Renal Membrane Research Articles

Abstract 020: Impact Of Sex Hormones On Renal Mitochondria Function In Sprague Dawley Rats

Introduction: Until females reach menopause, they are generally protected from renal disease. Mitochondrial function is involved in sexual dimorphisms, however, there is a gap in knowledge regarding the contribution of mitochondrial bioenergetics to renal disease susceptibility. Our studies identified sex differences in renal mitochondrial metabolism, respiration, and ROS production in young healthy rats. We hypothesized here that sex hormones mediate the sex differences in renal mitochondrial bioenergetics. Methods: Male and female Sprague Dawley rats were randomly divided into groups that underwent a gonadectomy or a sham surgery (4 weeks of age). At 6 months of age renal cortex and medulla were used for ex vivo experiments. Mitochondria were isolated and spectrofluorimetry was employed to measure membrane potential (TMRM) and H 2 O 2 production (Amplex Red) along with a Seahorse assay for oxygen consumption rate (OCR). OriginPro was used for analysis (two-way ANOVA with post hoc test). Results: Spectrofluorimetry revealed that oophorectomy reduced membrane potential in mitochondria from the renal medulla compared to shams (p=0.001); this effect was not observed in the medulla of orchiectomized males. Female sham rats had higher H 2 O 2 compared to male sham rats (cortex p<0.001, medulla p<0.001). Removing sex hormones’ influence lowered H 2 O 2 production in both the female cortex and medulla (p<0.001, p<0.001), while in males, the loss of sex hormones led to higher H 2 O 2 production in the medulla (p<0.001). Orchiectomy increased ATP-linked respiration in both the cortex and medulla compared to shams (p=0.02, p=0.04), while an oophorectomy did not alter respiration in either region. ATP-linked respiration was increased in the female shams compared to male shams for both regions (p=0.04, p=0.04). Basal OCR was similar between all groups, with cortical mitochondria exhibiting lower OCR compared to medullary mitochondria for all groups (p<0.001). Conclusions: We report that renal membrane potential, H 2 O 2 , and OCR are uniquely impacted by sex hormones in healthy rats 5 months post-gonadectomy. The observed dissimilarities resulting from sex hormones’ highlight the need to consider sex for the prevention and treatment of renal diseases.

Oxidative stress-induced by different stressors alters kidney tissue antioxidant markers and levels of creatinine and urea: the fate of renal membrane integrity

The cellular integrity of the kidney in homeostatic regulation has constantly been compromised by oxidative stress following exposure to varying nature of stressor present within the environment. The objective of the work was to evaluate the renal effect of the different stressor stimuli applied. Twenty-four adult female rats weighing averagely 160–200 g and within the ages of 12–14 weeks were used for experiment-1, while 12 offspring were utilized for experiment-2. Three stress models namely; restraint, mirror chamber and cat intruder stressors were used. Tissues were isolated from the animal and homogenized for tissue antioxidant assay. Serum was collected for assays of urea and creatinine for the kidney function test using ELISA. Data collected were analyzed for Mean ± SEM using One Way ANOVA. The present study revealed that exposure of rats to different stressors reduced relative kidney weights but did not significantly alter serum creatinine concentration in the Wistar rats, although the concentrations were slightly increased compared to controls. Urea concentration was significantly (p < 0.05) increased in rats exposed to restraint and intruder stressors. Exposure to a mirror chamber stressor did not significantly alter urea concentration. Offspring from parents of stressed female rats exhibited a significant (p < 0.05) increase in serum urea level, minimal increase in serum creatinine levels. GSH and GST levels showed no significant difference when compared to control group, whereas, GPx were significantly (p < 0.05) decreased irrespective of the stressor applied. SOD activity were significantly (p < 0.05) reduced in the group exposed to restraint or cat intruder stressor. CAT activities were significantly (p < 0.05) reduced in the rats exposed to restraint or cat intruder stressor. In all, the different stress model altered the antioxidant capacity of the kidney tissues. Exposure of rats to a stressful condition of the different nature of stressor has the tendency of compromising the functional integrity of the kidney, thus, with the potency of complicating female renal function.

