Blood pressure variability compromises vascular function in middle-aged mice.

Blood pressure variability (BPV) has emerged as a novel risk factor for cognitive decline and dementia, independent of alterations in average blood pressure (BP). However, the underlying consequences of large BP fluctuations on the neurovascular complex are unknown. We developed a novel mouse model of BPV in middle-aged mice based on intermittent Angiotensin II infusions. Using radio telemetry, we demonstrated that the 24-hr BP averages of these mice were similar to controls, indicating BPV without hypertension. Chronic (20-25 days) BPV led to a blunted bradycardic response and cognitive deficits. Two-photon imaging of parenchymal arterioles showed enhanced pressure-evoked constrictions (myogenic response) in BPV mice. Sensory stimulus-evoked dilations (neurovascular coupling) were greater at higher BP levels in control mice, but this pressure-dependence was lost in BPV mice. Our findings support the notion that large BP variations impair vascular function at the neurovascular complex and contribute to cognitive decline.

A novel mouse model carrying a gene trap insertion into the Hmgxb4 gene locus to examine Hmgxb4 expression in vivo.

HMG (high mobility group) proteins are a diverse family of nonhistone chromosomal proteins that interact with DNA and a wide range of transcriptional regulators to regulate the structural architecture of DNA. HMGXB4 (also known as HMG2L1) is an HMG protein family member that contains a single HMG box domain. Our previous studies have demonstrated that HMGXB4 suppresses smooth muscle differentiation and exacerbates endotoxemia by promoting a systemic inflammatory response in mice. However, the expression of Hmgxb4 in vivo has not fully examined. Herein, we generated a mouse model that harbors a gene trap in the form of a lacZ gene insertion into the Hmgxb4 gene. This mouse enables the visualization of endogenous HMGXB4 expression in different tissues via staining for the ß-galactosidase activity of LacZ which is under the control of the endogenous Hmgxb4 gene promoter. We found that HMGXB4 is widely expressed in mouse tissues and is a nuclear protein. Furthermore, the Hmgxb4 gene trap mice exhibit normal cardiac function and blood pressure. Measurement of ß-galactosidase activity in the Hmgxb4 gene trap mice demonstrated that the arterial injury significantly induces Hmgxb4 expression. In summary, the Hmgxb4 gene trap reporter mouse described here provides a valuable tool to examine the expression level of endogenous Hmgxb4 in both physiological and pathological settings in vivo.

NaHCO3 loading causes increased arterial pressure and kidney damage in rats with chronic kidney disease.

Sodium bicarbonate (NaHCO3) is commonly utilized as a therapeutic to treat metabolic acidosis in people with chronic kidney disease (CKD). While increased dietary sodium chloride (NaCl) is known to promote volume retention and increase blood pressure, the effects of NaHCO3 loading on blood pressure and volume retention in CKD remain unclear. In the present study, we compared the effects of NaCl and NaHCO3 loading on volume retention, blood pressure, and kidney injury in both 2/3 and 5/6 nephrectomy remnant kidney rats, a well-established rodent model of CKD. We tested the hypothesis that NaCl loading promotes greater volume retention and increases in blood pressure than equimolar NaHCO3. Blood pressure was measured 24 h daily using radio telemetry. NaCl and NaHCO3 were administered in drinking water ad libitum or infused via indwelling catheters. Rats were housed in metabolic cages to determine volume retention. Our data indicate that both NaHCO3 and NaCl promote hypertension and volume retention in remnant kidney rats, with salt-sensitivity increasing with greater renal mass reduction. Importantly, while NaHCO3 intake was less pro-hypertensive than equimolar NaCl intake, NaHCO3 was not benign. NaHCO3 loading significantly elevated blood pressure and promoted volume retention in rats with CKD when compared with control rats receiving tap water. Our findings provide important insight into the effects of sodium loading with NaHCO3 in CKD and indicate that NaHCO3 loading in patients with CKD is unlikely to be benign.

Galectin-3 Mediates Vascular Dysfunction in Obesity by Regulating NADPH Oxidase 1.

