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Data are available on request from the corresponding author, R. Pawlinski (rafal_pawlinski@med.unc.edu). The online version of this article contains a data supplement. The American Society of Hematology A hypercoagulable state, chronic inflammation, and increased risk of venous thrombosis and stroke are prominent features in patients with sickle cell disease (SCD). Coagulation factor XII (FXII) triggers activation of the contact system that is known to be involved in both thrombosis and inflammation, but not in physiological hemostasis. Therefore, we investigated whether FXII contributes to the prothrombotic and inflammatory complications associated with SCD. We found that when compared with healthy controls, patients with SCD exhibit increased circulating biomarkers of FXII activation that are associated with increased activation of the contact pathway. We also found that FXII, but not tissue factor, contributes to enhanced thrombin generation and systemic inflammation observed in sickle cell mice challenged with tumor necrosis factor α. In addition, FXII inhibition significantly reduced experimental venous thrombosis, congestion, and microvascular stasis in a mouse model of SCD. Moreover, inhibition of FXII attenuated brain damage and reduced neutrophil adhesion to the brain vasculature of sickle cell mice after ischemia/reperfusion induced by transient middle cerebral artery occlusion. Finally, we found higher FXII, urokinase plasminogen activator receptor, and αMβ2 integrin expression in neutrophils of patients with SCD compared with healthy controls. Our data indicate that targeting FXII effectively reduces experimental thromboinflammation and vascular complications in a mouse model of SCD, suggesting that FXII inhibition may provide a safe approach for interference with inflammation, thrombotic complications, and vaso-occlusion in patients with SCD. This work was funded in part by National Institutes of Health (NIH) R01s HL142604 (RP), HL157441 (RP, NK) and NIH U01 HL117659 (RP, NK). EMS is supported by NIH R01 HL 155193. MK was supported by R00HL144817 from NHLBI and UAB AMC21 MULTIPI6724 grant. GMV and JDB were supported by research funding from NIH 5R01 HL114567. TR acknowledges Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) grants P6 - KFO306, 80750187 - SFB841 and 470698011 -SFB877. DG was supported by NIH R35 HL140025. FG was supported by Royal Society Wolfson Foundation (RSWF\R3\183001). AG and MW were supported by NIH R44 HL126235. This work was supported by the NIH R01 HL137695 (E.X.S.), Merit Review Awards BX003851 (E.X.S.) and the Oscar D. Ratnoff Endowed Professorship (E.X.S.). The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH, the U.S. Department of Veterans Affairs, or the United States Government.

Sickle cell disease (SCD), an inherited blood disorder, is associated with prevalent chronic kidney disease (CKD), evidenced by persistent albuminuria. It is critical to understand the natural history of albuminuria and albumin to creatinine (ACR) ratio variance to optimally identify, diagnose and treat SCD pediatrics prior to CKD. Thus, we investigated natural course of albuminuria in pediatrics and murine SCD, and identify ACR level predictive of persistent albuminuria.Basic science study: As hyperfiltration occurs at 12 weeks of age in male and 20 weeks of age in female humanized sickle cell (HbSS) mice, we performed metabolic cage studies in 1-week intervals for 4 weeks before (4 weeks old mice, n=21) and after hyperfiltration phase (males: 16 weeks (n=10), females: 20 weeks old (n=8)), and measured albuminuria using GenWay Biotech ELISA kit. Clinical study: We performed a retrospective study of the UAB Pediatric Sickle Cell Kidney Cohort. We identified all participants with HbSS and HbSB0 thalassemia ≥ 5 years of age with ACR measurements performed at a steady-state outpatient clinic visit. We identified the date and age of participants with at least 1 ACR level ≥100mg/g to compare outcomes for albuminuria and to determine the natural course of ACR after an ACR measurement >100mg/g.We observed a significant difference in the trajectory of albuminuria over time prior and post hyperfiltration (p=0.02) in HbSS mice. Young HbSS mice presented with downward trend over time, with average values of albuminuria 47.6±27.2 (week 1), 32.6±20.3 (week 2), 32.0±24.3 (week 3), 31.3±16.1mg/24h (week 4), respectively. After hyperfiltration, older HbSS mice had an upward trend in albuminuria over time (week 1: 49.7±23.6, week 2: 74.5±66.9, week 3: 55.9±82.2, week 4: 73.0±86.4 mg/24h, respectively). Finally, prior to hyperfiltration the average albuminuria over a time of 4 weeks was 35.9±7.8 mg/24h (±21.7%), while following hyperfiltration it was 63.3±12.4 mg/24h (±19.6%). These findings mirror human data in which pediatric patients present with similar degree of natural variability in ACR. We identified 62 patients (17%) with persistent albuminuria, 46 (13%) with intermittent albuminuria, and 247 (70%) with not yet having albuminuria. Out of 107 patients with at least 1 abnormal ACR, 45 had 1 measurement >100mg/g, and 43 of these participants were categorized with persistent albuminuria (50x higher odds ratio than patients with ACR100mg/g showed significant variability. Regarding 1-year clinical trial design, the change in ACR showed a decline from 175 to 131 mg/g, accounting for 23% decline in albuminuria without any intervention.The natural course of albuminuria is an important characteristic of renal phenotype in SCD and should be considered while designing and assessing effectiveness of novel therapeutic approaches in preclinical and clinical studies targeting SCD nephropathy. Funded by NIH R25 DK 112731 and NHLBI–R00HL144817 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background: Sickle cell disease (SCD) is an autosomal recessive blood disorder caused by mutations in the β-globin gene, resulting in hemoglobin S (HbS) polymerization and red blood sickling. SCD patients carry two copies of the mutant HbS (HbSS) and develop anemia, iron overload and to end-organ damage. Heterozygous carriers (AS) are said to have sickle cell trait (SCT). While not associated with shortened life expectancy, individuals with SCT are at increased risk for a limited number of complications, including chronic kidney disease (CKD) that has yet to be fully elucidated. Our objective was to examine if mouse model of SCT is associated with worsening kidney function over time, mimicking an increased risk of CKD in SCT. Methods: We used male humanized sickle cell trait (HbAS) and age-matched genetic control (HbAA) mice (n=18). Analysis of urinary renal biomarkers (albuminuria, proteinuria, kidney injury marker 1 (KIM-1), osmolality) and time course of kidney function (determined via transcutaneous measurement of glomerular filtration rate (GFR)) were conducted in 4-week intervals from 12 to 40 weeks of age. Results: At 8 weeks of age, HbAS mice exhibited no differences in GFR, or proteinuria compared to HbAA controls. Significant increase in GFR, but not proteinuria, was observed in HbAS at 16 weeks of age compared to HbAA mice (241.6±5.3 ul/min vs 218.2±5 ul/min, respectively, p ˂0.05). Importantly, significant loss of kidney function measured by GFR (200 ± 3 vs 217.9 ± 3.2 ul/min, p Funding: NHLBI–R00HL144817 and NHLBI-R01 HL157441 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Progressive iron accumulation and renal impairment are prominent in both patients and mouse models of sickle cell disease (SCD). Endothelin A receptor (ETA) antagonism prevents this iron accumulation phenotype and reduces renal iron deposition in the proximal tubules of SCD mice. To better understand the mechanisms of iron metabolism in the kidney and the role of the ETA receptor in iron chelation and transport, we studied renal iron handling in a nonsickle cell iron overload model, heme oxygenase-1 ( Hmox-1−/−) knockout mice. We found that Hmox-1−/− mice had elevated plasma endothelin-1 (ET-1), cortical ET-1 mRNA expression, and renal iron content compared with Hmox-1+/+ controls. The ETA receptor antagonist, ambrisentan, attenuated renal iron deposition, without any changes to anemia status in Hmox-1−/− mice. This was accompanied by reduced urinary iron excretion. Finally, ambrisentan had an important iron recycling effect by increasing the expression of the cellular iron exporter, ferroportin-1 (FPN-1), and circulating total iron levels in Hmox-1−/− mice. These findings suggest that the ET-1/ETA signaling pathway contributes to renal iron trafficking in a murine model of iron overload.