Pathophysiological aspects of renal stone formation and stone types

Urinary stone formation is one of the oldest and most widespread diseases known to man. The disease has a multifactorial etiology that includes anatomic, environmental, genetic, infectious, metabolic, nutritional, and most importantly socio-economic factors. It is caused by a biochemical imbalance in urine between stone-forming inhibitors and promotors in a process known as lithogenesis. The mechanisms underlying the formation and development of urinary stones are not fully understood, but it can be said that they generally begin by increased urinary supersaturation of lithiasis promoters in the urine, followed by nucleation and aggregation. Subsequently, the crystals combine with other crystals in solution to form agglomerates that accumulate in the kidney. Free radical-mediated oxalate-induced renal membrane damage promotes crystal retention at the surface of the renal papilla, as well as crystal nucleation at lower supersaturation levels. In addition, stone type identification is of great interest in guiding the physician to an effective diagnosis, which allows to determine the causes in order to treat the disease and prevent recurrence. In this context, this present study reviews current knowledge on the pathophysiological aspects of kidney stone formation as well as the type of stones.

Role of inwardly rectifying K+ channel 5.1 (Kir5.1) in the regulation of renal membrane transport.

Kir5.1 interacts with Kir4.2 in proximal tubule and with Kir4.1 in distal convoluted tubule (DCT), connecting tubule (CNT) and cortical collecting duct (CCD) to form basolateral-K+-channels. Kir4.2/Kir5.1 and Kir4.1/Kir5.1 play an important role in regulating Na+/HCO3--transport of the proximal tubule and Na+/K+ -transport in the DCT/CNT/CCD. The main focus of this review is to provide an overview of the recent development in the field regarding the role of Kir5.1 regulating renal electrolyte transport in the proximal tubule and DCT. Loss-of-function-mutations of KCNJ16 cause a new form of tubulopathy, characterized by hypokalaemia, Na+-wasting, acid-base-imbalance and metabolic-acidosis. Abnormal bicarbonate transport induced by loss-of-function of KCNJ16-mutants is recapitulated in Kir4.2-knockout-(Kir4.2 KO) mice. Deletion of Kir5.1 also abolishes the effect of dietary Na+ and K+-intakes on the basolateral membrane voltage and NCC expression/activity. Long-term high-salt intake or high-K+-intake causes hyperkalaemic in Kir5.1-deficient mice. Kir4.2/Kir5.1 activity in the proximal tubule plays a key role in regulating Na+, K+ and bicarbonate-transport through regulating electrogenic-Na+-bicarbonate-cotransporter-(NBCe1) and type 3-Na+/H+-exchanger-(NHE3). Kir4.1/Kir5.1 activity of the DCT plays a critical role in mediating the effect of dietary-K+ and Na+-intakes on NCC activity/expression. As NCC determines the Na+ delivery rate to the aldosterone-sensitive distal nephron (ASDN), defective regulation of NCC during high-salt and high-K+ compromises renal K+ excretion and K+ homeostasis.

Lack of interaction of the fluorosurfactant C6O4 with human renal transporters: In vitro/in silico analysis

C6O4 is a water soluble perfluoroether carboxylic acid ammonium salt used as surfactant in the synthesis of fluoropolymers. Available experimental data in rats exposed by the oral route indicate it is eliminated in urine. Previous studies with various linear perfluorocarboxylic acids have suggested that these compounds are substrates of renal membrane transporters in rats and humans, and that the interaction with basal and apical membrane transporters can influence the elimination kinetic by these organisms and explain, in part, the observed differences in the respective half-lives. In particular, apical transporters may contribute to the reuptake of these exogenous compounds from the tubule lumen. The present study was designed to investigate the uptake of C6O4 in two renal cell lines transiently transfected with the human apical membrane transporters, organic anion transporter 4 (OAT4), and urate transporter 1 (URAT1). The uptake of the linear perfluorohexanoic acid (PFC6) was evaluated in parallel. While the uptake of the conjugated steroid estrone-3-sulfate (E3S), a known substrate for renal transporters, and of PFC6 was clearly observed in both cell types transfected with either OAT4 or URAT1, no significant uptake of C6O4 was measured under the same test conditions. The results of the transporter's functionality measured in vitro were consistent with molecular docking simulations. Both outward and inward models of the transporters showed a reduced interaction between C6O4 and URAT1 or OAT4. In contrast, more stable interactions were predicted for PFC6 and PFOA, as well as for the E3S substrate, as shown by the respective docking scores reflecting the binding strength and by the poses assumed in the transporter channels. Altogether, the in vitro and in silico modeling results showed a low reuptake potential and limited interactions of C6O4 molecule with two human apical membrane transporters, contrasting with the more efficient reuptake of PFC6 from the tubule lumen. These results suggest reabsorption from the proximal tubule by apical renal transporters is not likely to interfere with the elimination pathway of C6O4 in humans.

