Kidney Lysates Research Articles

IgT and IgD from sea bass (Dicentrarchus labrax): Localization of expressing and immunoreactive cells in lymphoid tissues

To investigate mucosal immunity, from a whole transcriptome of sea bass gills we identified and successively cloned complete cDNA sequences of IgT and IgD heavy chains. These sequences have been employed to prepare riboprobes for ISH, and to identify conserved regions of sea bass IgT/IgD in order to select sequences from which synthetic peptides have been prepared. The peptides have been used as immunogens in rabbits to obtain antisera against IgT (RaIgT1) and IgD (RaIgD1) and by employing either riboprobes and antisera, the pattern of IgT/IgD expressing tissues and the presence of IgT/IgD immunoreactive cells has been investigated. The results by qPCR showed that the IgT gene is mainly expressed in muscle, intestine and gills, and the IgD gene in head kidney. By ISH, the presence of IgT-expressing cells has been detected in the intestine, with their number increasing toward the posterior segment, and in the gills. These data have been confirmed by IHC employing the rabbit antiserum RaIgT1, and extended by revealing the presence of IgTimmunoreactive cells in head kidney and spleen, as well as intense reactivity in intestinal mucus. As expected, in mucosal tissues IgT-immunoreactive cells outnumbered IgM-immunoreactive cells. By IIF and flow cytometry with RaIgT1 we determined the number of IgT-positive cells in organs and tissues, being around 32 ± 12 % in head kidney leukocytes, 14 ± 6 % in GIALT and 23 ± 10 % in spleen leukocytes. At the microscope the IIF-positive cells had the morphology of lymphocytes. By western blotting of head kidney lysates, RaIgT1 stained a 180 kDa band in non-reducing gels and a 74 kDa band in reducing gels and, in addition, RaIgT1 showed cross-reaction with partially purified rainbow trout IgT. Leukocytes from the head kidney were enriched in RaIgT1-positive cells by magnetic sorting, and PCR analysis showed an enrichment of IgT and depletion of IgM expression, respectively, in RaIgT1-enriched cells. These data suggest we obtained an antibody against IgT cells of sea bass. Preliminary data employing the RaIgD1 showed by IHC the presence of immunoreactive cells in gills, spleen, head kidney, intestine, and work is in progress to highlights the characteristics of sea bass IgD expressing cells. Acknowledgements. The work was financially supported by EU-6FP project Targetfish, n°311993.

A Glyoxalase-1 Knockdown Does Not Have Major Short Term Effects on Energy Expenditure and Atherosclerosis in Mice

Objective. Glyoxalase-1 is an enzyme detoxifying methylglyoxal (MG). MG is a potent precursor of advanced glycation endproducts which are regarded to be a key player in micro- and macrovascular damage. Yet, the role of Glo1 in atherosclerosis remains unclear. In this study, the effect of Glo1 on mouse metabolism and atherosclerosis is evaluated. Methods. Glo1 knockdown mice were fed a high fat or a standard diet for 10 weeks. Body weight and composition were investigated by Echo MRI. The PhenoMaster system was used to measure the energy expenditure. To evaluate the impact of Glo1 on atherosclerosis, Glo1KD mice were crossed with ApoE-knockout mice and fed a high fat diet for 14 weeks. Results. Glo1 activity was significantly reduced in heart, liver, and kidney lysates derived from Glo1KD mice. Yet, there was no increase in methylglyoxal-derived AGEs in all organs analyzed. The Glo1 knockdown did not affect body weight or body composition. Metabolic studies via indirect calorimetry did not show significant effects on energy expenditure. Glo1KD mice crossed to ApoE−/− mice did not show enhanced formation of atherosclerosis. Conclusion. A Glo1 knockdown does not have major short term effects on the energy expenditure or the formation of atherosclerotic plaques.

Rab-GAP TBC1D4 (AS160) is dispensable for the renal control of sodium and water homeostasis but regulates GLUT4 in mouse kidney.

