 490 ng/kg/min). Contrary to our hypothesis, no increase in ANG II-induced blood pressure was observed in the NT/ANG or HT/ANG groups. Flow cytometry demonstrated that renal FoxP3(+) T regulatory cells were significantly decreased, and immunohistochemistry showed an increase in renal F4/80(+) macrophages in the HT/ANG group, suggesting a shift in the renal inflammatory environment despite no change in blood pressure. Renal mRNA expression of macrophage chemoattractant protein-1 (MCP-1), endothelin-1 (ET-1), and G protein-coupled estrogen receptor-1 (GPER-1) was significantly decreased in the HT/ANG group. The adoptive transfer of hypertensive splenocytes before ANG II infusion (HS/ANG) eliminated premenopausal protection from hypertension and significantly decreased splenic FoxP3(+) T regulatory cells compared with females that received normotensive splenocytes (NS/ANG). Expression of macrophage inflammatory protein 1α/chemokine (C-C motif) ligand 3 (MCP-1/CCL3), a potent macrophage chemokine, was elevated in the HS/ANG group; however, no increase in renal macrophage infiltration occurred. Together, these data show that in premenopausal females, T cells from hypertensive donors are not sufficient to induce robust ANG II-mediated hypertension; in contrast, transfer of hypertensive splenocytes (consisting of T/B lymphocytes, dendritic cells, and macrophages) is sufficient. Further work is needed to understand how innate and adaptive immune cells and estrogen signaling coordinate to cause differential hypertensive outcomes in premenopausal females. NEW & NOTEWORTHY Our study is the first to explore the role of hypertensive T cells versus hypertensive splenocytes in premenopausal protection from ANG II-induced hypertension. We show that the hypertensive status of T cell donors does not impact blood pressure in the recipient female. However, splenocytes, when transferred from hypertensive donors, significantly increased premenopausal recipient blood pressure following ANG II infusion, highlighting the importance of further investigation into estrogen signaling and immune cell activation in females.

There is a sharp rise in cardiovascular disease (CVD) risk and progression with the onset of menopause. The 4-vinylcyclohexene diepoxide (VCD) model of menopause recapitulates the natural, physiological transition through perimenopause to menopause. We hypothesized that menopausal female mice were more susceptible to CVD than pre- or perimenopausal females. Female mice were treated with VCD or vehicle for 20 consecutive days. Premenopausal, perimenopausal, and menopausal mice were administered angiotensin II (ANG II) or subjected to ischemia-reperfusion (I/R). Menopausal females were more susceptible to pathological ANG II-induced cardiac remodeling and cardiac injury from a myocardial infarction (MI), while perimenopausal, like premenopausal, females remained protected. Specifically, ANG II significantly elevated diastolic (130.9 ± 6.0 vs. 114.7 ± 6.2 mmHg) and systolic (156.9 ± 4.8 vs. 141.7 ± 5.0 mmHg) blood pressure and normalized cardiac mass (15.9 ± 1.0 vs. 7.7 ± 1.5%) to a greater extent in menopausal females compared with controls, whereas perimenopausal females demonstrated a similar elevation of diastolic (93.7 ± 2.9 vs. 100.5 ± 4.1 mmHg) and systolic (155.9 ± 7.3 vs. 152.3 ± 6.5 mmHg) blood pressure and normalized cardiac mass (8.3 ± 2.1 vs. 7.5 ± 1.4%) compared with controls. Similarly, menopausal females demonstrated a threefold increase in fibrosis measured by Picrosirus red staining. Finally, hearts of menopausal females (41 ± 5%) showed larger infarct sizes following I/R injury than perimenopausal (18.0 ± 5.6%) and premenopausal (16.2 ± 3.3, 20.1 ± 4.8%) groups. Using the VCD model of menopause, we provide evidence that menopausal females were more susceptible to pathological cardiac remodeling. We suggest that the VCD model of menopause may be critical to better elucidate cellular and molecular mechanisms underlying the transition to CVD susceptibility in menopausal women. NEW & NOTEWORTHY Before menopause, women are protected against cardiovascular disease (CVD) compared with age-matched men; this protection is gradually lost after menopause. We present the first evidence that demonstrates menopausal females are more susceptible to pathological cardiac remodeling while perimenopausal and cycling females are not. The VCD model permits appropriate examination of how increased susceptibility to the pathological process of cardiac remodeling accelerates from pre- to perimenopause to menopause.

Polycystic kidney disease (PKD) is the most common inheritable cause of kidney failure, and the underlying mechanisms remain incompletely uncovered. Renal nerves contribute to hypertension and chronic kidney disease—frequent complications of PKD. There is limited evidence that renal nerves may contribute to cardiorenal dysfunction in PKD and no investigations of the role of sympathetic versus afferent nerves in PKD. Afferent renal nerve activity (ARNA) is elevated in models of renal disease and fibrosis. However, it remains unknown if this is true in PKD. We tested the hypothesis that ARNA is elevated in a preclinical model of autosomal recessive PKD and that targeted renal nerve ablation would attenuate cystogenesis and cardiorenal dysfunction. We tested this by performing total renal denervation (T-RDNx) or afferent renal denervation (A-RDNx) denervation in 4-wk-old male and female PCK rats and then quantified renal and cardiovascular responses 6 wk following treatment. Cystogenesis was attenuated with A-RDNx and T-RDNx versus sham controls, highlighting a crucial role for renal afferent nerves in cystogenesis. In contrast, blood pressure was improved with T-RDNx but not A-RDNx. Importantly, treatments produced similar results in both males and females. Direct renal afferent nerve recordings revealed that ARNA was twofold greater in PCK rats versus noncystic controls and was directly correlated with cystic severity. To our knowledge, we are the first to demonstrate that PCK rats have greater ARNA than noncystic, age-matched controls. The findings of this study support a novel and crucial role for renal afferent innervation in cystogenesis in the PCK rat. NEW & NOTEWORTHY This is the first study to dissect the contributions of renal sympathetic and afferent innervation in the PCK rat, a preclinical model of autosomal recessive polycystic kidney disease. We demonstrated that resting afferent renal nerve activity is greater in the PCK rat than noncystic controls and that basal afferent renal nerve activity is directly correlated with the extent of renal cystogenesis.

Although it is known that the prevalence and severity of hypertension increases in women after menopause, the contribution of T cells to this process has not been explored. Although the immune system is both necessary and required for the development of angiotensin II (ANG II) hypertension in men, we have demonstrated that premenopausal women are protected from T cell-mediated hypertension. The goal of the current study was to test the hypotheses that 1) female protection against T cell-mediated ANG II hypertension is eliminated following progression into menopause and 2) T regulatory cells (Tregs) provide premenopausal protection against ANG II-induced hypertension. Menopause was induced in Rag-1−/− mice (via 4-vinylcyclohexene diepoxide), and all mice received a 14-day ANG II infusion. Donor CD3+ T cells were adoptively transferred 3 wk before ANG II infusion. In the absence of T cells, systolic blood pressure responses to ANG II were similar to those seen in premenopausal mice (Δ12 mmHg). After adoptive transfer of T cells, ANG II significantly increased systolic blood pressure in postmenopausal females (Δ28 mmHg). A significant increase in F4/80 positive renal macrophages, an increase in renal inflammatory gene expression, along with a reduction in renal expression of mannose receptor C-type 1, a marker for M2 macrophages, accompanied the increase in systolic blood pressure (SBP). Flow cytometric analysis identified that Tregs were significantly decreased in the spleen and kidneys of Rag-1−/− menopausal mice versus premenopausal females, following ANG II infusion. In a validation study, an anti-CD25 antibody was used to deplete Tregs in premenopausal mice, which induced a significant increase in SBP. These results demonstrate that premenopausal protection against T cell-mediated ANG II hypertension is eliminated once females enter menopause, suggesting that a change in hormonal status upregulates macrophage-induced proinflammatory and T cell-dependent responses. Furthermore, we are the first to report that the presence of Tregs are required to suppress ANG II hypertension in premenopausal females. NEW & NOTEWORTHY Whether progression into menopause eliminated female protection against T cell-mediated hypertension was examined. Menopausal mice without T cells remained protected against angiotensin II (ANG II) hypertension; however, in the presence of T cells, blood pressure responses to ANG II increased significantly in menopause. Underlying mechanisms examined were anti-inflammatory protection provided by T regulatory cells in premenopausal females and renal inflammatory processes involving macrophage infiltration and cytokine activation.