Peroxiredoxin-2 (Prx-2) is a critical antioxidant protein in red blood cells (RBC). Prx-2 is oxidized to a disulfide covalently-bound dimer by H(2)O(2), and then reduced back by the NADPH dependent thioredoxin – thioredoxin reductase system. Reduction of oxidized Prx-2 is relatively slow in RBCs. Since Prx-2 is highly abundant, Prx-2s’ peroxidase catalytic cycle is not considered to be limiting under normal conditions. However, whether Prx-2 recycling becomes limiting when RBCs are exposed to stress is not known. Using three different model systems characterized by increased oxidative damage to RBCs spanning the physiologic (endogenous RBCs of different ages), therapeutic (cold-stored RBCs in blood banks) and pathologic (RBCs from sickle cell disease (SCD) patients and humanized SCD mice) spectrum, basal levels of Prx-2 oxidation and Prx-2 recycling kinetics after addition of H(2)O(2) were determined. Reduction of oxidized Prx-2 was significantly slower in older versuin older versus younger RBCs, in RBCs stored for 4-5 weeks compared to 1 week, and in RBC from pediatric SCD patients compared to RBCs from control non-SCD patients. Similarly, the rate of Prx-2 recycling was slower in humanized SCD mice compared to WT mice. Treatment of RBC with carbon monoxide (CO) to limit heme-peroxidase activity had no effect on Prx-2 recycling kinetics. Treatment with glucose attenuated slowed Prx-2 recycling in older RBCs and SCD RBCs, but not stored RBCs. In conclusion, reduction of oxidized Prx-2 can be further slowed in RBCs, which may limit the protection afforded by this antioxidant protein in settings associated with erythrocyte stress.

BACKGROUND: Sickle cell disease (SCD) is characterized by chronic hemolytic anemia, vaso-occlusive crises, chronic inflammation, and activation of coagulation. The clinical complications such as painful crisis, stroke, pulmonary hypertension, nephropathy and venous thromboembolism lead to cumulative organ damage and premature death. High molecular weight kininogen (HK) is a central cofactor for the kallikrein-kinin and intrinsic coagulation pathways, which contributes to both coagulation and inflammation. OBJECTIVE: We hypothesize that HK contributes to the hypercoagulable and pro-inflammatory state that causes end-organ damage and early mortality in sickle mice. METHODS: We evaluated the role of HK in the Townes mouse model of SCD. RESULTS/CONCLUSIONS: We found elevated plasma levels of cleaved HK in sickle patients compared to healthy controls, suggesting ongoing HK activation in SCD. We used bone marrow transplantation to generate wild type and sickle cell mice on a HK-deficient background. We found that short-term HK deficiency attenuated thrombin generation and inflammation in sickle mice at steady state, which was independent of bradykinin signaling. Moreover, long-term HK deficiency attenuates kidney injury, reduces chronic inflammation, and ultimately improves survival of sickle mice.

Despite advancements in disease management, sickle cell nephropathy, a major contributor to mortality and morbidity in patients, has limited therapeutic options. Previous studies indicate hydroxyurea, a commonly prescribed therapy for sickle cell disease (SCD), can reduce renal injury in SCD but the mechanisms are uncertain. Because SCD is associated with reduced nitric oxide (NO) bioavailability, we hypothesized that hydroxyurea treatment would improve NO bioavailability in the humanized sickle cell mouse. Humanized male 12-wk-old sickle (HbSS) and genetic control (HbAA) mice were treated with hydroxyurea or regular tap water for 2 wk before renal and systemic NO bioavailability as well as renal injury were assessed. Untreated HbSS mice exhibited increased proteinuria, elevated plasma endothelin-1 (ET-1), and reduced urine concentrating ability compared with HbAA mice. Hydroxyurea reduced proteinuria and plasma ET-1 levels in HbSS mice. Untreated HbSS mice had reduced plasma nitrite and elevated plasma arginase concentrations compared with HbAA mice. Hydroxyurea treatment augmented plasma nitrite and attenuated plasma arginase in HbSS mice. Renal vessels isolated from HbSS mice also had elevated nitric oxide synthase 3 (NOS3) and arginase 2 expression compared with untreated HbAA mice. Hydroxyurea treatment did not alter renal vascular NOS3, however, renal vascular arginase 2 expression was significantly reduced. These data support the hypothesis that hydroxyurea treatment augments renal and systemic NO bioavailability by reducing arginase activity as a potential mechanism for the improvement on renal injury seen in SCD mice.

Timing of food intake has become a critical factor in determining overall cardiometabolic health. We hypothesized that timing of food intake entrains circadian rhythms of blood pressure (BP) and renal excretion in mice. Male C57BL/6J mice were fed ad libitum or reverse feeding (RF) where food was available at all times of day or only available during the 12-h lights-on period, respectively. Mice eating ad libitum had a significantly higher mean arterial pressure (MAP) during lights-off compared to lights-on (113 ± 2 mmHg vs 100 ± 2 mmHg, respectively; P 0.05); however, RF induced a diurnal rhythm of MAP (79 ± 3 mmHg vs 95 ± 2 mmHg, lights-off vs lights-on phase; P, Graphical Abstract Graphical Abstract