The Rab GTPase-activating protein TBC1D4 (AS160) controls trafficking of the glucose transporter GLUT4 in adipocytes and skeletal muscle cells. TBC1D4 is also highly abundant in the renal distal tubule, although its role in this tubule is so far unknown. In vitro studies suggest that it is involved in the regulation of renal transporters and channels such as the epithelial sodium channel (ENaC), aquaporin-2 (AQP2), and the Na+-K+-ATPase. To assess the physiological role of TBC1D4 in the kidney, wild-type (TBC1D4+/+) and TBC1D4-deficient (TBC1D4-/-) mice were studied. Unexpectedly, neither under standard nor under challenging conditions (low Na+/high K+, water restriction) did TBC1D4-/- mice show any difference in urinary Na+ and K+ excretion, urine osmolarity, plasma ion and aldosterone levels, and blood pressure compared with TBC1D4+/+ mice. Also, immunoblotting did not reveal any change in the abundance of major renal sodium- and water-transporting proteins [Na-K-2Cl cotransporter (NKCC2) NKCC2, NaCl cotransporter (NCC), ENaC, AQP2, and the Na+-K+-ATPase]. However, the abundance of GLUT4, which colocalizes with TBC1D4 along the distal nephron of TBC1D4+/+ mice, was lower in whole kidney lysates of TBC1D4-/- mice than in TBC1D4+/+ mice. Likewise, primary thick ascending limb (TAL) cells isolated from TBC1D4-/- mice showed an increased basal glucose uptake and an abrogated insulin response compared with TAL cells from TBC1D4+/+ mice. Thus, TBC1D4 is dispensable for the regulation of renal Na+ and water transport, but may play a role for GLUT4-mediated basolateral glucose uptake in distal tubules. The latter may contribute to the known anaerobic glycolytic capacity of distal tubules during renal ischemia.

Abstract 162: Detection of N-alpha-acetyltransferase Activity Towards Endogenous Inhibitor of Nitric Oxide Synthase Asymmetric Dimethylarginine in the Liver and Kidneys

Background: Endogenous inhibitor of nitric oxide synthase asymmetric dimethylarginine (ADMA) has been proposed as a risk factor and mediator of cardiovascular diseases. ADMA can undergo hydrolysis by dimethylarginine dimethylaminohydrolase or be processed through an alternative pathway by alanine:glyoxylate aminotransferase 2. We and other have also shown that ADMA can be N-acetylated to form asymmetrical Nα-acetyldimethylarginine (Ac-ADMA). The goal of the study was to characterize this novel pathway in vivo and identify the organs, which are responsible for the ADMA-acetylating activity. Results: We infused ADMA in mice for three days. Control mice received saline infusion. Half of the mice underwent bilateral nephrectomy 24 hours before completion of the infusion, the rest had a sham surgery. ADMA infusion resulted in a 3.5 fold rise in plasma Ac-ADMA levels in the sham-operated mice (1.35 ± 0.45 nmol/L vs. 4.77 ± 0.88 nmol/L). In nephrectomized mice plasma Ac-ADMA levels were markedly increased even after saline infusion (45.08 ± 9.25 nmol/L) and further raised 5-fold after ADMA infusion (229.65 ± 91.30 nmol/L). We assessed tissue distribution of Ac-ADMA in mice and found the highest levels in pancreas, small intestine, liver and kidney. We incubated the lysates of those organs with ADMA for 24 hours and estimated production of Ac-ADMA. Only liver and kidney lysates showed significant increase in Ac-ADMA levels upon addition of ADMA (from 0.03 ± 0.01 nmol/μg protein to 0.18 ± 0.06 nmol/μg protein and from 0.06 ± 0.01 nmol/μg protein to 0.11 ± 0.02 nmol/μg protein). Conclusions: We showed that overload of ADMA leads to increase in plasma Ac-ADMA levels. This observation is consistent with the hypothesis that Ac-ADMA is formed from ADMA in vivo. The striking rise in plasma Ac-ADMA concentrations after bilateral nephrectomy suggests that Ac-ADMA is predominantly eliminated via the kidneys. We demonstrated that liver and kidney are the organs, in which acetylation of ADMA takes place. The high levels of Ac-ADMA in pancreas and intestine may be due to accumulation of Ac-ADMA in those organs. In the next step we want to identify the enzyme responsible for acetylation of ADMA and explore the clinical implications of this novel pathway of ADMA metabolism.

Natural killer T (NKT) cells accelerate Shiga toxin type 2 (Stx2) pathology in mice.