Therapeutic Drug Monitoring of Antifungal Agents in Critically Ill Patients: Is There a Need for Dose Optimisation?

Invasive fungal infections are an important cause of morbidity and mortality, especially in critically ill patients. Increasing resistance rates and inadequate antifungal exposure have been documented in these patients, due to clinically relevant pharmacokinetic (PK) and pharmacodynamic (PD) alterations, leading to treatment failure. Physiological changes such as third spacing (movement of fluid from the intravascular compartment to the interstitial space), hypoalbuminemia, renal failure and hepatic failure, as well as common interventions in the intensive care unit, such as renal replacement therapy and extracorporeal membrane oxygenation, can lead to these PK and PD alterations. Consequently, a therapeutic target concentration that may be useful for one patient may not be appropriate for another. Regular doses do not take into account the important PK variations in the critically ill, and the need to select an effective dose while minimising toxicity advocates for the use of therapeutic drug monitoring (TDM). This review aims to describe the current evidence regarding optimal PK/PD indices associated with the clinical efficacy of the most commonly used antifungal agents in critically ill patients (azoles, echinocandins, lipid complexes of amphotericin B, and flucytosine), provide a comprehensive understanding of the factors affecting the PK of each agent, document the PK parameters of critically ill patients compared to healthy volunteers, and, finally, make recommendations for therapeutic drug monitoring (TDM) of antifungals in critically ill patients.

Hematobiochemical and histopathological alterations in Nile Tilapia (Oreochromis niloticus) exposed to ethidium bromide: The protective role of Spirulinaplatensis

Ethidium bromide (EtBr) is one of the contaminants recorded in aquatic environments whose effects have been investigated; however, there is still limited knowledge about its remediation. This study examined the potential protective effects of Spirulina platensis (SP) against the effects of EtBr toxicity in the Nile tilapia (Oreochromis niloticus) fry. Fry were divided to five groups, viz., a control and four treatment groups of low-dose EtBr (10 μg/L), low-dose EtBr with SP (10 μg/L EtBr + 200 mg/L SP), high-dose EtBr (100 μg/L), and high-dose EtBr with SP (100 μg/L EtBr + 200 mg/L SP); the exposure period was 2 weeks. Low and high doses of EtBr induced alterations in some hematological, biochemical, and histopathological parameters. Necrotic hepatocytes, degenerated area, vacuolated hepatocytes, pyknotic nuclei, constricted and dilated blood sinusoids, and infiltration of inflammatory cells were observed. Lipid peroxidation concentration was not significantly different in groups exposed to low doses of EtBr and EtBr with SP, but it was increased in groups exposed to high doses of EtBr and EtBr with SP, compared with the control group. After feeding with SP, most histological and histochemical parameters restored to normal values. Therefore, SP may possess the ability to preserve the structural integrity of the hepatic and renal membranes.