BACKGROUND: Obesity is associated with increased risk of cardiovascular disease, but underlying mechanisms remain elusive. Metabolic dysfunction, especially hyperglycemia, is thought to be a major contributor, but how glucose impacts vascular function is unclear. GAL3 (galectin-3) is a sugar-binding lectin upregulated by hyperglycemia, but its role as a causative mechanism of cardiovascular disease remains poorly understood. Therefore, the objective of this study was to determine the role of GAL3 in regulating microvascular endothelial vasodilation in obesity. METHODS: GAL3 was measured and found to be markedly increased in the plasma of overweight and obese patients, as well as in the microvascular endothelium of diabetic patients. To investigate causative mechanisms in cardiovascular disease, mice deficient in GAL3 were bred with obese db/db mice to generate lean, lean GAL3 knockout, obese, and obese GAL3 knockout genotypes. Endothelial cell-specific GAL3 knockout mice with novel AAV-induced obesity recapitulated whole-body knockout studies to confirm cell specificity. RESULTS: Deletion of GAL3 did not alter body mass, adiposity, or plasma indices of glycemia and lipidemia, but levels of plasma reactive oxygen species as assessed by plasma thiobarbituric acid reactive substances were normalized in obese GAL3 knockout mice. Obese mice exhibited profound endothelial dysfunction and hypertension, both of which were rescued by GAL3 deletion. Isolated microvascular endothelial cells from obese mice had increased expression of NOX1 (nicotinamide adenine dinucleotide phosphate oxidase 1), which we have previously shown to contribute to increased oxidative stress and endothelial dysfunction, which was normalized in microvascular endothelium from mice lacking GAL3. Cell-specific deletion confirmed that endothelial GAL3 regulates obesity-induced NOX1 overexpression and subsequent microvascular function. Furthermore, improvement of metabolic syndrome by increasing muscle mass, improving insulin signaling, or treating with metformin decreased microvascular GAL3, and thereby NOX1, expression levels. CONCLUSIONS: Deletion of GAL3 normalizes microvascular endothelial function in obese db/db mice, likely through a NOX1-mediated mechanism. Pathological levels of GAL3, and in turn NOX1, are amenable to improvements in metabolic status, presenting a potential therapeutic target to ameliorate pathological cardiovascular consequences of obesity.

Aldosterone Antagonism Is More Effective at Reducing Blood Pressure and Excessive Renal ENaC Activity in AngII-Infused Female Rats Than in Males.

BACKGROUND: AngII (angiotensin II)-dependent hypertension causes comparable elevations of blood pressure (BP), aldosterone levels, and renal ENaC (epithelial Na+ channel) activity in male and female rodents. Mineralocorticoid receptor (MR) antagonism has a limited antihypertensive effect associated with insufficient suppression of renal ENaC in male rodents with AngII-hypertension. While MR blockade effectively reduces BP in female mice with salt-sensitive and leptin-induced hypertension, MR antagonism has not been studied in female rodents with AngII-hypertension. We hypothesize that overstimulation of renal MR signaling drives redundant ENaC-mediated Na+ reabsorption and BP increase in female rats with AngII-hypertension. METHODS: We employ a combination of physiological, pharmacological, biochemical, and biophysical approaches to compare the effect of MR inhibitors on BP and ENaC activity in AngII-infused male and female Sprague Dawley rats. RESULTS: MR blockade markedly attenuates AngII-hypertension in female rats but has only a marginal effect in males. Spironolactone increases urinary sodium excretion and urinary sodium-to-potassium ratio in AngII-infused female, but not male, rats. The expression of renal MR and HSD11ß2 (11ß-hydroxysteroid dehydrogenase type 2) that determines the availability of MR to aldosterone is significantly higher in AngII-infused female rats than in males. ENaC activity is ≈2× lower in spironolactone-treated AngII-infused female rats than in males. Reduced ENaC activity in AngII-infused female rats on spironolactone correlates with increased interaction with ubiquitin ligase Nedd4-2 (neural precursor cell expressed developmentally down-regulated protein 4-2), targeting ENaC for degradation. CONCLUSIONS: MR-ENaC axis is the primary determinant of excessive renal sodium reabsorption and an attractive antihypertensive target in female rats with AngII-hypertension, but not in males.

A Non-Systemic Phosphodiesterase-5 Inhibitor Suppresses Colon Proliferation in Mice.