Shiga toxin-producing Escherichia coli (STEC) is a leading cause of childhood renal disease Hemolytic Uremic Syndrome (HUS). The involvement of renal cytokines and chemokines is suspected to play a critical role in disease progression. In current article, we tested the hypothesis that NKT cells are involved in Stx2-induced pathology in vivo. To address this hypothesis we compared Stx2 toxicity in WT and CD1 knockout (KO) mice. In CD1KO mice, which lack natural killer T (NKT) cells, Stx2-induced pathologies such as weight loss, renal failure, and death were delayed. In WT mice, Stx2-specific selective increase in urinary albumin occurs in later time points, and this was also delayed in NKT cell deficient mice. NKT cell-associated cytokines such as IL-2, IL-4, IFN-γ, and IL-17 were detected in kidney lysates of Stx2-injected WT mice with the peak around 36 h after Stx2 injection. In CD1KO, there was a delay in the kinetics, and increases in these cytokines were observed 60 h post Stx2 injection. These data suggest that NKT cells accelerate Stx2-induced pathology in mouse kidneys. To determine the mechanism by which NKT cells promote Stx2-associated disease, in vitro studies were performed using murine renal cells. We found that murine glomerular endothelial cells and podocytes express functional CD1d molecules and can present exogenous antigen to NKT cells. Moreover, we observed the direct interaction between Stx2 and the receptor Gb3 on the surface of mouse renal cells by 3D STORM-TIRF which provides single molecule imaging. Collectively, these data suggest that Stx2 binds to Gb3 on renal cells and leads to aberrant CD1d-mediated NKT cell activation. Therefore, strategies targeting NKT cells could have a significant impact on Stx2-associated renal pathology in STEC disease.

Fasting upregulates adipose triglyceride lipase and hormone-sensitive lipase levels and phosphorylation in mouse kidney.

Circulating non-esterified fatty acids (NEFA) rise during fasting and are taken up by the kidneys, either directly from the plasma or during re-uptake of albumin from glomerular filtrate, and are stored as triacylglycerol (TAG). Subsequent utilization of stored fatty acids requires their hydrolytic release from cellular lipid droplets, but relatively little is known about renal lipolysis. We found that total [(3)H]triolein hydrolase activity of kidney lysates was significantly increased by 15% in the fasted state. Adipose triglyceride lipase (Atgl) and hormone-sensitive lipase (Hsl) mRNA expression was time-dependently increased by fasting, along with other fatty acid metabolism genes (Pparα, Cd36, and Aox). ATGL and HSL protein levels were also significantly induced (by 239 ± 7% and 322 ± 8%, respectively). Concomitant with changes in total protein levels, there was an increase in ATGL phosphorylation at the AMPK-regulated serine 406 site in the 14-3-3 binding motif, and an increase in HSL phosphorylation at serines 565 and 660 that are regulated by AMPK and PKA, respectively. Using immunofluorescence, we further demonstrate nearly ubiquitous expression of ATGL in the renal cortex with a concentration on the apical/lumenal surface of some cortical tubules. Our findings suggest a role for ATGL and HSL in kidney lipolysis.

Renal effects of chronic pharmacological manipulation of CB2 receptors in rats with diet-induced obesity.

In diabetic nephropathy agonism of CB2 receptors reduces albuminuria and podocyte loss; however, the role of CB2 receptors in obesity-related nephropathy is unknown. The aim of this study was to determine the role of CB2 receptors in a model of diet-induced obesity (DIO) and characterize the hallmark signs of renal damage in response to agonism (AM1241) and antagonism (AM630) of CB2 receptors. Male Sprague Dawley rats were fed a high-fat diet (HFD: 40% digestible energy from lipids) for 10 weeks. In another cohort, after 9 weeks on a HFD, rats were injected daily with either 3 mg·kg(-1) AM1241, 0.3 mg·kg(-1) AM630 or saline for 6 weeks. Ten weeks on a HFD significantly reduced renal expression of CB2 receptors and renal function. Treatment with AM1241 or AM630 did not reduce weight gain or food consumption in DIO. Despite this, AM1241 significantly reduced systolic BP, peri-renal adipose accumulation, plasma leptin, urinary protein, urinary albumin, urinary sodium excretion and the fibrotic markers TGF-β1, collagen IV and VEGF in kidney lysate. Treatment with AM630 of DIO rats significantly reduced creatinine clearance and increased glomerular area and kidney weight (gross and standardized for body weight). Diastolic BP, glucose tolerance, insulin sensitivity, plasma creatinine, plasma TGF-β1 and kidney expression of fibronectin and α-smooth muscle actin were not altered by either AM1241 or AM630 in DIO. This study demonstrates that while agonism of CB2 receptors with AM1241 treatment for 6 weeks does not reduce weight gain in obese rats, it leads to improvements in obesity-related renal dysfunction. This article is part of a themed section on Endocannabinoids. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.7/issuetoc.