Phosphatidylserine eversion regulated by phospholipid scramblase activated by TGF-\u03b21/Smad signaling in the early stage of kidney stone formation

The mechanism underlying phosphatidylserine eversion in renal tubule cells following calcium oxalate-mediated damage remains unclear; therefore, we investigated the effects of TGF-β1/Smad signaling on phosphatidylserine eversion in the renal tubule cell membrane during the early stage of kidney stone development. In a rat model of early stage of calcium oxalate stone formation, phosphatidylserine eversion on the renal tubular cell membrane was detected by flow cytometry, and the expression of TGF-β1 (transforming growth factor-β1), Smad7, and phospholipid scramblase in the renal tubular cell membrane was measured by western blotting. We observed that the TGF-β1/Smad signaling pathway increased phosphatidylserine eversion at the organism level. The results of in vitro studies demonstrated that oxalate exposure to renal tubule cells induced TGF-β1 expression, increasing phospholipid scramblase activity and phosphatidylserine eversion in the renal tubule cell membrane. These results indicate that TGF-β1 stimulates phosphatidylserine eversion by increasing the phospholipid scramblase activity in the renal tubule cell membrane during the early stage of kidney stone development. The results of this study form a basis for further detailed research on the development of therapeutic agents that specifically treat urolithiasis and exert fewer adverse effects.

Renal Parenchymal Hypoxia in Young Children in the Period of Complete Remission of Acute Uncomplicated Pyelonephritis without Renal Impairment

Introduction. After establishing the diagnosis of pyelonephritis, there is a need to determine, whether renal parenchymal hypoxia remains in the period of remission. The least studied methods up to now are functional ones, which allow to verify the presence and degree of tissue hypoxia in secondary pyelonephritis and to evaluate the results of the medical measures to restore kidney function. Objective: to clarify the presence of renal parenchymal hypoxia and membrane destruction of the renal epithelium in young children with pyelonephritis without anatomic congenital malformations of the urinary system during its remission.Materials and methods of the study. The study involved 65 children with acute uncomplicated pyelonephritis, who after nephro-urological examination did not reveal anatomical malformations of the urinary system. General clinical examination was carried out, markers of tissue hypoxia and morphofunctional state of cell membranes of renal epithelium were determined.Discussion of the study results. The ability of urine to prevent crystal formation was significantly reduced to oxalates and phosphates in 40.0 and 47.7 % of the surveyed children. In one-third of children with pyelonephritis, oxalate excretion was significantly higher than in healthy children, hypophosphaturia was detected in 52.3 % of cases and the tendency to reduce the excretion of urates in 10.8 % of pediatric patients with pyelonephritis. The content of lipid peroxidation products in urine was increased in 41.5 % of the surveyed children with pyelonephritis, and appearance of polar lipids was observed in 46.2 % of patients.Conclusions. To predict the formation and for the purpose of early diagnosis of renal parenchymal hypoxia and the processes of nephrothelial membrane destruction in young children with pyelonephritis, it is recommended to use such markers, as indicators of urine ability to prevent crystal formation, daily excretion of salts, excretion of lipid peroxidation products and polar lipids in the urine. It is recommended to apply the methods to correct these changes.

Macroscopic, Histomorphological and Histochemical Studies on the Kidneys of Guinea Fowl (Numida meleagris)

Background: Kidneys, the paired organ which essentially plays for excreting nitrogenous wastes, excessive water, inorganic salts and toxic substances produced during the process of body metabolism. Maintenance of osmotic regulation and homeostatic fluid balance of the body has also be performed by kidneys. Each of the kidneys filtrate carried to cloaca by respective ureter from where the urine leaves the body. The present macroscopic, histomorphological and histochemical studies undertaken on the Kidneys of Guinea fowl (Numida meleagris) is nothing but the attempt to widen our thrust area for that bird which is nonexistent as on documentation.Methods: The present studies were carried upon 12 pairs of in-vitro kidney samples in Guinea fowl. Gross morphology and morphometrical observations were carried on fresh specimens. Histological studies were carried on fixed samples those were processed for dehydration, clearing, embedding into paraffin and sections of 5-6 μ thickness were obtained. Different staining procedure as to Haematoxylin and Eosin, Silver orcein, Aniline blue and Weigert’s staining methods were employed for different histological components. The histochemical and histoenzymic studies were also accomplished with Periodic acid Schiff‘s (PAS), Alcian blue methods (pH-2.5) and Gomori’s cobalt method for Alkaline phosphatase and Acid phosphatase. The Succinic dehydrogenase enzyme was also estimated on cryostat sections.Result: The observations on kidneys of Guinea fowl were accomplished after macroscopic, histomorphological and histochemical means. This reddish-brown colored paired organ of kidneys was retroperitoneally placed in synsacral fossa and had incomplete division of three parts. The wrapping material of capsule was the dense connective tissue. The outer cortex and inner medulla had different parts of nephrons. Two types of nephrons viz. mammalian type (with Henle loop) and reptilian type (without Henle loop) were observed. Collagen, elastic and reticular fibers were there in different components of the kidney. Histochemically it has determined the secretion of neutral as well as acidic mucopolysaccharide substances. PAS activity was observed at mesangial cells and basement membrane of DCT. The Alkaline phosphatase showed the positive activity in capsule, brush border of proximal and distal convoluted tubules. The Acid phosphatases were showing intense activity in parietal and visceral layer of renal corpuscles, lumen and basement membrane of PCT. The intense activity of Succinic dehydrogenase showed at PCT, DCT, collecting tubule whereas renal corpuscles showed feeble activity.

