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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.

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.

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.

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.

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.

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.

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.

Impairment in the ability of the skin to properly store Na

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.

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.

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.

Podocytes are highly specialized cells that wrap around glomerular capillaries and comprise a key component of the glomerular filtration barrier. They are uniquely sensitive to insulin; like skeletal muscle and fat cells, they exhibit insulin-stimulated glucose uptake and express glucose transporters. Podocyte insulin signaling is mediated by protein kinase G type I (PKGI), and it leads to changes in glomerular permeability to albumin. Here, we investigated whether large-conductance Ca 2 + -activated K + channels (BK Ca ) were involved in insulin-mediated, PKGIα-dependent filtration barrier permeability. Insulin-induced glomerular permeability was measured in glomeruli isolated from Wistar rats. Transepithelial albumin flux was measured in cultured rat podocyte monolayers. Expression of BK Ca subunits was detected by RT-PCR. BK Ca , PKGIα, and upstream protein expression were examined in podocytes with Western blotting and immunofluorescence. The BK Ca –PKGIα interaction was assessed with co-immunoprecipitation. RT-PCR showed that primary cultured rat podocytes expressed mRNAs that encoded the pore-forming α subunit and four accessory β subunits of BK Ca . The BK Ca inhibitor, iberiotoxin (ibTX), abolished insulin-dependent glomerular albumin permeability and PKGI-dependent transepithelial albumin flux. Insulin-evoked albumin permeability across podocyte monolayers was also blocked with BK Ca siRNA. Moreover, ibTX blocked insulin-induced disruption of the actin cytoskeleton and changes in the phosphorylation of PKG target proteins, MYPT1 and RhoA. These results indicated that insulin increased filtration barrier permeability through mobilization of BK Ca channels via PKGI in cultured rat podocytes. This molecular mechanism may explain podocyte injury and proteinuria in diabetes.

The increase in the permeability of the glomerular barrier filtration to albumin is a well-known feature of diabetic microvasculature and a negative prognostic factor for vascular complications. However, the underlying mechanisms are incompletely understood. We demonstrated recently that superoxide anion generation increases dimerization of protein kinase G type Iα (PKGIα) subunits, leading to podocyte dysfunction. Here we investigated whether high insulin concentration is involved in PKGI-dependent hyperpermeability of the diabetic glomerular filtration barrier. We assessed changes in insulin-induced glomerular permeability by measuring glomerular capillary permeability to albumin in isolated glomeruli from Wistar and obese and lean Zucker rats and transmembrane albumin flux in cultured rat podocytes. Expression of PKGIα and upstream proteins was confirmed in the podocytes using Western blotting and immunofluorescence. Insulin (300nM, 5min) increased NAD(P)H-dependent glomerular albumin permeability in Wistar rats and PKGI-dependent transmembrane albumin flux in cultured podocytes. Podocyte exposure to insulin in non-reducing conditions increased PKGIα interprotein disulfide bond formation, altered the phosphorylation of the PKG target proteins MYPT1 and MLC, and disrupted the actin cytoskeleton. The role of NADPH oxidase (NOX) in insulin-induced reactive oxygen species (ROS) generation and insulin-evoked increases in albumin permeability in podocytes was confirmed with NOX2 and NOX4 siRNA. Glomerular albumin permeability was increased in hyperinsulinemic Zucker obese rats with isolated glomeruli showing increased expression of PKGIα and NOX4. Taken together, these data demonstrate that insulin increases glomerular barrier albumin permeability via a PKGI-dependent mechanism involving NAD(P)H-dependent generation of superoxide anion. These findings reveal a role for insulin in the pathophysiology of diabetic glomerular nephropathy.