Background: Premenopausal women have a lower risk of hypertension compared to age-matched men and postmenopausal women. P2Y2 and P2Y4 purinoceptor can be considered potential contributors to hypertension due to their emerging roles in regulating renal tubular Na+ transport. Activation of these receptors inhibits epithelial Na+ channel activity (ENaC) via a phospholipase C (PLC)-dependent pathway resulting in natriuresis. We recently reported that activation of P2Y2 and P2Y4 receptors in the renal medulla by UTP promotes natriuresis in male and ovariectomized (OVX) rats, but not in ovary-intact females. This led us to hypothesize that ovary-intact females have greater basal renal medullary activity of P2 (P2Y2 and P2Y4) receptors regulating Na+ excretion compared to male and OVX rats. Methods: To test our hypothesis, we determined (i) the effect of inhibiting medullary P2 receptors by suramin (750 μg/kg/min) on urinary Na+ excretion in anesthetized male, ovary-intact female and OVX Sprague Dawley rats, (ii) mRNA expression and protein abundance of P2Y2 and P2Y4 receptors and (iii) mRNA expression of their downstream effectors (PLC-1d and ENaCa) in renal inner medullary tissues obtained from these three groups. We also subjected cultured mouse inner medullary collecting duct cells (segment 3, mIMCD3) to different concentrations of 17ß-estradiol (E2, 0, 10, 100 and 1000 nM) to test whether E2 increases mRNA expression of P2Y2 and P2Y4 receptors.Results: Acute P2 inhibition attenuated urinary Na+ excretion in ovary-intact females, but not in male or OVX rats. We found that P2Y2 and P2Y4 mRNA expression was higher in the inner medulla from females compared to males or OVX. Inner medullary lysates showed that ovary-intact females have higher P2Y2 receptor protein abundance, compared to males, however, OVX did not eliminate this sex difference. We also found that E2 dose-dependently upregulated P2Y2 and P2Y4 mRNA expression in mIMCD3. Conclusion: These data suggest that ovary-intact females have enhanced P2Y2 and P2Y4-dependent regulation of Na+ handling in the renal medulla, compared to male and OVX rats. We speculate that the P2 pathway contributes to facilitated renal Na+ handling in premenopausal females.

Acute psychosocial stress has been linked to the onset of vaso-occlusive pain crises in patients with sickle cell disease (SCD), however mechanistic insight is lacking. Studies suggest that histamine is a stress-responsive factor and promotes inflammation. In SCD, histamine levels are elevated in association with vaso-occulsive crisis, however, the impact of acute stress is unknown. We hypothesized that acute stress in a humanized SCD mouse model stimulates the histamine pathway, inflammatory mediator release, and a pressor response. Acute stress was induced using cage switch stress (CSS) in male humanized SCD (HbSS) or control (HbAA) mice (n=6-8/group) with blood pressure (BP) monitored by radiotelemetry and mice terminated at baseline (BL, no CSS) and 30 min post-CSS for plasma measurements. Plasma histamine was unchanged in HbAA mice but was elevated in HbSS in response to CSS (nM; HbAA: 90.2±21.3; 104.3±10.2; HbSS: 91.1±13.6; 174.3±19.2*, *pgenotype =0.0001, *pgenotype genotype

The diurnal rhythms of sodium handling and blood pressure are thought to be regulated by clock genes, such as Bmal1. However, little is known about the regulation of these factors by Bmal1, especially in rats. Using a novel whole-body Bmal1 knockout rat model ( Bmal1 − /− ), we hypothesized that time of day regulation of sodium excretion is dependent on Bmal1. Using telemetry to continuously record mean arterial pressure, we observed that male and female Bmal1 −/− rats had significantly reduced mean arterial pressure over the course of 24 hours compared with littermate controls. The circadian mean arterial pressure pattern remained intact in both sexes of Bmal1 −/− rats, which is in contrast to the Bmal1 −/− mouse model. Male Bmal1 −/− rats had no significant difference in baseline sodium excretion between 12-hour active and inactive periods, indicating a lack of diurnal control independent of maintained mean arterial pressure rhythms. Female Bmal1 −/− rats, however, had significantly greater sodium excretion during the active versus inactive period similar to controls. Thus, we observed a clear dissociation between circadian blood pressure and control of sodium excretion that is sex dependent. These findings are consistent with a more robust ability of females to maintain control of sodium excretion, and furthermore, demonstrate a novel role for Bmal1 in control of diurnal blood pressure independent of sodium excretion.

High salt intake (HS) is associated with obesity and insulin resistance. ET-1, a peptide released in response to HS, inhibits the actions of insulin on cultured adipocytes through ET-1 type B (ETB) receptors; however, the in vivo implications of ETB receptor activation on lipid metabolism and insulin resistance is unknown. We hypothesized that activation of ETB receptors in response to HS intake promotes dyslipidemia and insulin resistance. In normal salt (NS) fed rats, no significant difference in body mass or epididymal fat mass was observed between control and ETB deficient rats. After 2 weeks of HS, ETB-deficient rats had significantly lower body mass and epididymal fat mass compared to controls. Nonfasting plasma glucose was not different between genotypes; however, plasma insulin concentration was significantly lower in ETB-deficient rats compared to controls, suggesting improved insulin sensitivity. In addition, ETB-deficient rats had higher circulating free fatty acids in both NS and HS groups, with no difference in plasma triglycerides between genotypes. In a separate experiment, ETB-deficient rats had significantly lower fasting blood glucose and improved glucose and insulin tolerance compared to controls. These data suggest that ET-1 promotes adipose deposition and insulin resistance via the ETB receptor.

Impairment in the ability of the skin to properly store Na+ nonosmotically (without water) has recently been hypothesized as contributing to salt-sensitive hypertension. Our laboratory has shown that endothelial production of endothelin-1 (ET-1) is crucial to skin Na+ handling. Furthermore, it is well established that loss of endothelin type B receptor (ETB) receptor function impairs Na+ excretion by the kidney. Thus we hypothesized that rats lacking functional ETB receptors (ETB-def) will have a reduced capacity of the skin to store Na+ during chronic high-salt (HS) intake. We observed that ETB-def rats exhibited salt-sensitive hypertension with an approximate doubling in the diurnal amplitude of mean arterial pressure compared with genetic control rats on a HS diet. Two weeks of HS diet significantly increased skin Na+ content relative to water; however, there was no significant difference between control and ETB-def rats. Interestingly, HS intake led to a 19% increase in skin Na+ and 16% increase in water content (relative to dry wt.) during the active phase (zeitgeber time 16) versus inactive phase (zeitgeber time 4, P < 0.05) in ETB-def rats. There was no significant circadian variation in total skin Na+ or water content of control rats fed normal or HS. These data indicate that ETB receptors have little influence on the ability to store Na+ nonosmotically in the skin during long-term HS intake but, rather, appear to regulate diurnal rhythms in skin Na+ content and circadian blood pressure rhythms associated with a HS diet.