Background Menopause is associated with an increase in the prevalence and severity of hypertension in women. Although premenopausal females are protected against T cell‐dependent immune activation and development of angiotensin II (Ang II) hypertension, this protection is lost in postmenopausal females. Therefore, the current study hypothesized that specific CD4 + T cell pathways are regulated by sex hormones and Ang II to mediate progression from premenopausal protection to postmenopausal hypertension. Methods and Results Menopause was induced in C57BL/6 mice via repeated 4‐vinylcyclohexene diepoxide injections, while premenopausal females received sesame oil vehicle. A subset of premenopausal mice and all menopausal mice were infused with Ang II for 14 days (Control, Ang II, Meno/Ang II). Proteomic and phosphoproteomic profiles of CD4 + T cells isolated from spleens were examined. Ang II markedly increased CD4 + T cell protein abundance and phosphorylation associated with DNA and histone methylation in both premenopausal and postmenopausal females. Compared with premenopausal T cells, Ang II infusion in menopausal mice increased T cell phosphorylation of MP2K2, an upstream regulator of ERK, and was associated with upregulated phosphorylation at ERK targeted sites. Additionally, Ang II infusion in menopausal mice decreased T cell phosphorylation of TLN1, a key regulator of IL‐2Rα and FOXP3 expression. Conclusions These findings identify novel, distinct T cell pathways that influence T cell‐mediated inflammation during postmenopausal hypertension.

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Estrogen has been implicated in the regulation of growth and immune function in the kidney, which expresses the full-length estrogen receptor-α (ERα66), its ERα splice variants, and estrogen receptor-β (ERβ). Thus, we hypothesized that these splice variants may inhibit the glomerular enlargement that occurs early in type 1 diabetes (T1D). T1D was induced by streptozotocin (STZ) injection in 8- to 12-wk-old female mice lacking ERα66 (ERα66KO) or all ERα variants (αERKO), and their wild-type (WT) littermates. Basal renal ERα36 protein expression was reduced in the ERα66KO model and was downregulated by T1D in WT mice. T1D did not alter ERα46 or ERβ in WT-STZ; however, ERα46 was decreased modestly in ERα66KO mice. Renal hypertrophy was evident in all diabetic mice. F4/80-positive immunostaining was reduced in ERα66KO compared with WT and αERKO mice but was higher in STZ than in Control mice across all genotypes. Glomerular area was greater in WT and αERKO than in ERα66KO mice, with T1D-induced glomerular enlargement apparent in WT-STZ and αERKO-STZ, but not in ERα66KO-STZ mice. Proteinuria and hyperfiltration were evident in ERα66KO-STZ and αERKO-STZ, but not in WT-STZ mice. These data indicate that ERα splice variants may exert an inhibitory influence on glomerular enlargement and macrophage infiltration during T1D; however, effects of splice variants are masked in the presence of the full-length ERα66, suggesting that ERα66 acts in opposition to its splice variants to influence these parameters. In contrast, hyperfiltration and proteinuria in T1D are attenuated via an ERα66-dependent mechanism that is unaffected by splice variant status.

Oxidative stress and mitochondrial dysfunction exacerbate acute kidney injury (AKI), but their role in any associated progress to chronic kidney disease (CKD) remains unclear. Antioxidant therapies often benefit AKI, but their benefits in CKD are controversial since clinical and preclinical investigations often conflict. Here we examined the influence of the antioxidant N-acetyl-cysteine (NAC) on oxidative stress and mitochondrial function during AKI (20-min bilateral renal ischemia plus reperfusion/IR) and progression to chronic kidney pathologies in mice. NAC (5% in diet) was given to mice 7 days prior and up to 21 days post-IR (21d-IR). NAC treatment resulted in the following: prevented proximal tubular epithelial cell apoptosis at early IR (40-min postischemia), yet enhanced interstitial cell proliferation at 21d-IR; increased transforming growth factor-β1 expression independent of IR time; and significantly dampened nuclear factor-like 2-initiated cytoprotective signaling at early IR. In the long term, NAC enhanced cellular metabolic impairment demonstrated by increased peroxisome proliferator activator-γ serine-112 phosphorylation at 21d-IR. Intravital multiphoton microscopy revealed increased endogenous fluorescence of nicotinamide adenine dinucleotide (NADH) in cortical tubular epithelial cells during ischemia, and at 21d-IR that was not attenuated with NAC. Fluorescence lifetime imaging microscopy demonstrated persistent metabolic impairment by increased free/bound NADH in the cortex at 21d-IR that was enhanced by NAC. Increased mitochondrial dysfunction in remnant tubular cells was demonstrated at 21d-IR by tetramethylrhodamine methyl ester fluorimetry. In summary, NAC enhanced progression to CKD following AKI not only by dampening endogenous cellular antioxidant responses at time of injury but also by enhancing persistent kidney mitochondrial and metabolic dysfunction.

Antibody use is a critical component of cardiovascular physiology research, and antibodies are used to monitor protein abundance (immunoblot analysis) and protein expression and localization (in tissue by immunohistochemistry and in cells by immunocytochemistry). With ongoing discussions on how to improve reproducibility and rigor, the goal of this review is to provide best practice guidelines regarding how to optimize antibody use for increased rigor and reproducibility in both immunoblot analysis and immunohistochemistry approaches. Listen to this article’s corresponding podcast at http://ajpheart.podbean.com/e/guidelines-on-antibody-use-in-physiology-studies/ .

In this paper we investigate category-specific effects through the lens of Welsh mutation. Smith (2011) and Moreton et al. (2017) show that English distinguishes nouns and proper nouns in an experimental blending task. Here we show that Welsh distinguishes nouns, verbs, personal names, and place names in the mutation system. We demonstrate these effects experimentally in a translation task designed to elicit mutation intuitions and in several corpus studies. In addition, we show that these effects correlate with lexical frequency. Deeper statistical analysis and a review of the English data suggests that frequency is a more explanatory factor than part of speech in both languages. We therefore argue that these category-specific effects can be reduced to lexical frequency effects.