Silencing of MiRNA-126 in Kidney Ischemia Reperfusion is Associated with Elevated SDF-1 Levels and Mobilization of Sca-1+/Lin- Progenitor Cells

Integrity of the capillary network in the kidney is essential in the recovery from ischemia/ reperfusion injury (IRI), a phenomenon central to kidney transplantation and acute kidney injury. MicroRNA- 126 (miR-126) is known to be important in maintaining vascular homeostasis by facilitating vascular regeneration and modulating the mobilization of vascular progenitor cells. Stromal cell-derived factor 1 (SDF-1), important in the mobilization of vascular progenitor cells, is a direct target of miR-126 and modulation of miR-126 was previously shown to affect the number of circulating Sca-1(+)/Lin(-) vascular progenitor cells in a mouse model for hind limb ischemia. Here, we assessed the in vivo contribution of miR-126 to progenitor cell mobilization and kidney function following IRI in mice. A three day follow up of blood urea levels following kidney IRI demonstrated that systemic antagomir silencing of miR-126 did not impact the loss or subsequent restoration of kidney function. However, whole kidney lysates displayed elevated gene expression levels of Sdf-1, Vegf-A and eNOS after IRI as a result of systemic silencing of miR-126. Furthermore, FACS-analysis on whole blood three days after surgery revealed a marked up regulation of the number of circulating Sca-1(+)/Lin(-) progenitor cells in the antagomir-126 treated mice, in an ischemia dependent manner. Our data indicate that silencing of miR-126 can enhance renal expression of Sdf-1 after IRI, leading to the mobilization of vascular progenitor cells into the circulation.

Chloride channel ClC-5 binds to aspartyl aminopeptidase to regulate renal albumin endocytosis

ClC-5 is a chloride/proton exchanger that plays an obligate role in albumin uptake by the renal proximal tubule. ClC-5 forms an endocytic complex with the albumin receptor megalin/cubilin. We have identified a novel ClC-5 binding partner, cytosolic aspartyl aminopeptidase (DNPEP; EC 3.4.11.21), that catalyzes the release of N-terminal aspartate/glutamate residues. The physiological role of DNPEP remains largely unresolved. Mass spectrometric analysis of proteins binding to the glutathione-S-transferase (GST)-ClC-5 C terminus identified DNPEP as an interacting partner. Coimmunoprecipitation confirmed that DNPEP and ClC-5 also associated in cells. Further experiments using purified GST-ClC-5 and His-DNPEP proteins demonstrated that the two proteins bound directly to each other. In opossum kidney (OK) cells, confocal immunofluorescence studies revealed that DNPEP colocalized with albumin-containing endocytic vesicles. Overexpression of wild-type DNPEP increased cell-surface levels of ClC-5 and albumin uptake. Analysis of DNPEP-immunoprecipitated products from rat kidney lysate identified β-actin and tubulin, suggesting a role for DNPEP in cytoskeletal maintenance. A DNase I inhibition assay showed a significant decrease in the amount of G actin when DNPEP was overexpressed in OK cells, suggesting a role for DNPEP in stabilizing the cytoskeleton. DNPEP was not present in the urine of healthy rats; however, it was readily detected in the urine in rat models of mild and heavy proteinuria (diabetic nephropathy and anti-glomerular basement membrane disease, respectively). Urinary levels of DNPEP were found to correlate with the severity of proteinuria. Therefore, we have identified another key molecular component of the albumin endocytic machinery in the renal proximal tubule and describe a new role for DNPEP in stabilizing the actin cytoskeleton.

Hyperoxia-Induced Protein Alterations in Renal Rat Tissue: A Quantitative Proteomic Approach to Identify Hyperoxia-Induced Effects in Cellular Signaling Pathways.