Phosphodiesterase-5 inhibitors (PDE5i) are under investigation for repurposing for colon cancer prevention. A drawback to conventional PDE5i are their side-effects and drug-drug interactions. We designed an analog of the prototypical PDE5i sildenafil by replacing the methyl group on the piperazine ring with malonic acid to reduce lipophilicity, and measured its entry into the circulation and effects on colon epithelium. This modification did not affect pharmacology as malonyl-sildenafil had a similar IC50 to sildenafil but exhibited an almost 20-fold reduced EC50 for increasing cellular cGMP. Using an LC-MS/MS approach, malonyl-sildenafil was negligible in mouse plasma after oral administration but was detected at high levels in the feces. No bioactive metabolites of malonyl-sildenafil were detected in the circulation by measuring interactions with isosorbide mononitrate. The treatment of mice with malonyl-sildenafil in the drinking water resulted in a suppression of proliferation in the colon epithelium that is consistent with results previously published for mice treated with PDE5i. A carboxylic-acid-containing analog of sildenafil prohibits the systemic delivery of the compound but maintains sufficient penetration into the colon epithelium to suppress proliferation. This highlights a novel approach to generating a first-in-class drug for colon cancer chemoprevention.

Galectin-3 Mediates Vascular Dysfunction in Obesity by Regulating NADPH Oxidase 1.

Rationale: Obesity increases the risk of cardiovascular disease (CVD) through mechanisms that remain incompletely defined. Metabolic dysfunction, especially hyperglycemia, is thought to be a major contributor but how glucose impacts vascular function is unclear. Galectin-3 (GAL3) is a sugar binding lectin upregulated by hyperglycemia but its role as a causative mechanism of CVD remains poorly understood. Objective: To determine the role of GAL3 in regulating microvascular endothelial vasodilation in obesity. Methods and Results: GAL3 was markedly increased in the plasma of overweight and obese patients, as well as in the microvascular endothelium of diabetic patients. To investigate a role for GAL3 in CVD, mice deficient in GAL3 were bred with obese db/db mice to generate lean, lean GAL3 knockout (KO), obese, and obese GAL3 KO genotypes. GAL3 KO did not alter body mass, adiposity, glycemia or lipidemia, but normalized elevated markers of reactive oxygen species (TBARS) in plasma. Obese mice exhibited profound endothelial dysfunction and hypertension, both of which were rescued by GAL3 deletion. Isolated microvascular endothelial cells (EC) from obese mice had increased NOX1 expression, which we have previously shown to contribute to increased oxidative stress and endothelial dysfunction, and NOX1 levels were normalized in EC from obese mice lacking GAL3. EC-specific GAL3 knockout mice made obese using a novel AAV-approach recapitulated whole-body knockout studies, confirming that endothelial GAL3 drives obesity-induced NOX1 overexpression and endothelial dysfunction. Improved metabolism through increased muscle mass, enhanced insulin signaling, or metformin treatment, decreased microvascular GAL3 and NOX1. GAL3 increased NOX1 promoter activity and this was dependent on GAL3 oligomerization. Conclusions: Deletion of GAL3 normalizes microvascular endothelial function in obese db/db mice, likely through a NOX1-mediated mechanism. Pathological levels of GAL3 and in turn, NOX1, are amenable to improvements in metabolic status, presenting a potential therapeutic target to ameliorate pathological cardiovascular consequences of obesity.

Perinatal intermittent hypoxia increases early susceptibility to ANG II-induced hypertension in adult male but not in female Sprague-Dawley rats.

Prenatal, perinatal, and adulthood exposure to chronic intermittent hypoxia (IH) increases blood pressure in rodents. Males exposed to chronic IH have higher blood pressure versus females. However, it is unknown if this same-sex difference exists with acute perinatal IH. We tested the hypothesis that acute perinatal IH increases baseline blood pressure and enhances sensitivity to angiotensin II (ANG II)-induced hypertension in male Sprague-Dawley rats. Male and female pups were randomized to control (room air) or IH (10 min of ∼10% O2 for 3 times/day) for the first 8 days of life. IH decreased oxygen saturation, as confirmed via a pulse oximeter. Pups were weaned at postnatal day 21. Blood pressure was measured via telemetry beginning at 14 wk of age and analyzed separately into light and dark phases to assess circadian rhythm. Osmotic minipumps to deliver ANG II were implanted at 15 wk of age. Perinatal IH exposure did not alter baseline blood pressure. One week of ANG II treatment increased blood pressure in light and dark periods in males exposed to IH versus control; there was no effect in females. Blood pressure among the groups was comparable following 2 wk of ANG II infusion. Perinatal IH did not change the circadian rhythm. Following ANG II treatment, indexes of renal injury were measured. Perinatal IH did not alter kidney size, structure, nephron number, or creatinine clearance. These data indicate that acute perinatal IH enhances early ANG II-induced hypertension in males, independent of nephron loss or decreases in body weight or kidney function.NEW & NOTEWORTHY The impact of acute intermittent hypoxia (IH) in early life on blood pressure in adulthood is unknown. This study used a new model exposing female and male rat pups to acute IH in the first 8 days of life, without exposing the dam. Although baseline blood pressure was not altered in adulthood, IH increased susceptibility to angiotensin II hypertension only in males, supporting increased susceptibility of males exposed to IH to a second cardiovascular stressor.