T cells are required for the development of hypertension in male and postmenopausal female mice while premenopausal females are protected from T cell mediated hypertension. To better understand sex differences in immune-cell mediated hypertensive responses, we sought to determine if there were any significant differences in the immune cell profiles of premenopausal female (F), VCD-treated postmenopausal female (PMF), and male (M) mice. Spleens were collected from all mice and processed for flow cytometric analysis of T cell populations (n=8/group). Analysis of splenic T cell populations revealed no significant difference in the frequency of CD3+ or CD4+ T cells between groups (CD3+: F 33.4%, PMF 30.3%, M 30.2% of total lymphocytes, CD4+: F 64.8%, PMF 70.7%, M 67.5% of CD3+ cells). However, postmenopausal females had a significantly lower frequency of splenic CD8+ T cells compared to both males and premenopausal females (CD8+: F 27.9%, PMF 19.5%*, M 25.3% of CD3+ cells *p

T cells are involved in hypertension pathogenesis in both males and postmenopausal females while premenopausal females are resistant to T cell-mediated Ang II-induced hypertension. The goals of this study were (1) to identify T cell specific proteomic pathways associated with postmenopausal susceptibility to hypertension (2) to identify T cell specific transcriptomic changes associated with premenopausal protection from hypertension. Proteomic analysis was performed on splenic CD4 + T cells isolated from premenopausal and postmenopausal females (VCD, 160 mg/kg/day i.p. 20d) following Ang II infusion (800 ng/kg/min 14d). 384 proteins from CD4 + T cells were identified as differentially expressed following Ang II infusion in premenopausal females. 285 proteins from CD4 + T cells were identified as differentially expressed between premenopausal and postmenopausal females following Ang II infusion. Gene ontology (GO) analysis of pre vs. postmenopausal proteins identified enriched pathways associated with RNA binding, chaperone activity and cellular stress responses. Transcriptomic changes were analyzed, via RNAseq, on isolated splenic CD4 + T cells from premenopausal females, with and without Ang II infusion. Thirty-four genes were identified as differentially expressed in CD4 + T cells following Ang II infusion. GO analysis of Ang II upregulated genes revealed an enrichment of five distinct molecular functions, including antioxidant activity. In a subsequent study to validate the RNAseq, we confirmed that Ang II increased CD4 + T cell mRNA expression of calprotectin (S100a8/S100a9), a calcium and zinc binding protein complex that contributes to antioxidant defense (S100a8: Con 1.0 ± 0.4 vs Ang II 5.0 ± 0.8*; S100a9: Con 1.0 ± 0.4 vs Ang II 6.0 ± 0.8*, *P

PURPOSE OF REVIEW: Despite enhanced screening and therapeutic management, hypertension remains the most prevalent chronic disease in the United States and the leading cause of heart disease, chronic kidney disease, and stroke in both men and women. It is widely accepted that hypertension is a pro-inflammatory disease and that the immune system plays a vital role in mediating hypertensive outcomes and end organ damage. Despite known discrepancies in the risk of hypertension development between men and women, preclinical models of immune-mediated hypertension were historically developed solely in male animals, leading to a lack of sex-specific clinical practice guidelines or therapeutic targets. RECENT FINDINGS: Following the NIH policy on the consideration of sex as a biological variable in 2015, significant advancements have been made into sex-specific disease mechanisms in inflammation and hypertension. SUMMARY: This review article serves to critically evaluate recent advancements in the field of sex-specific immune-mediated hypertension.

Although it is known that the prevalence and severity of hypertension increases in women after menopause, the contribution of T cells to this process has not been explored. Although the immune system is both necessary and required for the development of angiotensin II (ANG II) hypertension in men, we have demonstrated that premenopausal women are protected from T cell-mediated hypertension. The goal of the current study was to test the hypotheses that 1) female protection against T cell-mediated ANG II hypertension is eliminated following progression into menopause and 2) T regulatory cells (Tregs) provide premenopausal protection against ANG II-induced hypertension. Menopause was induced in Rag-1(−/−) mice (via 4-vinylcyclohexene diepoxide), and all mice received a 14-day ANG II infusion. Donor CD3(+) T cells were adoptively transferred 3 wk before ANG II infusion. In the absence of T cells, systolic blood pressure responses to ANG II were similar to those seen in premenopausal mice (Δ12 mmHg). After adoptive transfer of T cells, ANG II significantly increased systolic blood pressure in postmenopausal females (Δ28 mmHg). A significant increase in F4/80 positive renal macrophages, an increase in renal inflammatory gene expression, along with a reduction in renal expression of mannose receptor C-type 1, a marker for M2 macrophages, accompanied the increase in systolic blood pressure (SBP). Flow cytometric analysis identified that Tregs were significantly decreased in the spleen and kidneys of Rag-1(−/−) menopausal mice versus premenopausal females, following ANG II infusion. In a validation study, an anti-CD25 antibody was used to deplete Tregs in premenopausal mice, which induced a significant increase in SBP. These results demonstrate that premenopausal protection against T cell-mediated ANG II hypertension is eliminated once females enter menopause, suggesting that a change in hormonal status upregulates macrophage-induced proinflammatory and T cell-dependent responses. Furthermore, we are the first to report that the presence of Tregs are required to suppress ANG II hypertension in premenopausal females. NEW & NOTEWORTHY Whether progression into menopause eliminated female protection against T cell-mediated hypertension was examined. Menopausal mice without T cells remained protected against angiotensin II (ANG II) hypertension; however, in the presence of T cells, blood pressure responses to ANG II increased significantly in menopause. Underlying mechanisms examined were anti-inflammatory protection provided by T regulatory cells in premenopausal females and renal inflammatory processes involving macrophage infiltration and cytokine activation.

Purpose Recent studies suggest that the neural retinal response to light is compromised in diabetes. Electroretinogram studies suggest that the dim light retinal rod pathway is especially susceptible to diabetic damage. The purpose of this study was to determine whether diabetes alters rod pathway signaling. Methods Diabetes was induced in C57BL/6J mice by three intraperitoneal injections of streptozotocin (STZ; 75 mg/kg), and confirmed by blood glucose levels > 200 mg/dL. Six weeks after the first injection, whole-cell voltage clamp recordings of spontaneous and light-evoked inhibitory postsynaptic currents from rod bipolar cells were made in dark-adapted retinal slices. Light-evoked excitatory currents from rod bipolar and AII amacrine cells, and spontaneous excitatory currents from AII amacrine cells were also measured. Receptor inputs were pharmacologically isolated. Immunohistochemistry was performed on whole mounted retinas. Results Rod bipolar cells had reduced light-evoked inhibitory input from amacrine cells but no change in excitatory input from rod photoreceptors. Reduced light-evoked inhibition, mediated by both GABAA and GABAC receptors, increased rod bipolar cell output onto AII amacrine cells. Spontaneous release of GABA onto rod bipolar cells was increased, which may limit GABA availability for light-evoked release. These physiological changes occurred in the absence of retinal cell loss or changes in GABAA receptor expression levels. Conclusions Our results indicate that early diabetes causes deficits in the rod pathway leading to decreased light-evoked rod bipolar cell inhibition and increased rod pathway output that provide a basis for the development of early diabetic visual deficits.

Activation of T cell-dependent pro-inflammatory responses are required for Ang II hypertension in male mice. However, females are protected from T cell-mediated hypertension and may suppress hypertension by directly preventing Ang II-induced pro-inflammatory T cell activation. Here we sought to determine whether transferring T cells from hypertensive donor mice eliminates female protection against T cell-mediated hypertension. Splenic CD3 + T cells were transferred from normotensive (NT) or Ang II-hypertensive (HT) C57BL/6J male donors to female Rag-1 -/- (NT T cell female-NTF; HT T cell female-HTF) or male Rag-1 -/- (HT T cell male-HTM) recipient mice. Blood pressure was monitored (tail cuff) for 5 weeks post-transfer. Ang II (490ng/kg/min) was infused into recipient mice for 14 days during weeks 4 and 5 post-transfer (NTFA; HTFA; HTMA). Ang II significantly increased MAP in donor male mice (NT 114 vs HT 157 mmHg, p+ 4 mmHg*, HTF 88 + 6 mmHg*, NTFA 101 + 5 mmHg*, HTFA 103 + 5 mmHg*, HTMA 138 + 3 mmHg, *p0.05). However, regulatory T cells were significantly reduced in male recipients compared to all female groups (Foxp3: NTF 21.6%*, NTFA 22.2%*, HTF 22.8%*, HTFA 22.6%*, HTMA 15.3%, *p