Introduction. In renal tissue as well as in other organs, supranormal oxygen pressure may lead to deleterious consequences on a cellular level. Additionally, hyperoxia-induced effect in cells and related free radicals may potentially contribute to renal failure. The aim of this study was to analyze time-dependent alterations of rat kidney protein expression after short-term normobaric hyperoxia using proteomics and bioinformatic approaches. Material and Methods. N = 36 Wistar rats were randomized into six different groups: three groups with normobaric hyperoxia (exposure to 100% oxygen for 3 h) and three groups with normobaric normoxia (NN; room air). After hyperoxia exposure, kidneys were removed immediately, after 3 days and after 7 days. Kidney lysates were analyzed by two-dimensional gel electrophoresis followed by peptide mass fingerprinting using tandem mass spectrometry. Statistical analysis was performed with DeCyder 2D software (p < 0.01). Biological functions of differential regulated proteins were studied using functional network analysis (Ingenuity Pathways Analysis and PathwayStudio). Results. Expression of 14 proteins was significantly altered (p < 0.01): eight proteins (MEP1A_RAT, RSSA_RAT, F16P1_RAT, STML2_RAT, BPNT1_RAT, LGMN_RAT, ATPA_RAT, and VDAC1_RAT) were downregulated and six proteins (MTUS1_RAT, F16P1_RAT, ACTG_RAT, ACTB_RAT, 2ABA_RAT, and RAB1A_RAT) were upregulated. Bioinformatic analyses revealed an association of regulated proteins with inflammation. Conclusions. Significant alterations in renal protein expression could be demonstrated for up to 7 days even after short-term hyperoxia. The identified proteins indicate an association with inflammation signaling cascades. MEP1A and VDAC1 could be promising candidates to identify hyperoxic injury in kidney cells.

Development and characterization of monoclonal antibodies to detect klotho.

Although antibodies are commercially available to allow investigation into the biology of the age-regulating protein Klotho, problems with antibody specificity and application functionality are significant barriers to progress. Chief among these limitations is the inability of current tools to allow in vivo validation of binding partners originally identified through transfection of tagged proteins. To overcome this barrier, we generated a series of hybridoma cell lines by immunizing rats with a GST-KL1 fusion protein. Purified antibodies generated from these cell lines differentially detect human or mouse Klotho protein via Western blot, immunocyto/histochemistry, and immunoprecipitation. Specificity of antibody binding to Klotho was confirmed by mass spectrometry following immunoprecipitation. With this confidence in antibody specificity, co-immunoprecipitation was utilized to validate the interaction of Klotho/FGFR and Klotho/wnt7a in mouse kidney lysates.

Short Forms of Ste20-related Proline/Alanine-rich Kinase (SPAK) in the Kidney Are Created by Aspartyl Aminopeptidase (Dnpep)-mediated Proteolytic Cleavage

The Ste20-related kinase SPAK regulates sodium, potassium, and chloride transport in a variety of tissues. Recently, SPAK fragments, which lack the catalytic domain and are inhibitory to Na(+) transporters, have been detected in kidney. It has been hypothesized that the fragments originate from alternative translation start sites, but their precise origin is unknown. Here, we demonstrate that kidney lysate possesses proteolytic cleavage activity toward SPAK. Ion exchange and size exclusion chromatography combined with mass spectrometry identified the protease as aspartyl aminopeptidase. The presence of the protease was verified in the active fractions, and recombinant aspartyl aminopeptidase recapitulated the cleavage pattern observed with kidney lysate. Identification of the sites of cleavage by mass spectrometry allowed us to test the function of the smaller fragments and demonstrate their inhibitory action toward the Na(+)-K(+)-2Cl(-) cotransporter, NKCC2.