Sex differences in apoptosis do not contribute to sex differences in blood pressure or renal T cells in spontaneously hypertensive rats.

Apoptosis is a physiological and anti-inflammatory form of cell death that is indispensable for normal physiology and homeostasis. Several studies have reported aberrant activation of apoptosis in various tissues at the onset of hypertension. However, the functional significance of apoptosis during essential hypertension remains largely undefined. The current study was designed to test the hypothesis that apoptosis contributes to sex differences in blood pressure and the T cell profile in spontaneously hypertensive rats (SHR). Apoptosis was measured in kidney, aorta and spleen of 13-week-old adult hypertensive male and female SHR. Female SHR had greater renal and aortic apoptosis compared to age-matched males; apoptosis in the spleen was comparable between the sexes. Based on well-established sex differences in hypertension, we tested the hypothesis that greater apoptosis in female SHR contributes to the lower BP and pro-inflammatory profile compared to males. Male and female SHR were randomized to receive vehicle or ZVAD-FMK, a cell permeable pan-caspase inhibitor, in established hypertension from 13 to 15 weeks of age or at the onset of hypertension from 6 to 12 weeks or age. Treatment with ZVAD-FMK lowered renal apoptosis in both studies, yet neither BP nor renal T cells were altered in either male or female SHR. These results suggest that apoptosis does not contribute to the control or maintenance of BP in male or female SHR or sex differences in renal T cells.

Splenectomy increases blood pressure and abolishes sex differences in renal T-regulatory cells in spontaneously hypertensive rats.

Over the past decade there has been increasing support for a role of the immune system in the development of hypertension. Our lab has previously reported that female spontaneously hypertensive rats (SHRs) have a blood pressure (BP)-dependent increase in anti-inflammatory renal regulatory T cells (Tregs), corresponding to lower BP compared with males. However, little is known regarding the mechanism for greater renal Tregs in females. The current study was designed to test the hypothesis that the greater relative abundance of renal Tregs in female SHR is due to greater Treg production. To test this hypothesis, T cell profiles were measured in the spleen by flow cytometry in male and female SHR at 5 and 14 weeks of age. Splenic Tregs did not differ between males and females, suggesting sex differences in renal Tregs is not due to differences in production. To assess the role of the spleen in sex differences in renal Tregs and BP control, rats were randomized to receive sham surgery (CON) or splenectomy (SPLNX) at 12 weeks of age and implanted with telemeters to measure BP. After 2 weeks, kidneys were harvested for flow cytometric analysis of T cells. Splenectomy increased BP in both sexes after 2 weeks. Renal Tregs decreased in both sexes after splenectomy, abolishing the sex differences in renal Tregs. In conclusion, splenic Tregs were comparable in male and female SHRs, suggesting that sex differences in renal Tregs is due to differences in renal Treg recruitment, not Treg production.

Adverse Maternal and Fetal Outcomes in a Novel Experimental Model of Pregnancy after Recovery from Renal Ischemia-Reperfusion Injury.