Psychiatric patients treated with lithium (Li(+)) may develop nephrogenic diabetes insipidus (NDI). Although the etiology of Li(+)-induced NDI (Li-NDI) is poorly understood, it occurs partially due to reduced aquaporin-2 (AQP2) expression in the kidney collecting ducts. A mechanism postulated for this is that Li(+) inhibits adenylyl cyclase (AC) activity, leading to decreased cAMP, reduced AQP2 abundance, and less membrane targeting. We hypothesized that Li-NDI would not develop in mice lacking AC6. Whole-body AC6 knockout (AC6(-/-)) mice and potentially novel connecting tubule/principal cell-specific AC6 knockout (AC6(loxloxCre)) mice had approximately 50% lower urine osmolality and doubled water intake under baseline conditions compared with controls. Dietary Li(+) administration increased water intake and reduced urine osmolality in control, AC6(-/-), and AC6(loxloxCre) mice. Consistent with AC6(-/-) mice, medullary AQP2 and pS256-AQP2 abundances were lower in AC6(loxloxCre) mice compared with controls under standard conditions, and levels were further reduced after Li(+) administration. AC6(loxloxCre) and control mice had a similar increase in the numbers of proliferating cell nuclear antigen-positive cells in response to Li(+). However, AC6(loxloxCre) mice had a higher number of H(+)-ATPase B1 subunit-positive cells under standard conditions and after Li(+) administration. Collectively, AC6 has a minor role in Li-NDI development but may be important for determining the intercalated cell-to-principal cell ratio.

Aging of the world population and a concomitant increase in age-related diseases and disabilities mandates the search for strategies to increase healthspan, the length of time an individual lives healthy and productively. Due to the age-related decline of the immune system, infectious diseases remain among the top 5–10 causes of mortality and morbidity in the elderly, and improving immune function during aging remains an important aspect of healthspan extension. Calorie restriction (CR) and more recently rapamycin (rapa) feeding have both been used to extend lifespan in mice. Preciously few studies have actually investigated the impact of each of these interventions upon in vivo immune defense against relevant microbial challenge in old organisms. We tested how rapa and CR each impacted the immune system in adult and old mice. We report that each intervention differentially altered T-cell development in the thymus, peripheral T-cell maintenance, T-cell function and host survival after West Nile virus infection, inducing distinct but deleterious consequences to the aging immune system. We conclude that neither rapa feeding nor CR, in the current form/administration regimen, may be optimal strategies for extending healthy immune function and, with it, lifespan.

There is extensive evidence that activation of the immune system is both necessary and required for the development of angiotensin II (Ang II)–induced hypertension in males. The purpose of this study was to determine whether sex differences exist in the ability of the adaptive immune system to induce Ang II–dependent hypertension and whether central and renal T-cell infiltration during Ang II–induced hypertension is sex dependent. Recombinant activating gene-1 ( Rag-1 ) –/– mice, lacking both T and B cells, were used. Male and female Rag-1 –/– mice received adoptive transfer of male CD3 + T cells 3 weeks before 14-day Ang II infusion (490 ng/kg per minute). Blood pressure was monitored via tail cuff. In the absence of T cells, systolic blood pressure responses to Ang II were similar between sexes (Δ22.1 mm Hg males versus Δ18 mm Hg females). After adoptive transfer of male T cells, Ang II significantly increased systolic blood pressure in males (Δ37.7 mm Hg; P Hg). Flow cytometric analysis of total T cells and CD4 + , CD8 + , and regulatory Foxp3 + -CD4 + T-cell subsets identified that renal lymphocyte infiltration was significantly increased in males versus females in both control and Ang II–infused animals ( P + -positive T cells in the subfornical organ region of the brain was increased in males when compared with that in females. These results suggest that female Rag-1 –/– mice are protected from male T-cell–mediated increases in Ang II–induced hypertension when compared with their male counterparts, and this protection may involve sex differences in the magnitude of T-cell infiltration of the kidney and brain.

Nephrogenic diabetes insipidus (NDI) is the most common renal side effect in patients undergoing lithium therapy for bipolar affective disorders. Approximately 2 million US patients take lithium of whom ∼50% will have altered renal function and develop NDI ( 2 , 37 ). Lithium-induced NDI is a defect in the urinary concentrating mechanism. Lithium therapy also leads to proliferation and abundant renal cysts (microcysts), commonly in the collecting ducts of the cortico-medullary region. The mTOR pathway integrates nutrient and mitogen signals to control cell proliferation and cell growth (size) via the mTOR Complex 1 (mTORC1). To address our hypothesis that mTOR activation may be responsible for lithium-induced proliferation of collecting ducts, we fed mice lithium chronically and assessed mTORC1 signaling in the renal medulla. We demonstrate that mTOR signaling is activated in the renal collecting ducts of lithium-treated mice; lithium increased the phosphorylation of rS6 (Ser240/Ser244), p-TSC2 (Thr1462), and p-mTOR (Ser2448). Consistent with our hypothesis, treatment with rapamycin, an allosteric inhibitor of mTOR, reversed lithium-induced proliferation of medullary collecting duct cells and reduced levels of p-rS6 and p-mTOR. Medullary levels of p-GSK3β were increased in the renal medullas of lithium-treated mice and remained elevated following rapamycin treatment. However, mTOR inhibition did not improve lithium-induced NDI and did not restore the expression of collecting duct proteins aquaporin-2 or UT-A1.

Prior to menopause, women are protected against cardiovascular disease (CVD) compared to age-matched men; this protection is gradually lost after menopause. Loss of estrogen is detrimental; yet, estrogen replacement as cardioprotective remains controversial. Gonadectomized rodents demonstrate benefit of estrogen replacement for CVD. Human studies that show benefit of hormone replacement therapy are challenged by studies that do not show benefit. A novel model of menopause utilizing 4-vinylcyclohexene diepoxide (VCD) induces gradual ovarian failure, preserving the “perimenopause” transitional period and androgen secreting capacity of residual ovarian tissue. We hypothesize that menopausal females are hypersensitive to CVD-induced pathological cardiac remodeling. To address this hypothesis, we instigated menopause in 2 month-old females by daily (i.p.) injections of VCD (160mg/kg, 20 consecutive days); control mice received sesame oil as vehicle. Vaginal cytology was used to monitor estrous cycles and determine when cycling ceased. Mice were considered menopausal after 15 consecutive days in persistent diestrus, non-cycling. Female mice from cycling, perimenopausal and menopausal groups received the hypertensive agent angiotensin II (Ang II, 800 ng/kg/min via alzet s.q. mini-pump, 14 days). Menopause did not impact systolic blood pressure (SBP) measured via tail cuff (control: 115.1±6.0 mmHg, n=7; menopause: 115.0±5.2 mmHg, n=10). Ang II infusion induced a significant exacerbation of hypertension (SBP) in menopausal females (156.9±4.8 mmHg; n=10) compared to Ang II controls (114.7±5.0 mmHg; n=8). This hypersensitivity to Ang II-induced hypertension was attenuated by estrogen supplementation started during perimenopause (0.1 mg, 60 day release). In addition, menopausal females demonstrated worsened pathological cardiac remodeling measured by functional (echocardiography), cellular (myocardial fibrosis) and molecular (fetal gene program) assessments. Using a novel model of menopause (VCD) combined with infusion of the hypertensive agent Ang II, we demonstrated that menopausal mice are more susceptible, or hypersensitive to pathological cardiac remodeling compared to cycling and perimenopausal mice.