Speed JS, Hyndman KA, Roth K, Heimlich JB, Kasztan M, Fox BM, Johnston JG, Becker BK, Jin C, Gamble KL, Young ME, Pollock JS, Pollock DM. High dietary sodium causes dyssynchrony of the renal molecular clock in rats. Am J Physiol Renal Physiol 314: F89–F98, 2018. First published September 27, 2017; doi:10.1152/ajprenal.00028.2017.—Dyssynchrony of circadian rhythms is associated with various disorders, including cardiovascular and metabolic diseases. The cell autonomous molecular clock maintains circadian control; however, environmental factors that may cause circadian dyssynchrony either within or between organ systems are poorly understood. Our laboratory recently reported that the endothelin (ET-1) B (ETB) receptor functions to facilitate Na+ excretion in a time of day-dependent manner. Therefore, the present study was designed to determine whether high salt (HS) intake leads to circadian dyssynchrony within the kidney and whether the renal endothelin system contributes to control of the renal molecular clock. We observed that HS feeding led to region-specific alterations in circadian clock components within the kidney. For instance, HS caused a significant 5.5-h phase delay in the peak expression of Bmal1 and suppressed Cry1 and Per2 expression in the renal inner medulla, but not the renal cortex, of control rats. The phase delay in Bmal1 expression appears to be mediated by ET-1 because this phenomenon was not observed in the ETB-deficient rat. In cultured inner medullary collecting duct cells, ET-1 suppressed Bmal1 mRNA expression. Furthermore, Bmal1 knockdown in these cells reduced epithelial Na+ channel expression. These data reveal that HS feeding leads to intrarenal circadian dyssynchrony mediated, in part, through activation of ETB receptors within the renal inner medulla.

P-selectin (Psel) is an adhesion molecule expressed on platelets and endothelial cells, which interacts with its ligand PSGL-1 on leukocytes and a PSGL-1-like molecule on sickle red blood cells (RBCs). Psel mediates the formation of the multicellular aggregates that promote vaso-occlusive crisis (VOC) and acute painful episodes in sickle cell disease (SCD). Crizanlizumab, a monoclonal antibody blocking Psel, reduces the frequency of VOC in SCD patients. In SCD mice, Psel contributes to heme-induced microvascular stasis, and thrombus and platelet-neutrophil aggregate formation in the lung and liver. Unexpectedly, Psel deficiency promotes liver senescence in a mouse model of SCD, suggesting a possible detrimental effect of Psel inhibition. In this study, we further investigated the long-term effects of Psel deficiency on thromboinflammation and end-organ damage in murine SCD. We used Townes wild type (AA) and sickle (SS) mice (n=12-27) that either express Psel (Psel +/+) or lack Psel (Psel -/-) at 11-12 months of age. Kidney damage was evaluated by histology and urine analysis. Heart function was determined using echocardiography. Complete blood counts and plasma biomarkers of thrombin generation (thrombin anti-thrombin [TAT] complexes) and inflammation (interleukin [IL]-6) were also analyzed. In addition, in a pilot experiment, we analyzed the effect of Psel deficiency on experimental venous thrombosis in SS mice. SS Psel +/+ mice exhibited renal damage, urinary concentrating defect, and reduced creatinine clearance, which was not improved in SS Psel -/- mice. Psel deficiency attenuated glomerular (GLM) congestion (0.17±0.05 vs 0.39±0.06, p We observed cardiac hypertrophy (elevated heart weight to tibia length ratio) in SS mice, regardless of Psel expression. Echocardiography of left ventricle (LV) revealed that SS mice had increased LV mass and LV internal diameter with no change in ejection fraction or fractional shortening compared to AA mice. None of these parameters were affected by Psel expression. Furthermore, hypertrophy observed in other organs (kidney, liver, lung and spleen) of SS Psel +/+ mice was also not affected by Psel deficiency. Consistent with previous studies, SS Psel +/+ mice had elevated plasma levels of TAT and IL-6 compared to AA Psel +/+ mice. Psel deficiency significantly reduced IL-6 (47±15 vs 20±10 ng/mL, P As expected, SS Psel +/+ mice exhibited anemia as shown by reduced RBC number, hemoglobin (Hb) and hematocrit (Hct) compared to AA Psel +/+ animals. Interestingly, these parameters were further decreased in SS Psel -/- mice (p Our data suggest that despite improving acute vascular pathologies, including VOC and thromboinflammation, long term deficiency of Psel does not prevent end-organ damage. If fact, it may contribute to organ dysfunction and enhanced anemia. More mechanistic studies are needed to better understand the long-term effects of anti-Psel treatment in SCD patients. Disclosures Reeves: Incyte Corporation: Honoraria; Takeda: Honoraria; Bristol-Myers Squibb: Speakers Bureau; Pharma Essentia: Consultancy, Honoraria. Sundd: CSL Behring Inc: Research Funding; Bayer: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Hypertension is a prevalent pathology that increases risk for numerous cardiovascular diseases. Because the etiology of hypertension varies across patients, specific and effective therapeutic approaches are needed. The role of renal sympathetic nerves is established in numerous forms of hypertension, but their contribution to salt sensitivity and interaction with factors such as endothelin-1 are poorly understood. Rats deficient of functional ETB receptors (ETB-def) on all tissues except sympathetic nerves are hypertensive and exhibit salt-sensitive increases in blood pressure. We hypothesized that renal sympathetic nerves contribute to hypertension and salt sensitivity in ETB-def rats. The hypothesis was tested through bilateral renal sympathetic nerve denervation and measuring blood pressure during normal salt (0.49% NaCl) and high-salt (4.0% NaCl) diets. Denervation reduced mean arterial pressure in ETB-def rats compared with sham-operated controls by 12 ± 3 (SE) mmHg; however, denervation did not affect the increase in blood pressure after 2 wk of high-salt diet (+19 ± 3 vs. +16 ± 3 mmHg relative to normal salt diet; denervated vs. sham, respectively). Denervation reduced cardiac sympathetic-to-parasympathetic tone [low frequency-high frequency (LF/HF)] during normal salt diet and vasomotor LF/HF tone during high-salt diet in ETB-def rats. We conclude that the renal sympathetic nerves contribute to the hypertension but not to salt sensitivity of ETB-def rats.

We recently reported that natriuresis produced by renal medullary salt loading is dependent on endothelin (ET)-1 and purinergic (P2) receptors in male rats. Because sex differences in ET-1 and P2 signaling have been reported, we decided to test whether ovarian sex hormones regulate renal medullary ET-1 and P2-dependent natriuresis. The effect of medullary NaCl loading on Na+excretion was determined in intact and ovariectomized (OVX) female Sprague-Dawley rats with and without ET-1 or P2 receptor antagonism. Isosmotic saline (284 mosmol/kgH2O) was infused in the renal medullary interstitium of anesthetized rats during a baseline urine collection period, followed by isosmotic or hyperosmotic saline (1,800 mosmol/kgH2O) infusion. Medullary NaCl loading significantly enhanced Na+excretion in intact and OVX female rats. ETA+Bor P2 receptor blockade did not attenuate the natriuretic effect of medullary NaCl loading in intact females, whereas ETA+Bor P2 receptor blockade attenuated the natriuretic response to NaCl loading in OVX rats. Activation of medullary P2Y2and P2Y4receptors by UTP infusion had no significant effect in intact females but enhanced Na+excretion in OVX rats. Combined ETA+Breceptor blockade significantly inhibited the natriuretic response to UTP observed in OVX rats. These data demonstrate that medullary NaCl loading induces ET-1 and P2-independent natriuresis in intact females. In OVX, activation of medullary P2 receptors promotes ET-dependent natriuresis, suggesting that ovarian hormones may regulate the interplay between the renal ET-1 and P2 signaling systems to facilitate Na+excretion.