Abstract 318: The Role of PKC-alpha in NCC-Mediated Hypertension

The sodium-chloride cotransporter (NCC) is an essential regulator of sodium transport in the distal convoluted tubule (DCT); functional aberrations will lead to dysregulation of blood pressure. Although this role is well-accepted, the mechanisms accounting for increased distal nephron sodium reabsorption is not completely understood. Previously published data from the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) Study has demonstrated that a polymorphism in PKC-alpha may lead to an increased sensitivity to thiazide diuretics. To investigate whether PKC-alpha may be altering blood pressure via NCC, PKC-alpha KO (PKC KO) and PKC Control (Ctl) mice were used to measure blood pressure via radiotelemetry after normal and high salt (HS, 4%) dietary manipulations and Western blot analysis performed using kidney lysates. Using radiotelemetry (DSI) measurements obtained over an 18 hour ‘active’ time period, we report a significant increase in mean arterial pressure (MAP) in the PKC KO mice as compared to control (123.2±1.1 mmHg vs. 102.0±0.77 mmHg, p&lt;0.001). With HS feeding, MAP increased in both groups; in addition MAP remained significantly higher in the PKC KO mice as compared to the Ctl after 21 days (139.5±0.3 mmHg vs. 124.7±1.1 mmHg, p&lt;0.01). To determine a possible mechanism for this phenomenon, whole kidney lysate was used to assay for total NCC protein. Total NCC protein was almost 3 fold higher in the PKC KO mice as compared to Ctl (in arbitrary units, 208±16 vs. 61±30, p&lt;0.01). Herein we report that lack of PKC-alpha leads to an increase in MAP, which may be accounted for by increased total NCC expression and a subsequent increase in distal nephron reabsorption. We believe that PKC-alpha may be negative regulator of NCC protein expression, and that aberrations in this pathway will lead to hypertension.

Integration of Proteomics and Metabolomics to Unravel the Mechanism of Ischemia Reperfusion Injury in Donor Kidneys for Transplantation.

Increased acceptance of higher risk donor organs for transplantation, results in an increased risk of compromised outcomes after kidney transplantation. These organs are exposed to ischemia/reperfusion injury (IRI) which has detrimental effects on organ function and survival. Understanding the underlying pathophysiological events, at the molecular level, that occur following IRI is still not well defined. In this study, we used a rat model employing proteomcis and metabolomics approaches to evaluate the molecular changes associated with IRI. IRI was induced by clamping left kidney for 45 min in male Fischer rats, allowing for 4 hours of reperfusion (n=6). The right kidney served as an internal control, and healthy rodents used as additional controls (n=2). Renal cortical samples, containing the proximal convoluted tubules were micro-dissected for proteomics and metabolomics studies. Liquid chromatography tandem mass spectrometry based label free quantitative proteomics was employed to measure tryptic digested kidney lysates. NMR spectroscopy was used to measure aqueous fractions extracted from cortex of the same samples. In total, 1390 proteins were identified with 64 proteins shortlisted (>2 fold change, ANOVA P<0.05) between IRI and control groups. Among 64 differentially expressed proteins, 4 up-regulated proteins were associated with acute renal failure. Proteins associated with decreased transmembrane potential and depolarization of mitochondria were up-regulated as were enzymes involved in amino acid and fatty acid metabolism. 1H-NMR spectra were acquired and 29 metabolites identified. Principle component analysis using all spectra showed clear separation of IRI against controls. Amongst 29 identified metabolites, creatinine and leucine were up regulated in IRI vs healthy controls, consistent with acute renal injury and dysregulation of amino-acid metabolism. A decrease of glucose and AMP, NAD+, and increased lactate may indicate dependence on glycolysis and mitochondrial dysfunction during IRI. In summary, this study demonstrates successful use of proteomics and metabolomics to evaluate IRI in a rat model revealing specific molecular signatures. Preventing acute renal injury by preserving mitochondrial function through pre or post conditioning will lead to improved organ quality and improve transplant outcome.

The calcium-activated chloride channel Anoctamin 1 contributes to the regulation of renal function