NBCe2 (Slc4a5) Is Expressed in the Renal Connecting Tubules and Cortical Collecting Ducts and Mediates Base Extrusion.

Arterial hypertension, is a common disorder with multiple and variable etiologies. Single nucleotide polymorphism analyses have detected an association between variants in the gene encoding the electrogenic Na+:HCO3– cotransporter NBCe2 (Slc4a5), and salt-sensitive hypertension. Mice with genetic deletion of NBCe2 are hypertensive, and the cause of the blood pressure (BP) increase is believed to arise from a lack of renal NBCe2 function. The exact cellular expression of NBCe2 in the kidney tubular system is, however, not determined. Here, we find NBCe2 to be expressed predominantly in isolated connecting tubules (CNT) and cortical collecting ducts (CD) by RT-PCR. In isolated renal CNT and CCD, genetic deletion of NBCe2 leads to decreased net base extrusion. To determine the role of renal NBCe2 in the development of hypertension, we generated CNT and intercalated cell NBCe2 knockout mice by crossing an Slc4a5 lox mouse with mice expressing cre recombinase under the V-ATPase B1 subunit promotor. Although the mice displayed changes in the expression of renal membrane transporters, we did not detect hypertension in these mice by tail cuff recordings. In conclusion, while global NBCe2 deletion certainly causes hypertension this study cannot confirm the role of renal NBCe2 expression in blood pressure regulation.

Distribution of the organic anion transporters Oat1 and Oat3 between renal membrane microdomains in obstructive jaundice.

Relation between the renal function and the membrane environment where the organic anion transporters Oat1 and Oat3 are localized is scarce. The aim of this study was to examine the Oat1 and Oat3 distribution in different cellular fractions under physiological conditions as well as the effects of extrahepatic cholestasis on membrane distribution of both proteins. Besides, the potential role of jaundice serum on the Oat1 and Oat3 expression in suspensions of renal tubular cells was evaluated. Cellular and membrane fractions of renal cortex were obtained from control rats to evaluate Oat1 and Oat3 protein expressions. Other rats were subjected to bile duct ligation (BDL) or Sham operation to determine the membrane distribution of Oat1 and Oat3 between lipid raft domains (LRD) and non-LRD. Incubation of renal cortical cells with serum from Sham and BDL were also performed to study Oat1 and Oat3 protein expressions. In physiological conditions, Oat1 and Oat3 were concentrated in LRD. The pathology induced a shift of Oat1 from LRD to non-LRD, while Oat3 showed no changes in its distribution. In cells exposed to BDL serum, Oat1 protein expression in membranes significantly increased. For Oat3, no difference between groups was observed. The Oat1 redistribution to non-LRD in BDL could be favoring the increase in renal transport of organic anions previously observed. This change was specific to Oat1. The in vitro experiment allows to conclude that some component present in BDL serum is responsible for the alterations observed in Oat1 expression in cortical membranes.