BACKGROUND: Recent clinical studies report that women with a history of AKI have an increased incidence of maternal and fetal adverse outcomes during pregnancy, despite fully recovering renal function prior to conception. The mechanisms contributing to such adverse outcomes in pregnancy after AKI are not yet understood. METHODS: To develop a rodent model to investigate fetal and maternal outcomes in female animals with a history of AKI, we used ischemia-reperfusion injury as an experimental model of AKI in female Sprague Dawley rats. The 12-week-old animals underwent warm bilateral ischemia-reperfusion surgery involving clamping of both renal arteries for 45 minutes or sham surgery (control). Rats were allowed to recover for 1 month prior to mating. Recovery from ischemia-reperfusion injury was confirmed by measurements of plasma creatinine and urinary protein excretion. We assessed maternal and fetal outcomes during late pregnancy on gestational day 20. RESULTS: After recovery from ischemia-reperfusion injury, compared with healthy sham-surgery controls, dams exhibited pregnancy-induced renal insufficiency with increases in plasma creatinine and urea, along with increased urinary protein excretion. Additionally, recovered ischemia-reperfusion dams experienced worse fetal outcomes compared with controls, with intrauterine growth restriction leading to higher rates of fetal demise and smaller pups. CONCLUSIONS: In this rat model, despite biochemical resolution of ischemia-reperfusion injury, subsequent pregnancy resulted in maternal renal insufficiency and significant impairments in fetal growth. This mirrors findings in recent reports in the clinical population, indicating that this model may be a useful tool to further explore the alterations in kidney function after AKI in women.

Decreased parenchymal arteriolar tone uncouples vessel-to-neuronal communication in a mouse model of vascular cognitive impairment.

Chronic hypoperfusion is a key contributor to cognitive decline and neurodegenerative conditions, but the cellular mechanisms remain ill-defined. Using a multidisciplinary approach, we sought to elucidate chronic hypoperfusion-evoked functional changes at the neurovascular unit. We used bilateral common carotid artery stenosis (BCAS), a well-established model of vascular cognitive impairment, combined with an ex vivo preparation that allows pressurization of parenchymal arterioles in a brain slice. Our results demonstrate that mild (~ 30%), chronic hypoperfusion significantly altered the functional integrity of the cortical neurovascular unit. Although pial cerebral perfusion recovered over time, parenchymal arterioles progressively lost tone, exhibiting significant reductions by day 28 post-surgery. We provide supportive evidence for reduced adenosine 1 receptor-mediated vasoconstriction as a potential mechanism in the adaptive response underlying the reduced baseline tone in parenchymal arterioles. In addition, we show that in response to the neuromodulator adenosine, the action potential frequency of cortical pyramidal neurons was significantly reduced in all groups. However, a significant decrease in adenosine-induced hyperpolarization was observed in BCAS 14 days. At the microvascular level, constriction-induced inhibition of pyramidal neurons was significantly compromised in BCAS mice. Collectively, these results suggest that BCAS uncouples vessel-to-neuron communication-vasculo-neuronal coupling-a potential early event in cognitive decline.

Greater T Regulatory Cells in Females Attenuate DOCA-Salt-Induced Increases in Blood Pressure Versus Males.

Hypertension is the most common risk factor for cardiovascular disease, causing over 18 million deaths a year. Although the mechanisms controlling blood pressure (BP) in either sex remain largely unknown, T cells play a critical role in the development of hypertension. Further evidence supports a role for the immune system in contributing to sex differences in hypertension. The goal of the current study was to first, determine the impact of sex on the renal T-cell profiles in DOCA-salt hypertensive males and females and second, test the hypothesis that greater numbers of T regulatory cells (Tregs) in females protect against DOCA-salt-induced increases in BP and kidney injury. Male rats displayed greater increases in BP than females following 3 weeks of DOCA-salt treatment, although increases in renal injury were comparable between the sexes. DOCA-salt treatment resulted in an increase in proinflammatory T cells in both sexes; however, females had more anti-inflammatory Tregs than males. Additional male and female DOCA-salt rats were treated with anti-CD25 to decrease Tregs. Decreasing Tregs significantly increased BP only in females, thereby abolishing the sex difference in the BP response to DOCA-salt. This data supports the hypothesis that Tregs protect against the development of hypertension and are particularly important for the control of BP in females.

Necrosis Contributes to the Development of Hypertension in Male, but Not Female, Spontaneously Hypertensive Rats.