Sickle cell disease (SCD)-associated nephropathy is a major source of morbidity and mortality in patients because of the lack of efficacious treatments targeting renal manifestations of the disease. Here, we describe a long-term treatment strategy with the selective endothelin-A receptor (ETA) antagonist, ambrisentan, designed to interfere with the development of nephropathy in a humanized mouse model of SCD. Ambrisentan preserved GFR at the level of nondisease controls and prevented the development of proteinuria, albuminuria, and nephrinuria. Microscopy studies demonstrated prevention of podocyte loss and structural alterations, the absence of vascular congestion, and attenuation of glomerulosclerosis in treated mice. Studies in isolated glomeruli showed that treatment reduced inflammation and oxidative stress. At the level of renal tubules, ambrisentan treatment prevented the increased excretion of urinary tubular injury biomarkers. Additionally, the treatment strategy prevented tubular brush border loss, diminished tubular iron deposition, blocked the development of interstitial fibrosis, and prevented immune cell infiltration. Furthermore, the prevention of albuminuria in treated mice was associated with preservation of cortical megalin expression. In a separate series of identical experiments, combined ETA and ETB receptor antagonism provided only some of the protection observed with ambrisentan, highlighting the importance of exclusively targeting the ETA receptor in SCD. Our results demonstrate that ambrisentan treatment provides robust protection from diverse renal pathologies in SCD mice, and suggest that long-term ETA receptor antagonism may provide a strategy for the prevention of renal complications of SCD.

Existing methods to measure glomerular permeability are limited to relative measures using changes in size of isolated glomeruli in response to changes in oncotic pressure. Further, these techniques are not easily adapted for use with human glomeruli. In the current issue, Desideri and colleagues validate a sophisticated new technique with great promise for future understanding of the glomerular filtration barrier.

Vaso-occlusive crisis often occurs in sickle cell disease (SCD). Since psychosocial stress leads to impaired vascular function and inflammation, we hypothesized that acute psychosocial stress elicits an exaggerated blood pressure response and/or increased circulating inflammatory mediators in a mouse model of SCD. We utilized adult male (16-20 wks) humanized Townes sickle cell (HbSS) and control (HbAA) mice with telemetry (n=8) to monitor changes in systolic blood pressure (SBP), diastolic blood pressure (DBP), spectral analysis (low frequency/high frequency, LF/HF ratio) of blood pressure variability, heart rate (HR), and locomotor activity (LA) in response to cage switch stress (CSS). CSS-induced increases in DBP were significantly attenuated in HbSS versus HbAA (HbSS: 23.3±1.8mmHg, HbAA: 31.6±1.7mmHg, p=0.004), while the increase in SBP was similar in HbSS and HbAA. CSS increased LF/HF in both strains, although HbSS was significantly less than HbAA (CSS HbAA : 3.21±0.2; CSS HbSS : 1.68±0.2, p

We previously reported that humanized sickle cell (HbSS) mice develop spontaneous nephropathy, a major cause of morbidity and mortality in sickle cell disease (SCD). Because sex-dependent protective mechanisms in SCD have been reported, we examined the course of nephropathy in male and female HbSS mice to determine contributors and/or predictors of disease severity. In male HbSS mice, glomerular filtration rate was characterized by a rapid onset of hyperfiltration and subsequent progressive decline of renal function over 20 weeks. Early tubular injury presented with increased excretion of kidney injury marker 1 (KIM-1), progressive loss of tubular brush border, and interstitial fibrosis that preceded the onset of glomerular damage, suggesting a tubuloglomerular mechanism of kidney injury in these mice. Additionally, we observed a strong association between the magnitude of hyperfiltration and the degree of long-term kidney injury in male HbSS mice. Unlike males, female HbSS mice did not demonstrate a significant loss of renal function or severe kidney damage during the time course of the study. These results suggest that magnitude of hyperfiltration predicts the onset of chronic kidney damage in male HbSS mice, whereas protective mechanisms in female HbSS mice delay the onset of SCD nephropathy.

Deranged histone deacetylase (HDAC) activity causes uncontrolled proliferation, inflammation, fibrosis, and organ damage. It is unclear whether deranged HDAC activity results in acute kidney injury in the renal hypoperfusion model of bilateral ischemia-reperfusion injury (IRI) and whether in vivo inhibition is an appropriate therapeutic approach to limit injury. Male mice were implanted with intraperitoneal osmotic minipumps containing vehicle, the class I HDAC inhibitor, MS275, or the pan-HDAC inhibitor, trichostatin A (TSA), 3 days before sham/bilateral IRI surgery. Kidney cortical samples were analyzed using histological, immunohistochemical, and Western blotting techniques. HDAC-dependent proliferation rate was measured in immortalized rat epithelial cells and primary mouse or human proximal tubule (PT) cells. There were dynamic changes in cortical HDAC localization and abundance following IRI including a fourfold increase in HDAC4 in the PT. HDAC inhibition resulted in a significantly higher plasma creatinine, increased kidney damage, but reduced interstitial fibrosis compared with vehicle-treated IRI mice. HDAC-inhibited mice had reduced interstitial α-smooth muscle actin, fibronectin expression, and Sirius red-positive area, suggesting that IRI activates HDAC-mediated fibrotic pathways. In vivo proliferation of the kidney epithelium was significantly reduced in TSA-treated, but not MS275-treated, IRI mice, suggesting class II HDACs mediate proliferation. Furthermore, HDAC4 activation increased proliferation of human and mouse PTs. Kidney HDACs are activated during IRI with isoform-specific expression patterns. Our data point to mechanisms whereby IRI activates HDACs resulting in fibrotic pathways but also activation of PT proliferation and repair pathways. This study demonstrates the need to develop isoform-selective HDAC inhibitors for the treatment of renal hypoperfusion-induced injury.

Hyperfiltration, highly prevalent early in sickle cell disease (SCD), is in part driven by an increase in ultrafiltration coefficient (Kf). The increase in Kf may be due to enlarged filtration surface area and/or increased glomerular permeability (Palb). Previous studies have demonstrated that endothelin-1 (ET-1) contributes to Palb changes in models of diabetes and SCD. Thus, we performed longitudinal studies of renal function to determine the relationship between ET-1 and glomerular size and Palb that may contribute to hyperfiltration in humanized sickle cell (HbSS) and control (HbAA) mice at 8–32 weeks of age. HbSS mice were characterized by significant increases in plasma and glomerular ET-1 expression in both sexes although this increase was significantly greater in males. HbSS glomeruli of both males and females presented with a progressive and significant increase in glomerular size, volume, and Kf. During the onset of hyperfiltration, plasma and glomerular ET-1 expression were associated with a greater increase in glomerular size and Kf in HbSS mice, regardless of sex. The pattern of Palb augmentation during the hyperfiltration was also associated with an increase in glomerular ET-1 expression, in both male and female HbSS mice. However, the increase in Palb was significantly greater in males and delayed in time in females. Additionally, selective endothelin A receptor (ETA) antagonist prevented hyperfiltration in HbSS, regardless of sex. These results suggest that marked sex disparity in glomerular hyperfiltration may be driven, in part, by ET-1-dependent ultra-structural changes in filtration barrier components contributing to glomerular hyperfiltration in HbSS mice.