Prior to menopause, women are protected against cardiovascular disease (CVD) compared to age-matched men; this protection is gradually lost after menopause. The mechanisms responsible for loss of protection and rise in CVD onset are unknown. A novel model of menopause utilizing 4-vinylcyclohexene diepoxide (VCD) induces gradual ovarian failure, preserving the “perimenopause” transitional period and androgen secreting capacity of residual ovarian tissue. Our central hypothesis states that menopausal females are hypersensitive to CVD-induced pathological cardiac remodeling. To address this hypothesis, we instigated menopause in 2 month-old females by daily (i.p.) injections of VCD (160mg/kg, 20 consecutive days); control mice received sesame oil as vehicle. Vaginal cytology was used to monitor estrous cycles and determine when cycling ceased. Mice were considered menopausal after 15 consecutive days in persistent diestrus, non-cycling. Female mice from cycling, perimenopausal and menopausal groups were subjected to 30 minutes of ischemia followed by 2 days of reperfusion (I/R). Following reperfusion, hearts were rapidly excised, sectioned and double-stained with Evans Blue and triphenyltetrazolium chloride to define the area at risk (AAR) and infarct size as area of necrosis (AON). On average, menopausal females displayed a significant (p

In females, menopause, the cessation of menstrual cycling, is associated with an increase in risk for several diseases such as cardiovascular disease, osteoporosis, diabetes, the metabolic syndrome, and ovarian cancer. The majority of women enter menopause via a gradual reduction of ovarian function over several years (perimenopause) and retain residual ovarian tissue. The VCD mouse model of menopause (ovarian failure in rodents) is a follicle-deplete, ovary-intact animal that more closely approximates the natural human progression through perimenopause and into the postmenopausal stage of life. In this review, we present the physiological parameters of how to use the VCD model and explore the VCD model and its application into the study of postmenopausal disease mechanisms, focusing on recent murine studies of diabetic kidney disease, the metabolic syndrome, and hypertension.

The phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α) occurs under many stress conditions in mammalian cells and is mediated by one of four eIF2α kinases: PERK, PKR, GCN2, and HRI. Cells of the renal medulla are regularly exposed to fluctuating concentrations of urea and sodium, the extracellular solutes responsible for the high osmolality in the renal medulla, and thus the kidneys ability to concentrate the urine in times of dehydration. Urea stress is known to initiate molecular responses that diverge from those seen in response to hypertonic stress (NaCl). We show that urea-inducible GCN2 activation initiates the phosphorylation of eIF2α and the downstream increase of activating transcription factor 3 (ATF3). Loss of GCN2 sensitized cells to urea stress, increasing the expression of activated caspase-3 and decreasing cell survival. Loss of GCN2 ablated urea-induced phosphorylation of eIF2α and reduced the expression of ATF3.

Activation of V2 receptors (V2R) during antidiuresis increases the permeability of the inner medullary collecting duct to urea and water. Extracellular osmolality is elevated as the concentrating capacity of the kidney increases. Osmolality is known to contribute to the regulation of collecting duct water (aquaporin-2; AQP2) and urea transporter (UT-A1, UT-A3) regulation. AQP1KO mice are a concentrating mechanism knockout, a defect attributed to the loss of high interstitial osmolality. A V2R-specific agonist, deamino-8-d-arginine vasopressin (dDAVP), was infused into wild-type and AQP1KO mice for 7 days. UT-A1 mRNA and protein abundance were significantly increased in the medullas of wild-type and AQP1KO mice following dDAVP infusion. The mRNA and protein abundance of UT-A3, the basolateral urea transporter, was significantly increased by dDAVP in both wild-type and AQP1KO mice. Semiquantitative immunoblots revealed that dDAVP infusion induced a significant increase in the medullary expression of the endoplasmic reticulum (ER) chaperone GRP78. Immunofluorescence studies demonstrated that GRP78 expression colocalized with AQP2 in principal cells of the papillary tip of the renal medulla. Using immunohistochemistry and immunogold electron microscopy, we demonstrate that vasopressin induced a marked apical targeting of GRP78 in medullary principal cells. Urea-sensitive genes, GADD153 and ATF4 (components of the ER stress pathway), were significantly increased in AQP1KO mice by dDAVP infusion. These findings strongly support an important role of vasopressin in the activation of an ER stress response in renal collecting duct cells, in addition to its role in activating an increase in UT-A1 and UT-A3 abundance.

Factors comprising the metabolic syndrome occur with increased incidence in postmenopausal women. To investigate the effects of ovarian failure on the progression of the metabolic syndrome, female B6C3F1 mice were treated with 4-vinylcyclohexene diepoxide (VCD) and fed a high-fat (HF) diet for 16 wk. VCD destroys preantral follicles, causing early ovarian failure and is a well-characterized model for the gradual onset of menopause. After 12 wk on a HF diet, VCD-treated mice had developed an impaired glucose tolerance, whereas cycling controls were unaffected [12 wk AUC HF mice 13,455 ± 643 vs. HF/VCD 17,378 ± 1140 mg/dl/min, P < 0.05]. After 16 wk on a HF diet, VCD-treated mice had significantly higher fasting insulin levels (HF 5.4 ± 1.3 vs. HF/VCD 10.1 ± 1.4 ng/ml, P < 0.05) and were significantly more insulin resistant (HOMA-IR) than cycling controls on a HF diet (HF 56.2 ± 16.7 vs. HF/VCD 113.1 ± 19.6 mg/dl·μU/ml, P < 0.05). All mice on a HF diet gained more weight than mice on a standard diet, and weight gain in HF/VCD mice was significantly increased compared with HF cycling controls. Interestingly, even without a HF diet, progression into VCD-induced menopause caused a significant increase in cholesterol and free fatty acids. Furthermore, in mice fed a standard diet (6% fat), insulin resistance developed 4 mo after VCD-induced ovarian failure. Insulin resistance following ovarian failure (menopause) was prevented by estrogen replacement. Studies here demonstrate that ovarian failure (menopause) accelerates progression into the metabolic syndrome and that estrogen replacement prevents the onset of insulin resistance in VCD-treated mice. Thus, the VCD model of menopause provides a physiologically relevant means of studying how sex hormones influence the progression of the metabolic syndrome.

Aquaporin (AQP) 1 null mice have a defect in the renal concentrating gradient because of their inability to generate a hyperosmotic medullary interstitium. To determine the effect of vasopressin on renal medullary gene expression, in the absence of high local osmolarity, we infused 1-deamino-8-d-arginine vasopressin (dDAVP), a V2 receptor (V2R)-specific agonist, in AQP1 null mice for 7 days. cDNA microarray analysis was performed on the renal medullary tissue, and 5,140 genes of the possible 12,000 genes on the array were included in the analysis. In the renal medulla of AQP1 null mice, 245 transcripts were identified as increased by dDAVP infusion and 200 transcripts as decreased (1.5-fold or more). Quantitative real-time PCR measurements confirmed the increases seen for cyclin D1, early growth response gene 1, and activating transcription factor 3, genes associated with changes in cell cycle/growth. Changes in mRNA expression were correlated with changes in protein expression by semiquantitative immunoblotting; cyclin D1 and ATF3 were increased significantly in abundance following dDAVP infusion in the renal medulla of AQP1 null mice (161 and 461%, respectively). A significant increase in proliferation of medullary collecting ducts cells, following V2R activation, was identified by proliferating cell nuclear antigen immunohistochemistry; colocalization studies with AQP2 indicated that the increase in proliferation was primarily observed in principal cells of the inner medullary collecting duct (IMCD). V2R activation, via dDAVP, increased AQP2 and AQP3 protein abundance in the cortical collecting ducts of AQP1 null mice. However, V2R activation did not increase AQP2 protein abundance in the IMCD of AQP1 null mice.