Necrosis is a pathological form of cell death that induces an inflammatory response, and immune cell activation contributes to the development and maintenance of hypertension. Necrosis was measured in kidney, spleen, and aorta of 12- to 13-week-old male and female SHRs (spontaneously hypertensive rats); male SHRs had greater renal necrotic cell death than female SHRs. Because male SHRs have a higher blood pressure (BP) and a more proinflammatory T-cell profile than female SHRs, the current studies tested the hypothesis that greater necrotic cell death in male SHRs exacerbates increases in BP and contributes to the proinflammatory T-cell profile. Male and female SHRs were randomized to receive vehicle or Necrox-5-a cell permeable inhibitor of necrosis-from 6 to 12 weeks of age or from 11 to 13 weeks of age. In both studies, Necrox-5 decreased renal necrosis and abolished the sex difference. Treatment with Necrox-5 beginning at 6 weeks of age attenuated maturation-induced increases in BP in male SHR; BP in female SHR was not altered by Necrox-5 treatment. Necrox-5 decreased proinflammatory renal T cells in both sexes, although sex differences were maintained. Administration of Necrox-5 for 2 weeks in SHR with established hypertension resulted in a small but significant decrease in BP in males with no effect in females. These results suggest that greater necrotic cell death in male SHR exacerbates maturation-induced increases in BP with age contributing to sex differences in BP. Moreover, although necrosis is proinflammatory, it is unlikely to explain sex differences in the renal T-cell profile.

Postprandial effects on electrolyte homeostasis in the kidney.

Insulin is known to be an important regulator of a number of different channels and transporters in the kidney, but its role in the kidney to prevent Na+ and volume loss during the osmotic load after a meal has only recently been validated. With increasing numbers of people suffering from diabetes and hypertension, furthering our understanding of insulin signaling and renal Na+ handling in both normal and diseased states is essential for improving patient treatments and outcomes. The present review is focused on postprandial effects on Na+ reabsorption in the kidney and the role of the epithelial Na+ channels as an important channel contributing to insulin-mediated Na+ reclamation.

Reconstitution of autophagy ameliorates vascular function and arterial stiffening in spontaneously hypertensive rats.

Insufficient autophagy has been proposed as a mechanism of cellular aging, as this leads to the accumulation of dysfunctional macromolecules and organelles. Premature vascular aging occurs in hypertension. In fact, many factors that contribute to the deterioration of vascular function as we age are accelerated in clinical and experimental hypertension. Previously, we have reported decreased autophagy in arteries from spontaneously hypertensive rats (SHRs); however, the effects of restoring autophagic activity on blood pressure and vascular function are currently unknown. We hypothesized that reconstitution of arterial autophagy in SHRs would decrease blood pressure and improve endothelium-dependent relaxation. We treated 14- to 18-wk-old Wistar rats (n = 7 vehicle and n = 8 trehalose) and SHRs (n = 7/group) with autophagy activator trehalose (2% in drinking water) for 28 days. Blood pressure was measured by radiotelemetry, and vascular function and structure were measured in isolated mesenteric resistance arteries (MRAs) using wire and pressure myographs, respectively. Treatment with trehalose had no effect on blood pressure in SHRs; however, isolated MRAs presented enhanced relaxation to acetylcholine, in a cyclooxygenase- and reactive oxygen species-dependent manner. Similarly, trehalose treatment shifted the relaxation to the Rho kinase (ROCK) inhibitor Y-27632 to the right, indicating reduced ROCK activity. Finally, trehalose treatment decreased arterial stiffness as indicated by the slope of the stress-strain curve. Overall these data indicate that reconstitution of arterial autophagy in SHRs improves endothelial and vascular smooth muscle function, which could synergize to prevent stiffening. As a result, restoration of autophagic activity could be a novel therapeutic for premature vascular aging in hypertension.NEW & NOTEWORTHY This work supports the concept that diminished arterial autophagy contributes to premature vascular aging in hypertension and that therapeutic reconstitution of autophagic activity can ameliorate this phenotype. As vascular age is a new clinically used index for cardiovascular risk, understanding this mechanism may assist in the development of new drugs to prevent premature vascular aging in hypertension.

Increased ENaC activity during kidney preservation in Wisconsin solution.