Background In patients with diabetes mellitus, hyperfiltration precedes the development of albuminuria. Pediatric sickle cell anemia (SCA) patients have a high prevalence of hyperfiltration and albuminuria during early childhood and adolescence. We tested the hypothesis that hyperfiltration precedes the development of albuminuria in a longitudinal pediatric SCA cohort. Methods We identified 91 participants with HbSS or SB0 thalassemia 5-21 years of age enrolled in a longitudinal sickle cell nephropathy cohort study who had a cystatin C measured during early childhood (4-10 years of age). Early hyperfiltration was defined as a mean eGFR >180 mL/min/1.73m2 using cystatin C obtained from 4 to 10 years of age. Persistent albuminuria was defined as an albumin to creatinine ratio > 30 mg/g on two of three untimed urine specimens. Time to event analysis estimated survival curves for participants with and without hyperfiltration using Kaplan-Meier curves and used logrank test for categorical variables to assess the association with time to development of the first episode persistent albuminuria. Results Persistent albuminuria occurred more often and at an earlier age in participants with early hyperfiltration compared to those without early hyperfiltration (log-rank, P = .004). Participants who developed albuminuria have a significant increase in their eGFR during childhood (P = .003) as compared to participants who have not yet progressed to albuminuria (P = .26). For every 1 g/dL increase in hemoglobin, the hazard ratio for developing persistent proteinuria decreased by 0.56 (95% CI: 0.3, 1.06, P = .07). Conclusion Hyperfiltration precedes the development of persistent proteinuria in pediatric SCA patients. Intervention strategies should target lowering eGFR during early childhood.

Renal endothelin-1 (ET-1) and purinergic signaling systems regulate Na+reabsorption in the renal medulla. A link between the renal ET-1 and purinergic systems was demonstrated in vitro, however, the in vivo interaction between these systems has not been defined. To test whether renal medullary activation of purinergic (P2) receptors promotes ET-dependent natriuresis, we determined the effect of increased medullary NaCl loading on Na+excretion and inner medullary ET-1 mRNA expression in anesthetized adult male Sprague-Dawley rats in the presence and absence of purinergic receptor antagonism. Isosmotic saline (NaCl; 284 mosmol/kgH2O) was infused into the medullary interstitium (500 μl/h) during a 30-min baseline urine collection period, followed by isosmotic or hyperosmotic saline (1,800 mosmol/kgH2O) for two further 30-min urine collection periods. Na+excretion was significantly increased during intramedullary infusion of hyperosmotic saline. Compared with isosmotic saline, hyperosmotic saline infused into the renal medulla caused significant increases in inner medullary ET-1 mRNA expression. Renal intramedullary infusion of the P2 receptor antagonist suramin inhibited the increase in Na+excretion and inner medullary ET-1 mRNA expression induced by NaCl loading in the renal medulla. Activation of medullary P2Y2/4receptors by infusion of UTP increased urinary Na+excretion. Combined ETAand ETBreceptor blockade abolished the natriuretic response to intramedullary infusion of UTP. These data demonstrate that activation of medullary P2 receptors promotes ET-dependent natriuresis in male rats, suggesting that the renal ET-1 and purinergic signaling systems interact to efficiently facilitate excretion of a NaCl load.

Sickle cell disease (SCD) is a genetic hematologic disorder that is characterized by a variety of potentially life threatening acute and chronic complications. Currently, hydroxyurea is the only clinically approved pharmacological therapy for the treatment of SCD, and the continued prevalence of severe disease complications underscores the desperate need for the development of new therapeutic agents. Central features of the sickle cell disease milieu, including hypoxia, oxidative stress, and thrombosis, are established enhancers of endothelin-1 (ET-1) synthesis. This conceptual connection between ET-1 and SCD was confirmed by multiple studies that demonstrated markedly elevated plasma and urinary levels of ET-1 in SCD patients. Direct evidence for the involvement of ET-1 signaling in the development of SCD pathologies has come from studies using endothelin receptor antagonists in SCD mice. This review summarizes recent studies that have implicated ET-1 signaling as a mechanistic contributor to renal, vascular, pulmonary, and nociceptive complications of sickle cell disease and discusses the potential for the use of ET receptor antagonists in the treatment of SCD.

Podocytes are dynamic polarized cells that lie on the surface of glomerular capillaries and comprise an essential component of the glomerular filtration barrier. Insulin provoked a sustained, approximately 70%, increase in intracellular calcium concentration in podocytes. RT-PCR revealed the presence of mRNA encoding sarco/endoplasmic reticulum calcium ATPase isoforms 1-3, and plasma membrane Ca(2+) pump (PMCA) isoforms 1,3,4; mRNA levels were depressed by the addition of insulin. Inhibitors of PMCA, and the Na(+) -Ca(2+) exchanger, increased podocyte permeability to albumin, induced dimerization of protein kinase G type I alpha (PKGIα), and activation of PKGIα-dependent signaling. These data suggest the involvement of calcium and PKGIα signaling in insulin-enhanced filtration barrier permeability in podocytes.

Premenopausal women have a lower risk of hypertension compared to age-matched men. Recently, prominent roles have been assigned to P2Y 2 and P2Y 4 purinoceptor subtypes in promoting Na + excretion, implicating dysfunction of these receptors as potential contributors to hypertension. We recently reported that activation of P2Y 2 and P2Y 4 receptors in the renal medulla by UTP promotes Na + excretion in male rats. In intact females, UTP did not stimulate Na + excretion while ovariectomy unmasked UTP-induced natriuresis. These observations led us to hypothesize that intact females have higher basal renal medullary activity of P2Y 2 and P2Y 4 receptors in regulating Na + excretion compared to male and ovariectomized (OVX) rats. To test that, we determined (i) P2Y 2 and P2Y 4 mRNA and protein expression in the inner medulla from male, intact female and OVX Sprague Dawley rats and (ii) the effect of inhibiting medullary purinoceptors (P2 receptors) on Na + excretion in those rats. We found that P2Y 2 and P2Y 4 mRNA expression was higher in the inner medulla from females compared to males (1.00±0.09 vs. 0.70±0.05 and 1.00±0.22 vs. 0.29±0.05, respectively, P2 and P2Y 4 mRNA expression were eliminated by ovariectomy (0.60±0.06 and 0.29±0.04, respectively, p2 receptor, compared to males. In anesthetized rats, medullary P2 receptor inhibition by suramin (P2 receptor antagonist, 750 μg/kg/min) significantly attenuated Na + excretion in intact females (0.4±0.1 vs. 0.9±0.2 μmol/min, P2 ) increases the expression of P2Y 2 and P2Y 4 receptors, we subjected cultured mouse inner medullary collecting duct cells (mIMCD3) to different concentrations of E 2 (0, 10, 100 and 1000 nM). We found that E 2 dose-dependently increased the expression of P2Y 2 and P2Y 4 mRNA in mIMCD3. These data suggest that females have enhanced P2Y 2 and P2Y 4 -dependent regulation of Na + excretion in the renal medulla, compared to male and OVX rats, at least partially via an E 2 -dependent mechanism. This pathway may contribute to facilitated renal Na + handling in premenopausal females.