Erythrocytic membrane anionic charge, sialic acid content, and their correlations with urinary glycosaminoglycans in preeclampsia and eclampsia

Compared to healthy pregnant women, changes in erythrocytic membrane anionic charge (EAC) and urinary glycosaminoglycans (UGAGS) have been reported in African women with preeclampsia. A single previous study showed a decrease in erythrocytic membrane sialic acid (EMSA) in preeclampsia compared to healthy pregnancy; however, EMSA was not significantly different between women with preeclampsia and non-pregnant women. No study has focused on the relationships between EAC, EMSA, and UGAGS in preeclampsia and eclampsia compared to healthy pregnant and non-pregnant women of reproductive age. Moreover, the erythrocyte membrane contains sialoglycoproteins and proteoglycans involved in creating the negatively charged cell surface, disruption of which leads to erythrocyte aggregation seen in preeclampsia/eclampsia. However, the etiopathogenesis of preeclampsia and eclampsia remains unclear. Therefore, we evaluated the relationship between EAC, UGAGS, and EMSA in preeclampsia and eclampsia. Three groups of 30 women each were enrolled: Group A (non-pregnant women), Group B (healthy pregnant women without complications), and Group C (women with preeclampsia/eclampsia). EMSA was diminished under oxidative stress prevalent in eclampsia and preeclampsia which might have caused a decreased EAC. EAC was negatively correlated with UGAGS and positively correlated with EMSA (p < .001). EMSA was negatively correlated with UGAGS (p < .001). In conclusion, a loss of sialic acid from the erythrocyte membrane causes a significant decrease in the EAC which mirrors the decrease in the negative charge of the renal glomerular basement membrane and might lead to proteinuria and increased UGAGS excretion in preeclampsia and eclampsia.

Comparing Lipid and Detergent Environments as Models for Studying Renal Transmembrane Proteins

BackgroundMutations in transmembrane proteins cause kidney diseases such as Dent’s disease, Bartter syndrome, and polycystic kidney disease. 1 Understanding the structure and function of renal membrane proteins reveals how they contribute to kidney function as well as kidney disease. Recent studies have shown the importance of generating a native lipid environment when studying membrane proteins. 2 However, membrane proteins are often studied in the presence of detergent, which may modify their natural structure and lead to decreased function. Nanodiscs, a novel lipid bilayer system, provide a more native environment for protein reconstitution.MethodsIn this study we used two proteins found in the kidney, the Na+/K+‐ATPase pump and CFTR ion channel, to determine whether nanodiscs or detergent micelles serve as the better model for studying renal transmembrane proteins. To do this, we expressed and purified our own Membrane Scaffold Protein (MSP), a base protein for nanodiscs, in Escherichia coli. We generated empty nanodiscs with three different lipid types: 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC), 1‐palmitoyl‐2‐oleoyl‐glycero‐3‐phosphocholine (POPC), and 1‐Palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphoethanolamine (POPE).ResultsWe have and are continuing to reconstitute CFTR and Na+/K+‐ATPase into nanodiscs with different lipid compositions. We evaluate membrane protein stability using ATPase activity in detergent and different lipid nanodisc environments. Preliminary data show improved ATPase activity for CFTR in nanodiscs versus detergent. Higher ATPase activity of proteins reconstituted in nanodiscs would indicate that detergent structurally modifies these proteins and consequently decreases their function. Nanodiscs provide a more stable environment in which to study these and other proteins found in the kidneys, allowing for more accurate assessment of structure and function.

Effect of moonseed vine (Triclisia gilletii Staner) on ethane-1,2-diol-induced urolithiasis and its renotoxicity in Wistar albino rats