BACKGROUND: The invention of an effective kidney preservation solution capable of prolonging harvested kidney viability is the core of kidney transplantation procedure. Researchers have been working on upgrading the preservation solution quality aiming at prolonging storage time while maintaining utmost organ viability and functionality. For many years, the University of Wisconsin (UW) solution has been considered the gold standard solution for kidney preservation. However, the lifespan of kidney preservation in the UW solution is still limited. Its impact on the epithelial Na+ channel (ENaC) activity and its mediated processes is unknown and the primary goal of this study. METHODS: Kidneys harvested from 8 weeks old Sprague Dawley rats were divided into 4 groups depending upon the period of preservation in UW solution. Additional analysis was performed using dogs' kidneys. ENaC activity was measured using patch clamp technique; protein expression and mRNA transcription were tested through Western blot and RT-qPCR, respectively. A colorimetric LDH level estimation was performed at different time points during UW solution preservation. RESULTS: Kidney preservation in Wisconsin solution caused reduction of the kidney size and weight and elevation of LDH level. ENaC activity increased in both rat and dog kidneys preserved in the UW solution as assessed by patch clamp analysis. On the contrary, ENaC channel mRNA levels remained unchanged. CONCLUSIONS: ENaC activity is significantly elevated in the kidneys during preservation in UW solution, which might affect the immediate post-implantation allograft function and trajectory post-transplant.

Postprandial Effects on ENaC-Mediated Sodium Absorption.

Recent studies have suggested that postprandial increases in insulin directly contribute to reduced urinary sodium excretion. An abundance of research supports the ability of insulin to augment epithelial sodium channel (ENaC) transport. This study hypothesized that ENaC contributes to the increase in renal sodium reabsorption following a meal. To test this, we used fasted or 4 hour postprandial Sprague Dawley rats to analyze ENaC expression and activity. We also assessed total expression of additional sodium transporters (Na+-Cl- cotransporter (NCC), Na+-K+-2Cl- cotransporter (NKCC2), and Na+-K+-ATPase (NKA)) and circulating hormones involved in the renin-angiotensin-aldosterone system (RAAS). We found that after carbohydrate stimulus, ENaC open probability increased in split-open isolated collecting duct tubules, while ENaC protein levels remained unchanged. This was supported by a lack of change in phosphorylated Nedd4-2, an E3 ubiquitin ligase protein which regulates the number of ENaCs at the plasma membrane. Additionally, we found no differences in total expression of NCC, NKCC2, or NKA in the postprandial rats. Lastly, there were no significant changes in RAAS signaling between the stimulated and fasted rats, suggesting that acute hyperinsulinemia increases ENaC activity independent of the RAAS signaling cascade. These results demonstrate that insulin regulation of ENaC is a potential mechanism to preserve sodium and volume loss following a meal, and that this regulation is distinct from classical ENaC regulation by RAAS.

The TNF-derived TIP peptide activates the epithelial sodium channel and ameliorates experimental nephrotoxic serum nephritis.

In mice, the initial stage of nephrotoxic serum-induced nephritis (NTN) mimics antibody-mediated human glomerulonephritis. Local immune deposits generate tumor necrosis factor (TNF), which activates pro-inflammatory pathways in glomerular endothelial cells (GECs) and podocytes. Because TNF receptors mediate antibacterial defense, existing anti-TNF therapies can promote infection; however, we have previously demonstrated that different functional domains of TNF may have opposing effects. The TIP peptide mimics the lectin-like domain of TNF, and has been shown to blunt inflammation in acute lung injury without impairing TNF receptor-mediated antibacterial activity. We evaluated the impact of TIP peptide in NTN. Intraperitoneal administration of TIP peptide reduced inflammation, proteinuria, and blood urea nitrogen. The protective effect was blocked by the cyclooxygenase inhibitor indomethacin, indicating involvement of prostaglandins. Targeted glomerular delivery of TIP peptide improved pathology in moderate NTN and reduced mortality in severe NTN, indicating a local protective effect. We show that TIP peptide activates the epithelial sodium channel(ENaC), which is expressed by GEC, upon binding to the channel's α subunit. In vitro, TNF treatment of GEC activated pro-inflammatory pathways and decreased the generation of prostaglandin E2 and nitric oxide, which promote recovery from NTN. TIP peptide counteracted these effects. Despite the capacity of TIP peptide to activate ENaC, it did not increase mean arterial blood pressure in mice. In the later autologous phase of NTN, TIP peptide blunted the infiltration of Th17 cells. By countering the deleterious effects of TNF through direct actions in GEC, TIP peptide could provide a novel strategy to treat glomerular inflammation.