Epidemiological studies link arsenic exposure to increased risks of cancers of the skin, kidney, lung, bladder and liver. Additionally, a variety of non-cancerous conditions such as diabetes mellitus, hypertension, and cardiovascular disease have been associated with chronic ingestion of low levels of arsenic. However, the biological and molecular mechanisms by which arsenic exerts its effects remain elusive. Here we report increased renal hexokinase II (HKII) expression in response to arsenic exposure both in vivo and in vitro. In our model, HKII was up-regulated in the renal glomeruli of mice exposed to low levels of arsenic (10 ppb or 50 ppb) via their drinking water for up to 21 days. Additionally, a similar effect was observed in cultured renal mesangial cells exposed to arsenic. This correlation between our in vivo and in vitro data provides further evidence for a direct link between altered renal HKII expression and arsenic exposure. Thus, our data suggest that alterations in renal HKII expression may be involved in arsenic-induced pathological conditions involving the kidney. More importantly, these results were obtained using environmentally relevant arsenic concentrations.

The mammalian counterpart of the fish calcium-regulating hormone stanniocalcin-1 (STC1) inhibits monocyte chemotactic protein-1- and stromal-derived factor-1α (SDF-1α)-mediated chemotaxis and diminishes chemokinesis in macrophage-like RAW264.7 and U937 cells in a manner that may involve attenuation of the intracellular calcium signal. STC1 is strongly induced in the kidney following obstructive injury. We hypothesized that STC1 may serve to attenuate the influx of inflammatory cells to the site of tissue injury. In this study, we examined the effect of STC1 on the migration of freshly isolated human macrophages, neutrophils, and T and B lymphocytes through quiescent or IL-1β-treated human umbilical vein endothelial cell (HUVEC) monolayers. STC1 inhibited transmigration of macrophages and T lymphocytes through quiescent or IL-1β-activated HUVECs but did not attenuate the transmigration of neutrophils and B lymphocytes. STC1 regulates gene expression in cultured endothelial cells and is detected on the apical surface of endothelial cells in vivo. The data suggest that STC1 plays a critical role in transendothelial migration of inflammatory cells and is involved in the regulation of numerous aspects of endothelial function.

Compared to males, premenopausal females are resistant to the development of Ang II hypertension. In males, Ang II induces hypertension, in part, through mechanisms requiring T effector lymphocytes. Recently, our lab has demonstrated that females can prevent the T lymphocyte-dependent increase in blood pressure (SBP and MAP) and expression of pro-inflammatory cytokines in the kidney in response to Ang II infusion. Because Foxp3 + T regulatory cells suppress the pro-inflammatory and hypertensive actions of T effector cells, we sought to determine whether Foxp3 + T regulatory cells contribute to this resistance in females. Premenopausal (8 week old) 129SVE female mice were infused with Ang II (800ng/kg/min, 14d) and received 4 doses of the anti-CD25 antibody PC-61 to transiently deplete Foxp3 + T regulatory cells (every 84 hours beginning 12 hours prior to Ang II infusion, 250μg/dose, i.p., vehicle control). Blood pressure was measured before and after Ang II infusion via non-invasive tail cuff. Ang II induced a significant increase in systolic blood pressure in Foxp3 + -depleted mice, while resistance was retained in vehicle-treated mice (Con Δ5 ± 5mmHg, Ang II Δ10 ± 7mmHg, PC-61 Δ28 ± 9 * mmHg, * p+ splenic T cells compared to control (Con 1.7x10 6 cells, Ang II 2.3x10 6 cells, PC-61 8.3x10 5* cells, * P+ and CD4 + T cell counts. The number of Foxp3 + T cells residing in the kidney was also significantly reduced by PC-61 (Con 1,152 ± 368 cells, Ang II 686 ± 389 cells, PC-61 210 ± 35 * cells, * P+ -depleted mice (MCP-1- Con 1.0 ± 0.1, Ang II 1.6 ± 0.4, PC-61 1.8 ± 0.2 * ; ENaC-α- Con 1.0 ± 0.1, Ang II 1.6 ± 0.2, PC-61 2.1 ± 0.1 * , * P+ T regulatory cells play a significant role in mediating the resistance to Ang II hypertension in premenopausal female mice, and may influence renal inflammation and sodium retention during chronic Ang II infusion.

Premenopausal females are resistant to the development of hypertension, and this protection is lost after the onset of menopause, resulting in a sharp increase in disease onset and severity. However, it is unknown how a fluctuating ovarian hormone environment during the transition from perimenopause to menopause impacts the onset of hypertension, and whether interventions during perimenopause prevent disease onset after menopause. A gradual transition to menopause was induced by repeated daily injections of 4-vinylcyclohexene diepoxide (VCD). ANG II (800 ng·kg−1·min−1) was infused into perimenopausal and menopausal female mice for 14 days. A separate cohort of mice received 17β-estradiol replacement during perimenopause. ANG II infusion produced significantly higher mean arterial pressure (MAP) in menopausal vs. cycling females, and 17β-estradiol replacement prevented this increase. In contrast, MAP was not significantly different when ANG II was infused into perimenopausal and cycling females, suggesting that female resistance to ANG II-induced hypertension is intact during perimenopause. ANG II infusion caused a significant glomerular hypertrophy, and hypertrophy was not impacted by hormonal status. Expression levels of aquaporin-2 (AQP2), a collecting duct protein, have been suggested to reflect blood pressure. AQP2 protein expression was significantly downregulated in the renal cortex of the ANG II-infused menopause group, where blood pressure was increased. AQP2 expression levels were restored to control levels with 17β-estradiol replacement. This study indicates that the changing hormonal environment in the VCD model of menopause impacts the severity of ANG II-induced hypertension. These data highlight the utility of the ovary-intact VCD model of menopause as a clinically relevant model to investigate the physiological mechanisms of hypertension that occur in women during the transition into menopause.

The adrenal steroid hormone dehydroepiandrosterone (DHEA) and its sulfated derivative [DHEA(S)] have been extensively studied for their potential anti-aging effects. Associated with aging, DHEA levels decline in humans, whereas other adrenal hormones remain unchanged, suggesting that DHEA may be important in the aging process. However, the effect of DHEA(S) supplementation on cardiac function in the aged has not been investigated. Therefore, we administered to young and old female mice a 60-day treatment with exogenous DHEA(S) at a dose of 0.1 mg/ml in the drinking water and compared the effects on left ventricular diastolic function and the myocardial extracellular matrix composition. The left ventricular stiffness (β) was 0.30 ± 0.06 mmHg/μl in the older control mice compared with 0.17 ± 0.02 mmHg/μl in young control mice. Treatment with DHEA(S) decreased left ventricular stiffness to 0.12 ± 0.03 mmHg/μl in the older mice and increased left ventricular stiffness to 0.27 ± 0.04 mmHg/μl in young mice. The mechanism for the DHEA(S)-induced changes in diastolic function appeared to be associated with altered matrix metalloproteinase activity and the percentage of collagen cross-linking. We conclude that exogenous DHEA(S) supplementation is capable of reversing the left ventricular stiffness and fibrosis that accompanies aging, with a paradoxical increased ventricular stiffness in young mice.

Mice that lack the aquaporin-1 gene (AQP1) lack a functional countercurrent multiplier mechanism, fail to concentrate the inner medullary (IM) interstitium, and present with a urinary concentrating defect. In this study, we use DNA microarrays to identify the gene expression profile of the IM of AQP1 null mice and corresponding changes in gene expression resulting from a loss of a hypertonic medullary interstitium. An ANOVA analysis model, CARMA, was used to isolate the knockout effect while taking into account experimental variability associated with microarray studies. In this study 5,701 genes of the possible ∼12,000 genes on the array were included in the ANOVA; 531 genes were identified as demonstrating a >1.5-fold up- or downregulation between the wild-type and knockout groups. We randomly selected 35 genes for confirmation by real-time PCR, and 29 of the 35 genes were confirmed using this method. The overall pattern of gene expression in the AQP1 null mice was one of downregulation compared with gene expression in the renal medullas of the wild-type mice. Heat shock proteins 105 and 94, aldose reductase, adenylate kinase 2, aldolase B, aldehyde reductase 6, and p8 were decreased in the AQP1 null mice. Carboxylesterase 3, matrilin 2, lipocalin 2, and transforming growth factor-α were increased in IM of AQP1 null mice. In addition, we observed a loss of vasopressin type 2 receptor mRNA expression in renal medullas of the AQP1 null mice. Thus the loss of the hyperosmotic renal interstitium, due to a loss of the concentrating mechanism, drastically altered not only the phenotype of these animals but also their renal medullary gene expression profile.