BackgroundMoonseed vine (Triclisia gilletii Staner) in the family Menispermaceae is a robust creeper of up to 10 cm diameter, of the lowland dense rain forest. In Ondo State, located in the South Western part of Nigeria, the plant which is usually called Peshe is used for the management of renal-related ailments. The present study was undertaken to explore the efficacy of Triclisia gilletii, a folkloric therapy in the management of renal-related ailment.ResultsPhenols, steroids, saponins, and flavonoids are present in the TGME with a total antioxidant capacity of (30.36 ± 1.90 (mg GAE/g extract), LD50 greater than 5000 mg/kg b.w., and in vitro anti-nucleation activity (iC50 = 7.09 mg/mL). Calcium oxalate stone formation as a result of oxalate from ethane-1,2-diol was evident by hypocalcemia, and further electrolyte imbalance and decreased glomerular filtration rate. The enhanced oxidative milieu in hyperoxaluria was evident by increased MDA and PC and decreased enzymatic and non-enzymatic antioxidants as well as renal membrane enzymes activities. The renal histopathological study further emphasized oxalate-induced damage and the ameliorative potential of TGME.ConclusionThe abnormal biochemical, redox electrolyte, membrane integrity, and histological alterations were attenuated by TGME which affirms its usage as nephroprotectant.

Osmotic diuresis by SGLT2 inhibition stimulates vasopressin-induced water reabsorption to maintain body fluid volume.

Most of the filtered glucose is reabsorbed in the early proximal tubule by the sodium‐glucose cotransporter SGLT2. The glycosuric effect of the SGLT2 inhibitor ipragliflozin is linked to a diuretic and natriuretic effect that activates compensatory increases in fluid and food intake to stabilize body fluid volume (BFV). However, the compensatory mechanisms that are activated on the level of renal tubules remain unclear. Type 2 diabetic Goto‐Kakizaki (GK) rats were treated with vehicle or 0.01% (in diet) ipragliflozin with free access to fluid and food. After 8 weeks, GK rats were placed in metabolic cages for 24‐hr. Ipragliflozin decreased body weight, serum glucose and systolic blood pressure, and increased fluid and food intake, urinary glucose and Na+ excretion, urine volume, and renal osmolar clearance, as well as urine vasopressin and solute‐free water reabsorption (TcH2O). BFV, measured by bioimpedance spectroscopy, and fluid balance were similar among the two groups. Urine vasopressin in ipragliflozin‐treated rats was negatively and positively associated with fluid balance and TcH2O, respectively. Ipragliflozin increased the renal membrane protein expression of SGLT2, aquaporin (AQP) 2 phosphorylated at Ser269 and vasopressin V2 receptor. The expression of SGLT1, GLUT2, AQP1, and AQP2 was similar between the groups. In conclusion, the SGLT2 inhibitor ipragliflozin induced a sustained glucosuria, diuresis, and natriuresis, with compensatory increases in fluid intake and vasopressin‐induced TcH2O in proportion to the reduced fluid balance to maintain BFV. These results indicate that the osmotic diuresis induced by SGLT2 inhibition stimulates compensatory fluid intake and renal water reabsorption to maintain BFV.

A Comprehensive Analysis of Ontogeny of Renal Drug Transporters: mRNA Analyses, Quantitative Proteomics, and Localization.

Human renal membrane transporters play key roles in the disposition of renally cleared drugs and endogenous substrates, but their ontogeny is largely unknown. Using 184 human postmortem frozen renal cortical tissues (preterm newborns to adults) and a subset of 62 tissue samples, we measured the mRNA levels of 11 renal transporters and the transcription factor pregnane X receptor (PXR) with quantitative real‐time polymerase chain reaction, and protein abundance of nine transporters using liquid chromatography tandem mass spectrometry selective reaction monitoring, respectively. Expression levels of p‐glycoprotein, urate transporter 1, organic anion transporter 1, organic anion transporter 3, and organic cation transporter 2 increased with age. Protein levels of multidrug and toxin extrusion transporter 2‐K and breast cancer resistance protein showed no difference from newborns to adults, despite age‐related changes in mRNA expression. Multidrug and toxin extrusion transporter 1, glucose transporter 2, multidrug resistance‐associated protein 2, multidrug resistance‐associated protein 4 (MRP4), and PXR expression levels were stable. Using immunohistochemistry, we found that MRP4 localization in pediatric samples was similar to that in adult samples. Collectively, our study revealed that renal drug transporters exhibited different rates and patterns of maturation, suggesting that renal handling of substrates may change with age.