The role of calcium-activated chloride channels for renal function is unknown. By immunohistochemistry we demonstrate dominant expression of the recently identified calcium-activated chloride channels, Anoctamin 1 (Ano1, TMEM16A) in human and mouse proximal tubular epithelial (PTE) cells, with some expression in podocytes and other tubular segments. Ano1-null mice had proteinuria and numerous large reabsorption vesicles in PTE cells. Selective knockout of Ano1 in podocytes (Ano1-/-/Nphs2-Cre) did not impair renal function, whereas tubular knockout in Ano1-/-/Ksp-Cre mice increased urine protein excretion and decreased urine electrolyte concentrations. Purinergic stimulation activated calcium-dependent chloride currents in isolated proximal tubule epithelial cells from wild-type but not from Ano1-/-/Ksp-Cre mice. Ano1 currents were activated by acidic pH, suggesting parallel stimulation of Ano1 chloride secretion with activation of the proton-ATPase. Lack of calcium-dependent chloride secretion in cells from Ano1-/-/Ksp-Cre mice was paralleled by attenuated proton secretion and reduced endosomal acidification, which compromised proximal tubular albumin uptake. Tubular knockout of Ano1 enhanced serum renin and aldosterone concentrations, probably leading to enhanced compensatory distal tubular reabsorption, thus maintaining normal blood pressure levels. Thus, Ano1 has a role in proximal tubular proton secretion and protein reabsorption. The results correspond to regulation of the proton-ATPase by the Ano1-homolog Ist2 in yeast.

Cleaved prolactin increases activity of amiloride‐sensitive sodium channels in mouse collecting duct principal cells (893.3)

In the renal collecting duct, sodium reabsorption through the epithelial sodium channel (ENaC) creates an osmotic driving force for water reabsorption resulting in increased blood volume and pressure. Investigators have previously shown that prolactin (PRL) stimulated ENaC activity in bullfrog skin cells. However, studies in mammalian systems show no effect of PRL on ENaC function. PRL is present in two forms: a 23kDa full length form (PRL) and a 14‐16kDa cleaved form (PRL‐C). We hypothesized that these two PRL molecules may exert different effects on ENaC activity. To investigate this hypothesis, we used a cell culture model of principal cells from the cortical collecting duct (mpkCCD) which express ENaC subunits and exhibit amiloride‐sensitive current. We measured transepithelial voltage and resistance across cell monolayers before and after basolateral treatment with vehicle (water) or 1‐2 ug/mL of PRL or PRL‐C and calculated transepithelial current using Ohm’s law. We found that PRL produced only a small, variable effect on ENaC activity in mpkCCD cells. However, PRL‐C approximately doubled the amiloride‐sensitive current in these cells within 24 hrs. Compared to kidney lysates, mpkCCD cells exhibit a low abundance of the PRL receptor which may explain the need for large PRL concentrations in these cells. Our results indicate that cleaved PRL stimulates ENaC activity in mpkCCD cells. In future studies, we will examine the effect of PRL and PRL‐C on single channel properties of ENaC in isolated collecting ducts and the effect of these hormones on blood pressure in vivo.Grant Funding Source: Supported by NIH K12GM000680 and T32HL076118

Proteolytic cleavage of SPAK by a kidney protease (893.36)

The Ste20 kinase SPAK is a major regulator of sodium reabsorption in the distal nephron. Recently, SPAK fragments have been detected in the mouse kidney, and particularly in the thick ascending limb of Henle (TAL). These truncated fragments have been demonstrated to inhibit the activity of the Na+‐K+‐2Cl‐ cotransporter, NKCC2, and thereby decrease sodium transport across the TAL. Here we show that mouse kidney lysates possess protease activity that leads to the specific cleavage of a SPAK, as we observed a precise and reproducible cleavage pattern with a GST‐SPAK fusion protein, but not a GST‐OSR1 fusion protein. This activity is not observed with brain, liver, or spleen lysates. The activity is insensitive to PMSF, leupeptin, aprotinin, and pepstatin, but inhibited by DTT and EDTA. In an effort to identify the protease, we performed ion exchange chromatography. The proteolytic activity was detected in an elution fraction corresponding to 250 mM NaCl. The fraction was subjected to MS/MS analysis and 1119 proteins were identified at 95% threshold. Among these proteins, we identified 44 peptidases and proteases. Gel permeation chromatography experiments determined that the protease is larger than 100 kDa. Effort will be made to determine the identity of the protein involved in the cleavage of SPAK and to identify the sites of cleavage.Grant Funding Source: RO1 DK0935010

Endothelial nitric oxide synthase activation through obacunone-dependent arginase inhibition restored impaired endothelial function in ApoE-null mice