TonEBP is a transcription factor that, when activated by hypertonicity, increases transcription of genes, including those involved in organic osmolyte accumulation. Surprisingly, it is expressed in virtually all tissues, including many never normally exposed to hypertonicity. We measured TonEBP mRNA (real-time PCR) and protein (Western blot analysis) in tissues of control (plasma osmolality 294 ± 1 mosmol/kgH2O) and hyposmotic (dDAVP infusion plus water loading for 3 days, 241 ± 2 mosmol/kgH2O) rats to test whether the ubiquitous expression of TonEBP mRNA is osmotically regulated around the normal plasma osmolality. TonEBP protein is reduced by hyposmolality in thymus and liver, but not in brain, and is not detected in heart and skeletal muscle. TonEBP mRNA decreases in brain and liver but is unchanged in other tissues. There are no general changes in mRNA of TonEBP-mediated genes: aldose reductase (AR) does not change in any tissue, betaine transporter (BGT1) decreases only in liver, taurine transporter (TauT) only in brain and thymus, and inositol transporter (SMIT) only in skeletal muscle and liver. Heat shock protein (Hsp)70–1 and Hsp70–2 mRNA increase greatly in most tissues, which cannot be attributed to decreased TonEBP activity. The conclusions are as follows: 1) TonEBP protein or mRNA expression is reduced by hyposmolality in thymus, liver, and brain. 2) TonEBP protein and mRNA expression are differentially regulated in some tissues. 3) Although AR, SMIT, BGT1, and TauT are regulated by TonEBP in renal medullary cells, other sources of regulation may predominate in other tissues. 4) TonEBP abundance and activity are regulated by factors other than tonicity in some tissues.

We carried out semiquantitative immunoblotting of kidney to identify apical sodium transporter proteins whose abundances are regulated by angiotensin II. In NaCl-restricted rats (0.5 mEq Na/200 g BW/d), the type 1 angiotensin II receptor (AT 1 receptor) antagonist, candesartan, (1 mg/kg of body weight per day SC for 2 days) markedly decreased the abundance of the α subunit of the epithelial sodium channel (ENaC). This subunit has been shown to be rate-limiting for assembly of mature ENaC complexes. In addition, systemic infusion of angiotensin II increased αENaC protein abundance in rat kidney cortex. The decrease in αENaC protein abundance in response to AT 1 receptor blockade was associated with a fall in αENaC mRNA abundance (real-time RT-PCR), consistent with transcriptionally mediated regulation. The effect of AT 1 receptor blockade on αENaC expression was not blocked by spironolactone, suggesting a direct role of the AT 1 receptor in regulation of αENaC gene expression. Candesartan administration was also found to increase the abundances of the β and γ subunits. The increase in β and γENaC protein abundance was not associated with a significant increase in the renal abundances of the corresponding mRNAs, suggesting a posttranscriptional mechanism. Immunocytochemistry confirmed the increase in β and γENaC protein abundance and demonstrated candesartan-induced ENaC internalization in collecting duct cells. The results support the view that the angiotensin II receptor regulates ENaC abundance, consistent with a role for angiotensin II in regulation of collecting duct function.

We have used peptide-directed antibodies to each major renal Na transporter and channel proteins to screen renal homogenates for changes in Na transporter protein expression after initiation of dietary NaCl restriction. After equilibration on a NaCl-replete diet (2.0 meq · 200 g body wt−1· day−1), rats were switched to a NaCl-deficient diet (0.02 meq · 200 g body wt−1· day−1). Na excretion fell to 25% of baseline levels on day 1, followed by a further decrease

T lymphocytes are required for the development of Angiotensin II (Ang II) hypertension in male mice. Cycling female mice are protected against Ang II hypertension, and inducing ovarian failure (menopause) eliminates this protection. We have previously shown that female Rag-1 -/- mice with adoptively transferred T cells have significantly reduced renal T cell infiltration compared to males. Therefore, we hypothesized that an increase in renal T cell infiltration underlies the genesis of hypertension in menopausal female mice. 10 week old C57Bl/6 female mice received ip VCD injections for 20 consecutive days to induce ovarian failure. Cyclicity was monitored daily via vaginal cytology. Once in menopause, an osmotic minipump was implanted, releasing Ang II (800ng/kg/min) for 14 days. In a subset of mice, a 17-β estradiol pellet (0.1mg) was implanted subcutaneously. Upon harvest, T cell infiltration was measured via flow cytometry. Baseline blood pressure was similar among all groups. Ang II infusion elicited a significantly greater increase in MAP in menopausal mice compared to cycling control (Ang Δ11±10mmHg vs Meno/Ang Δ32±6, p2 Δ5±2mmHg). Despite elevated MAP, there was no significant increase in renal CD3 + , CD4 + or CD8 + T lymphocyte infiltration in Meno/Ang group vs control. However, 17-β estradiol replacement decreased IL-2 renal mRNA expression (0.45±0.13 fold), and increased IL-10 expression (2.41±0.73 fold) compared to Ang II-treated menopausal mice. Ang II induced glomerular hypertrophy in all groups, independent of hormonal status (Control 2156±54μm 2 vs. Ang 3970±383*μm 2 vs. Meno/Ang 3729±51*μm 2 vs. Meno/E 2 /Ang 3505±71*μm 2 , *p

Epithelial sodium channel (ENaC) subunit (α, β, and γ) mRNA and protein have been localized to the principal cells of the connecting tubule (CNT), cortical collecting duct (CCD), and outer medullary collecting duct (OMCD) in rat kidney. However, the subcellular localization of ENaC subunits in the principal cells of these cells is undefined. The cellular and subcellular localization of ENaC subunits in rat kidney was therefore examined. Immunocytochemistry demonstrated the presence of all three subunits in principal cells of the CNT, CCD, OMCD, and IMCD. In cortex and outer medulla, confocal microscopy demonstrated a difference in the subcellular localization of subunits. α-ENaC was localized mainly in a zone in the apical domains, whereas β- and γ-ENaC were found throughout the cytoplasm. Immunoelectron microscopy confirmed the presence of ENaC subunits in both the apical plasma membrane and intracellular vesicles. In contrast to the labeling pattern seen in cortex, α-ENaC labeling in IMCD cells was distributed throughout the cytoplasm. In the urothelium covering pelvis, ureters, and bladder, immunoperoxidase and confocal microscopy revealed differences the presence of all ENaC subunits. As seen in CCD, α-ENaC was present in a narrow zone near the apical plasma membrane, whereas β- and γ-ENaC were dispersed throughout the cytoplasm. In conclusion, all three subunits of ENaC are expressed throughout the collecting duct (CD), including the IMCD as well as in the urothelium. The intracellular vesicular pool in CD principal cells suggests ENaC trafficking as a potential mechanism for the regulation of Na+reabsorption.