Sickle cell disease (SCD) is an autosomal recessive hematologic disorder that afflicts millions of people across the globe. As longevity improves due to therapeutic advancements, end-organ damage, ...

Sickle cell disease is associated with increased prevalence of nephropathy and approximately 30% mortality from chronic kidney disease and renal failure. The spectrum of renal manifestations includ...

Impairment in the ability of the skin to properly store Na

Alterations in extracellular fluid volume regulation and sodium balance may result in the development and maintenance of salt-dependent hypertension, a major risk factor for cardiovascular disease. Numerous pathways contribute to the regulation of sodium excretion and blood pressure, including endothelin and purinergic signaling. Increasing evidence suggests a link between purinergic receptor activation and endothelin production within the renal collecting duct as a means of promoting natriuresis. A better understanding of the relationship between these two systems, especially in regard to sodium homeostasis, will fill a significant knowledge gap and may provide novel antihypertensive treatment options. Therefore, this review focuses on the cross talk between endothelin and purinergic signaling as it relates to the renal regulation of sodium and blood pressure homeostasis.

Podocytes are dynamic polarized cells on the surface of glomerular capillaries and an essential component of the glomerular filtration barrier. Insulin increases the activation of protein kinase G type Iα (PKGIα) subunits, leading to podocyte dysfunction. In addition, accumulating evidence suggests that TRPC6 channels are crucial mediators of podocyte calcium handling and involved in the regulation of glomerular filtration. Therefore, we investigated whether TRPC6 is involved in the regulation of filtration barrier permeability by insulin via the PKGIα-dependent manner. TRPC channel inhibitor SKF96365 abolished insulin-dependent glomerular albumin permeability and transepithelial albumin flux in cultured rat podocytes. Insulin-evoked albumin permeability across podocyte monolayers was also blocked using TRPC6 siRNA. The effect of insulin on albumin permeability was mimicked by treating podocytes with TRPC channel activator (oleolyl-2-acetyl-sn-glycerol, OAG). Insulin or OAG treatment rapidly increased the superoxide generation through activation of NADH oxidase. TRPC inhibitor SKF96365 or siRNA knockdown of TRPC6 attenuated insulin-dependent increase of ROS production. Furthermore, TRPC inhibitor or downregulation of TRPC6 blocked insulin-induced rearrangement of the actin cytoskeleton and attenuated oxidative activation of PKGIα and changes in the phosphorylation of PKG target proteins MYPT1 and MLC. Moreover insulin regulated the PKGIα interaction with TRPC6 in cultured rat podocytes. Taken together, our data suggest a key role of TRPC6 channels in the mediation of insulin-dependent activation of PKGIα signaling pathways. Overall, we have identified a potentially important mechanism that may explain disturbances in filtration barrier permeability in many diseases with increased expression of TRPC6 and chronic Ca2+ overload.

AIM: Chronic high salt intake exaggerates renal injury and inflammation, especially with the loss of functional ET(B) receptors. Tauroursodeoxycholic acid (TUDCA) is a chemical chaperone and bile salt that is approved for the treatment of hepatic diseases. Our aim was to determine whether TUDCA is reno-protective in a model of ET(B) receptor deficiency with chronic high salt-induced renal injury and inflammation. METHODS: ET(B)-deficient and transgenic control rats were placed on normal (0.8% NaCl) or high salt (8% NaCl) diet for 3 weeks, receiving TUDCA (400 mg/kg/day; i.p.) or vehicle. Histological and biochemical markers of kidney injury, renal cell death and renal inflammation were assessed. RESULTS: In ET(B)-deficient rats, high salt diet significantly increased glomerular and proximal tubular histological injury, proteinuria, albuminuria, excretion of tubular injury markers KIM-1 and NGAL, renal cortical cell death and renal CD4(+) T cell numbers. TUDCA treatment increased proximal tubule megalin expression as well as prevented high salt diet-induced glomerular and tubular damage in ET(B)-deficient rats, as indicated by reduced kidney injury markers, decreased glomerular permeability and proximal tubule brush border restoration, as well as reduced renal inflammation. However, TUDCA had no significant effect on blood pressure. CONCLUSIONS: TUDCA protects against the development of glomerular and proximal tubular damage, decreases renal cell death, and inflammation in the renal cortex in rats with ET(B) receptor dysfunction on a chronic high salt diet. These results highlight the potential use of TUDCA as a preventive tool against chronic high salt induced renal damage.

Introduction: Recent advances in preventive care, such as hydoxyurea and prophylactic antibiotics, have reduced the mortality of children with sickle cell disease (SCD) s in developed countries. Yet, the chronic hemolytic anemia and recurrent vaso-occlusive crises result in systemic inflammation and coagulopathy. Markers of coagulation activation correlate with painful crises, acute chest syndrome, stroke, venous thromboembolism, pulmonary hypertension, left ventricular diastolic heart disease, and sickle nephropathy. These complications result in end-organ failure that causes increased morbidity and mortality in adult SCD patients. We have shown that tissue factor (TF), the primary initiator of extrinsic coagulation, contributes to inflammation and coagulation in mouse models of SCD. (1,2). It has also been demonstrated that long-term reduction in thrombin protects from cardiopulmonary dysfunction and reduces mortality of sickle cell mice (3). Recent work from our laboratory demonstrates that high molecular weight kininogen (HK) promotes thrombin generation and inflammation in sickle mice. HK is proteolytically cleaved into bradykinin and cleaved HK fragments (HKf) by kallikrein and other proteases. HKf induces TF expression and activity on monocytes dependent on Mac-1 (CD11b/CD18). We found that Mac-1 inhibition attenuates thrombin generation and inflammation in sickle cell mice. Hypothesis: Long-term HK deficiency in sickle cell mice will attenuate TF-mediated coagulation and inflammation, and protect against end-organ damage and mortality. Methods and Results: To evaluate the effect of long-term HK deficiency on outcomes in sickle cell disease, we used bone marrow from Townes sickle (SS) and wild type (AA) mice to generate chimeras in lethally irradiated HK+/+ (WT) and HK-/- (KO) mice to create AA/WT, AA/KO, SS/WT, and SS/KO mice. Efficient reconstitution of bone marrow was confirmed by hemoglobin electrophoresis. Eight months after chimeras were generated, endpoints were assessed. SS/WT mice had early mortality (median survival 209 days, 6/23 mice survived to 250 days); HK deficiency significantly prolonged survival in SS mice (median survival 240 days, 24/29 mice survived to 250 days; p Conclusions: These data indicate that HK deficiency attenuates chronic inflammation, kidney failure, and heart hypertrophy, and improves survival of sickle cell mice. Disclosures No relevant conflicts of interest to declare.