Abstract 033: Identification of s Primary Renal AT 2 Receptor Defect in Spontaneously Hypertensive Rats (SHR)

Angiotensin III (Ang III) [des-aspartyl 1 -Ang II] is the predominant endogenous agonist for angiotensin type-2 receptor (AT 2 R)-induced natriuresis in rats. Hypertensive 12 wk-old SHR lack natriuretic responses to Ang III, and renal interstitial (RI) Ang III administration induces natriuresis in 4 wk-old Wistar-Kyoto rats (WKY), but not in 4 wk-old pre-hypertensive SHR. The defect in natriuresis could be explained by the concurrent observation that in WKY, but not SHR, Ang III induced AT 2 R translocation to renal proximal tubule apical plasma membranes and internalization/inactivation of Na + -transporters Na + - H + exchanger-3 (NHE-3) and Na + /K + ATPase (NKA). These findings suggest an Ang III/AT 2 R signaling defect in pre-hypertensive SHR. The present study sought to determine the cause of the defect: (1) accelerated intrarenal Ang III metabolism or (2) a primary AT 2 R (or post-receptor) signaling defect. Selective AT 2 R agonist Compound-21 (C-21) was infused interstitially [20, 40 and 60 ng/kg/min, each dose for 30 min] into experimental (left) kidneys of 4 wk-old WKY and SHR (N=7) pretreated for 24h with systemic infusion of Ang type-1 receptor (AT 1 R) antagonist candesartan (0.01 mg/kg/min by osmotic minipump); vehicle was infused into control (right) kidneys. Pre-infusion urinary Na + excretion (U Na V) was 0.022 ± 0.004 μmol/min in WKY and 0.024 ± 0.005 μmol/min in SHR (P=NS). In WKY, C-21 dose-dependently increased U Na V from 0.022 ± 0.004 to 0.058 ± 0.017, 0.078 ± 0.014 and 0.079 ± 0.079 μmol/min (P=0.01 from pre-infusion and time control kidneys). In contrast, SHR did not increase U Na V in response to C-21 [highest U Na V 0.033 ± 0.007 μmol/min (P=NS from pre-infusion and time control kidneys)]. Mean arterial pressure (MAP) was 64.6 ± 1.80 mmHg in WKY and 76.4 ± 2.2 mmHg (P&lt;0.01) in SHR and did not change with C-21 or vehicle in WKY and SHR. In a separate set of 4 wk-old WKY and SHR (N=8), RI Ang II and III concentrations were measured by HPLC and ELISA assay. RI Ang II and Ang III levels, respectively, in WKY were 55 ± 20 and 59 ± 17 pg/ml (P=NS) and in SHR were 37 ± 7 and 181± 44 pg/ml (P=0.01 vs SHR Ang II; P&lt;0.025 vs WKY Ang III). These findings exclude accelerated Ang III metabolism as a cause and identify a primary renal AT 2 R defect as responsible for increased Na + reabsorption in SHR.

What a Clinician Should Know About a Renal Replacement Membrane?

Conflicts of Interest Conflict of Interests: The authors declare to have no competing interests

Effects of high fat diet on kidney lipid content and the Na,K-ATPase activity

It is widely known that high fat diet (HFD) can contribute to the advent of health problems. Recent studies have indicated that obesity imposes a hemodynamic overload to the kidneys. In order to further investigate such injuries, two groups of six Swiss mice each were fed with a controlled AIN93G diet or a high fat (AIN93G modified) diet for eight weeks. Blood samples were collected to determine the hormonal, lipid profile, glucose, urea, and creatinine levels. Histopathological and immunohistochemical analysis were carried out to analysis the kidney damage. Fractions of renal membranes were prepared to assess the Na,K-ATPase activity, lipid peroxidation, total cholesterol, and phospholipid content. The results indicated that the blood lipid profile, urea and creatinine was not altered by the HFD. On the other hand, it was observed in HFD diet mice elevated glucose blood levels along with an augment on insulin and a decrease on corticosterone release. HFD provoked a reduction in the diameter of the convoluted tubules and cell volume in Bowman’s capsule and an increased number of positive cells with Na,K-ATPase, but reduced the Na,K-ATPase activity and the cholesterol content in the kidney cell membrane but favored the lipid peroxidation.