The Na+–H+ exchanger NHE3 and the thiazide-sensitive Na+–Cl− cotransporter NCC are the major apical sodium transporters in the proximal convoluted tubule and the distal convoluted tubule of the kidney, respectively. We investigated the mechanism of compensation that allows maintenance of sodium balance in NHE3 knockout mice and in NCC knockout mice. We used a so-called ‘targeted proteomics’ approach, which profiles the entire renal tubule with regard to changes in Na+ transporter and aquaporin abundance in response to the gene deletions. Specific antibodies to the Na+ transporters and aquaporins expressed along the nephron were utilized to determine the relative abundance of each transporter. Semiquantitative immunoblotting was used which gives an estimate of the percentage change in abundance of each transporter in knockout compared with wild-type mice. In NHE3 knockout mice three changes were identified which could compensate for the loss of NHE3-mediated sodium absorption. (a) The proximal sodium-phosphate cotransporter NaPi-2 was markedly upregulated. (b) In the collecting duct, the 70 kDa form of the γ-subunit of the epithelial sodium channel, ENaC, exhibited an increase in abundance. This is thought to be an aldosterone-stimulated form of γ-ENaC. (c) Glomerular filtration was significantly reduced. In the NCC knockout mice, amongst all the sodium transporters expressed along the renal tubule, only the 70 kDa form of the γ-subunit of the epithelial sodium channel, ENaC, exhibited an increase in abundance. In conclusion, both mouse knockout models demonstrated successful compensation for loss of the deleted transporter. More extensive adaptation occurred in the case of the NHE3 knockout, presumably because NHE3 is responsible for much more sodium absorption in normal mice than in NCC knockout mice. The rate of sodium excretion by the kidney is the rate of sodium filtration by the glomerulus minus the net rate of sodium reabsorption along the renal tubule. The renal tubule consists of several distinct segments, each with distinct sodium transporters. The sites of expression of the major apical Na+ transporters are summarized in Fig. 1. The predominant apical Na+ transporter in the proximal tubule is NHE3, the type 3 Na+–H+ exchanger (Biemesderfer et al. 1993). In the distal convoluted tubule, the predominant apical Na+ transporter is NCC, the thiazide-sensitive Na+–Cl− cotransporter (Kaplan et al. 1996). The genes for these two transporters have been knocked out in mice as described previously (Schultheis et al. 1998a,b). Figure 1 Sodium transporter distribution along the nephron In previous studies (Schultheis et al. 1998a) the most prominent effects in homozygous NHE3 knockout mice were in the gastro-intestinal tract, with dilatation of the small bowel and sustained diarrhoea. These mice exhibited a mild metabolic acidosis and had decreased arterial blood pressure. There was an increase in their circulating aldosterone levels, along with an increase in renin mRNA expression in the kidney. The most prominent effect in homozygous NCC knockout mice was decreased arterial blood pressure when placed on a low salt diet (Schultheis et al. 1998b). In addition, these mice displayed increased renin mRNA levels in the kidney, hypomagnesemia and hypocalciuria. These two knockout models are the focus of the present study. The other apical Na+ transporters described in Fig. 1 are: (1) NaPi-2, the sodium-phosphate cotransporter expressed in proximal tubule (Biber et al. 1998); (2) NKCC-2, the bumetanide-sensitive Na+-K+-2Cl− cotransporter expressed in the thick ascending limb of Henle's loop (Kaplan et al. 1996); and (3) ENaC, the amiloride-sensitive Na+ channel of the connecting tubule and the collecting duct, consisting of α-, β- and γ-subunits (Garty & Palmer, 1997). The major basolateral sodium transporter is the same in all segments, the Na+-K+-ATPase, which in the kidney consists of a complex of α1 and β1 subunit proteins (Lingrel et al. 1994). The Na+-K+-ATPase is the primary active transporter responsible for the maintenance of low intracellular sodium concentration and a negative intracellular electrical potential in all renal tubule epithelial cells. The electrochemical driving force for apical sodium entry through the various apical Na+ transporters described above is generated by the Na+-K+-ATPase in each segment. The cDNAs encoding the apical Na+ transporters described in Fig. 1 have been cloned, allowing the production of peptide-directed rabbit polyclonal antibodies to each (Fernandez-Llama et al. 1998; Kim et al. 1998, 1999, 2000; Masilamani et al. 1999). This ensemble of antibodies has allowed the development of a so-called ‘targeted proteomics’ approach (Masilamani et al. 1999; Kim et al. 2000) to the analysis of renal adaptation. This approach profiles the entire renal tubule with regard to changes in apical Na+ transporter and aquaporin abundance in response to specific systemic physiological perturbations. Here we use this transporter-profiling approach to investigate the mechanism of compensation that allows maintenance of sodium balance in the NHE3 and NCC knockout mice. We hypothesize that the deletion of an individual sodium transporter will result in compensatory increases in abundance of one or more of the remaining Na+ transporters. In NHE3 knockout mice three changes were identified which could compensate for the loss of NHE3-mediated sodium absorption. (a) The proximal sodium-phosphate cotransporter NaPi-2 was markedly upregulated. (b) In the collecting duct, the 70 kDa form of the γ-subunit of the epithelial sodium channel, ENaC, exhibited an increase in abundance. (c) Glomerular filtration was significantly reduced. In the NCC knockout mice, amongst all the sodium transporters expressed along the renal tubule, only the 70 kDa form of the γ-subunit of the epithelial sodium channel, ENaC, exhibited an increase in abundance.

The brain contains a subpopulation of glucosensing neurons that alter their firing rate in response to elevated glucose concentrations. In pancreatic beta-cells, glucokinase (GK), the rate-limiting enzyme in glycolysis, mediates glucose-induced insulin release by regulating intracellular ATP production. A similar role for GK is proposed to underlie neuronal glucosensing. Via in situ hybridization, GK mRNA was localized to hypothalamic areas that are thought to contain relatively large populations of glucosensing neurons (the arcuate, ventromedial, dorsomedial, and paraventricular nuclei and the lateral area). GK also was found in brain areas without known glucosensing neurons (the lateral habenula, the bed nucleus stria terminalis, the inferior olive, the retrochiasmatic and medial preoptic areas, and the thalamic posterior paraventricular, interpeduncular, oculomotor, and anterior olfactory nuclei). Conversely, GK message was not found in the nucleus tractus solitarius, which contains glucosensing neurons, or in ependymal cells lining the third ventricle, where others have described its presence. In the arcuate nucleus, >75% of neuropeptide Y-positive neurons also expressed GK, and most GK+ neurons also expressed KIR6.2 (the pore-forming subunit of the ATP-sensitive K+ channel). The anatomic distribution of GK mRNA was confirmed in micropunch samples of hypothalamus via reverse transcription-polymerase chain reaction (RT-PCR). Nucleotide sequencing of the recovered PCR product indicated identity with nucleotides 1092-1411 (within exon 9 and 10) of hepatic and beta-cell GK. The specific anatomic localization of GK mRNA in hypothalamic areas known to contain glucosensing neurons and the coexpression of KIR6.2 and NPY in GK+ neurons support a role for GK as a primary determinant of glucosensing in neuropeptide neurons that integrate multiple signals relating to peripheral energy metabolism.

The pore-forming protein of the human whipworm, Trichuris trichiura, has been postulated to facilitate invasion of the host gut and enable the parasite to maintain its syncytial environment. The data presented here describe the first, to our knowledge, molecular characterization of a pore-forming protein in any helminth and provide a unique demonstration of the functional interaction between a parasite antigen and host molecules. Immunological screening of a T. trichiura cDNA library with T. trichiura infection sera identified a clone of 1.4 kB, the cDNA consisting of 1495 base pairs encoding a protein of 50 kDa. The sequence has a highly repetitive nature containing nine four-disulphide-bonded core domains. Structural prediction analyses reveals an amphipathic nature. TT50 induced pore formation in bilayers in a manner identical to that of the native protein. IgG antibody isolated from T. trichiura infection serum was observed to abolish channel activity.