Circadian rhythms in physiologic functions are driven, at the molecular level, by a group of transcription factors that oscillate over a 24 hour period, collectively termed the molecular clock. Within the kidney, it has been shown that the molecular clock directly influences transcription of Na + transporters and channels, including ENaC. ENaC is regulated by endothelin-1 (ET-1), via ET B receptor activation, in response to high salt intake. Thus, we hypothesized that increases in dietary sodium regulate the renal molecular clock (which in turn would facilitate Na+ homeostasis) through an ET B dependent mechanism. To address this question, we examined the effect of high salt (HS) intake on renal clock gene ( Bmal1, Cry1, Per1, Per2 ) expression. Control and ET B receptor deficient (ET B def) rats (a model of elevated renal ENaC) were placed on either HS or normal salt (NS) for two weeks and euthanized every 4 hours beginning at Zeitgeber Time 0 (Lights on). In the inner medulla, HS causes a phase delay in Bmal1 (Fig 1A) expression in control but not ET B def rats (Fig 1B). In addition, HS suppressed the expression of Cry1 , and Per2 during the respective acrophase in both control and ET B def rats (Fig 1C-1F) with no significant effect on Per1 . In contrast, no significant difference in the expression of Bmal1, Cry1, Per2, or Per1 (Fig 1I-1P) was found in response to HS in the renal cortex of either control or ET B def. These data indicate that HS feeding desynchronizes the molecular clock within the kidney and provides evidence that peripheral clocks are regulated in a cell type specific manner, even within the same organ.

BACKGROUND Kidney noradrenergic innervation regulates tubular function. Adenosine triphosphate (ATP)-a co-transmitter of norepinephrine-acts on purinoceptors, including ion channel receptor, P2X. P2X receptor agonists, α,β-methylene ATP (α,β-meATP) and β,γ-methylene ATP (β,γ-meATP), induce natriuresis. Regarding the functional co-localization of adrenoceptors and P2X receptors, we evaluated rat renal tubular system sensitivity to natriuretic action of P2X receptor agonists in chronically denervated kidney. METHODS Clearance studies with α,β-meATP and β,γ-meATP (intravenous infusion rate, 2 µmol/kg + 20 nmol/kg/min) were performed after bilateral surgical kidney denervation (DNx) and sham-operation (Sham). Na/K-ATPase activity was measured in isolated rat renal proximal tubules. RESULTS In DNx compared with Sham, saline infusion significantly increased renal sodium and urine excretion and P2X receptor agonist infusion was significantly more natriuretic and diuretic. In DNx and Sham, respectively, α,β-meATP increased fractional excretion of sodium (FE(Na)) by 2 ± 0.3 and 0.6 ± 0.1% and urine (FE(V)) by 1.6 ± 0.3 and 0.9 ± 0.2%; β,γ-meATP had similar effects. In both groups of rats, natriuretic and diuretic actions were abolished by P2 receptor blocker (pyridoxal-phosphate-6-azophenyl-2',4'-disulphonate, PPADS), mean arterial blood pressure and glomerular filtration rate remained unchanged during infusion of P2X receptor agonists and antagonist and basal Na/K-ATPase activities in isolated proximal tubules were similar. Both α,β-meATP and β,γ-me-ATP decreased the Na/K-ATPase activity, with 20% inhibition (P < 0.05) in denervated and innervated rats; these inhibitory effects were abolished in the presence of PPADS. CONCLUSIONS Decreased renal sympathetic activity enhances the natriuretic effect of P2X receptor stimulation. This effect is probably not related to altered Na/K-ATPase activity in renal proximal tubules.

Purinoceptors (adrengeric receptors and P2 receptors) are expressed on the cellular components of the glomerular filtration barrier, and their activation may affect glomerular permeability to albumin, which may ultimately lead to albuminuria, a well-established risk factor for the progression of chronic kidney disease and development of cardiovascular diseases. We investigated the mechanisms underlying the in vitro and in vivo purinergic actions on glomerular filter permeability to albumin by measuring convectional albumin permeability ( Palb) in a single isolated rat glomerulus based on the video microscopy method. Primary cultured rat podocytes were used for the analysis of Palb, cGMP accumulation, PKG-Iα dimerization, and immunofluorescence. In vitro, natural nucleotides (ATP, ADP, UTP, and UDP) and nonmetabolized ATP analogs (2-meSATP and ATP-γ-S) increased Palbin a time- and concentration-dependent manner. The effects were dependent on P2 receptor activation, nitric oxide synthase, and cytoplasmic guanylate cyclase. ATP analogs significantly increased Palb, cGMP accumulation, and subcortical actin reorganization in a PKG-dependent but nondimer-mediated route in cultured podocytes. In vivo, 2-meSATP and ATP-γ-S increased Palbbut did not significantly affect urinary albumin excretion. Both agonists enhanced the clathrin-mediated endocytosis of albumin in podocytes. A product of adenine nucleotides hydrolysis, adenosine, increased the permeability of the glomerular barrier via adrenergic receptors in a dependent and independent manner. Our results suggest that the extracellular nucleotides that stimulate an increase of glomerular Palbinvolve nitric oxide synthase and cytoplasmic guanylate cyclase with actin reorganization in podocytes.

Purpose of review The role of endothelin-1 (ET-1) in the kidney has been under study for many years; however, the complex mechanisms by which endothelin controls the physiology/pathophysiology of this organ are not fully resolved. This review aims to summarize recent findings in the field, especially regarding glomerular and tubular damage, Na/water homeostasis and sex differences in ET-1 function. Recent findings Podocytes have been recently identified as a target of ET-1 in the glomerular filtration barrier via ETA receptor activation. Activation of the ETA receptor by ET-1 leads to renal tubular damage by promoting endoplasmic reticulum stress and apoptosis in these cells. In addition, high flow rates in the nephron in response to high salt intake induce ET-1 production by the collecting ducts and promote nitric oxide-dependent natriuresis through epithelial sodium channel inhibition. Recent evidence also indicates that sex hormones regulate the renal ET-1 system differently in men and women, with estrogen suppressing renal ET-1 production and testosterone upregulating that production. Summary Based on the reports reviewed in here, targeting of the renal endothelin system is a possible therapeutic approach against the development of glomerular injury. More animal and clinical studies are needed to better understand the dimorphic control of this system by sex hormones.