Endothelial arginase constrains the activity of endothelial nitric oxide synthase (eNOS) by substrate depletion and reduces nitric oxide bioavailability. During the screening course of arginase inhibitor, we found obacunone as an arginase inhibitor. We tested the hypothesis that obacunone regulates vascular endothelial NO production. Obacunone incubation inhibited arginase I and II activities in liver and kidney lysates, respectively, in dose-dependent manner. Obacunone reciprocally increased nitrite/nitrate (NOx) production in HUVECs. In isolated aortic rings, obacunone increased intracellular l-arginine concentration and enhanced eNOS coupling, leading to increased NO and decreased superoxide production, with no changes in protein expression. Vasoconstriction response to U46619 was attenuated in obacunone-treated aortic vessels compared to that in untreated vessels. Endothelium-dependent vasorelaxant response to acetylcholine was significantly increased in obacunone-treated vessels and was modulated by the NO-dependent signaling cascade. The dose-dependent vasorelaxant response to Ach was reduced in the aortic vessels of ApoE−/− mice fed a high-cholesterol diet. Obacunone incubation increased vasorelaxation to the level of a WT mouse, although the endothelium-independent response to sodium nitroprusside was identical among the groups. Therefore, obacunone may help treat cardiovascular diseases derived from endothelial dysfunction and may be useful for designing pharmaceutical compounds.

Ankyrin-3 is a novel binding partner of the voltage-gated potassium channel Kv1.1 implicated in renal magnesium handling

The voltage-gated potassium channel, Kv1.1, was recently identified as a causative gene in isolated dominant hypomagnesemia. The channel is situated in the distal convoluted tubule, where it participates in maintaining a favorable electrical gradient for driving magnesium ion into the cell through the transient receptor potential melastatin 6 channel. Pull-down experiments coupled to mass spectrometry using the carboxy-terminal domain of Kv1.1 as bait were used in mouse kidney lysates. Ankyrin-3 (ANK3) was identified as a binding partner of Kv1.1 and was enriched in isolated distal convoluted tubules as compared to whole kidney. Electrophysiology studies performed in HEK293 cells expressing Kv1.1 showed that ANK3 significantly inhibited Kv1.1-mediated currents (267 compared to 125 pA/pF) for control and ANK3, respectively. Finally, to evaluate a potential role of ANK3 in magnesium handling, the intrarenal abundance of ANK3 was measured in mice fed a low-, normal-, or high-magnesium diet for 10 days. Mice maintained on high dietary magnesium significantly doubled their fractional urinary excretion of magnesium, which coincided with a 1.8-fold increase in the renal expression of ANK3 compared to mice on a normal- or low-magnesium diet. Thus, our observations demonstrate a novel role for ANK3 in modulating the biophysical properties of Kv1.1. Such regulation appears to be particularly important in conditions of high dietary magnesium.

Characterization of a short isoform of the kidney protein podocin in human kidney

BackgroundSteroid resistant nephrotic syndrome is a severe hereditary disease often caused by mutations in the NPHS2 gene. This gene encodes the lipid binding protein podocin which localizes to the slit diaphragm of podocytes and is essential for the maintenance of an intact glomerular filtration barrier. Podocin is a hairpin-like membrane-associated protein that multimerizes to recruit lipids of the plasma membrane. Recent evidence suggested that podocin may exist in a canonical, well-studied large isoform and an ill-defined short isoform. Conclusive proof of the presence of this new podocin protein in the human system is still lacking.MethodsWe used database analyses to identify organisms for which an alternative splice variant has been annotated. Mass spectrometry was employed to prove the presence of the shorter isoform of podocin in human kidney lysates. Immunofluorescence, sucrose density gradient fractionation and PNGase-F assays were used to characterize this short isoform of human podocin.ResultsMass spectrometry revealed the existence of the short isoform of human podocin on protein level. We cloned the coding sequence from a human kidney cDNA library and showed that the expressed short variant was retained in the endoplasmic reticulum while still associating with detergent-resistant membrane fractions in sucrose gradient density centrifugation. The protein is partially N-glycosylated which implies the presence of a transmembranous form of the short isoform.ConclusionsA second isoform of human podocin is expressed in the kidney. This isoform lacks part of the PHB domain. It can be detected on protein level. Distinct subcellular localization suggests a physiological role for this isoform which may be different from the well-studied canonical variant. Possibly, the short isoform influences lipid and protein composition of the slit diaphragm complex by sequestration of lipid and protein interactors into the endoplasmic reticulum.