bluuebunny/crossref_metadata_2025 huggingface dataset:
Hormone-regulated transepithelial Na+transport in mammalian CCD cells requires SGK1 expression
Authors: Helms My N. , Fejes-Tóth Géza , Náray-Fejes-Tóth Anikó
Published: March 2015

To study the role of serum and glucocorticoid-inducible kinase-1 (SGK1) in mammalian cells, we compared Na+transport rates in wild-type (WT) M1 cortical collecting duct cells with M1 populations stably expressing human full-length SGK1, NH2-terminal truncated (ΔN-60) SGK1, “kinase-dead” (K127M) SGK1, and cells that have downregulated levels of SGK1 mRNA (antisense SGK1). Basal rates of transepithelial Na+transport were highest in full-length SGK1 populations, compared among the above populations. Dexamethasone treatment increased Na+transport in WT and full-length SGK1 cells 2.7- and 2-fold, respectively. Modest stimulation of Na+absorption was detected after dexamethasone treatment in ΔN-60 SGK1 populations. However, ΔN-60 SGK1 transport rates remained substantially lower than WT values. Importantly, a combination of high insulin, dexamethasone, and serum failed to significantly stimulate Na+transport in antisense or K127M SGK1 cells. Additionally, expression of antisense SGK1 significantly decreased transepithelial resistance values. Overall, we concluded that SGK1 is a critical component in corticosteroid-regulated Na+transport in mammalian cortical collecting duct cells. Furthermore, our data suggest that the NH2terminus of SGK1 may contain a Phox homology-like domain that may be necessary for effective Na+transport.
Alterations in renal mitochondrial respiration in response to the reactive oxoaldehyde methylglyoxal
Authors: Rosca Mariana G. , Monnier Vincent M. , Szweda Luke I. , Weiss Miriam F.
Published: March 2015

Chronic hyperglycemia has been linked to alterations in mitochondrial function, suggesting an important role in the pathophysiology of the complications of diabetes mellitus. In the diabetic kidney, ultrastructural changes in mitochondria are associated with impaired tubular function. The goal of this study was to determine if methylglyoxal (MGO), a dicarbonyl compound reaching high levels in hyperglycemic conditions, has direct toxicity for renal mitochondria. Intact mitochondria isolated from the renal cortex of rats were incubated with MGO to determine 1) its effect on mitochondrial respiration, 2) the conditions under which MGO exerts these effects, and 3) the potential mitochondrial targets of MGO influence. This study demonstrates that MGO has an inhibitory effect on both the tricarboxylic acid cycle and the electron respiratory chain. The modifications appear to be specific to certain mitochondrial proteins. Alterations of these proteins lead to disturbances in mitochondria that may play an important role in renal cellular toxicity and in the development of diabetic nephropathy.
New ideas about aldosterone signaling in epithelia
Authors: Stockand James D.
Published: March 2015

The systemic actions of aldosterone are well documented; however, in comparison, our understanding of the cellular and molecular mechanisms by which aldosterone orchestrates these actions is rudimentary. Aldosterone exerts most of its physiological actions by modifying gene expression. It is now apparent that aldosterone represses almost as many genes as it induces. Several aldosterone-sensitive genes, including serum and glucocorticoid-inducible kinase ( sgk) and small, monomeric Kirsten Ras GTP-binding protein (Ki- ras) have recently been identified. The molecular mechanisms and elements bestowing corticosteroid sensitivity on these and many other genes are becoming clear. Induction of Ki-Ras and Sgk is necessary and sufficient for some portion of aldosterone action in epithelia. These two signaling factors are components of a converging pathway with phosphatidylinositol 3-kinase positioned between them that enables both stabilizing the epithelial Na+channel (ENaC) in the open state as well as increasing the number of ENaC in the apical membrane. This aldosterone-induced signaling pathway contains many potential sites for feedback regulation and cross talk from other cascades and potentially impinges directly on the activity of transport proteins and/or cellular differentiation to modify electrolyte transport.
Impaired urine concentration and absence of tissue ACE: involvement of medullary transport proteins
Authors: Klein Janet D. , Le Quach D. , Cole Justin M. , Disher Kevin , Mongiu Anne K. , Wang Xiaodan , Bernstein Kenneth E. , Sands Jeff M.
Published: March 2015

ACE.2 mice lack all tissue angiotensin-converting enzyme (ACE) but have 33% of normal plasma ACE activity. They exhibit the urine-concentrating defect and hyperkalemia present in mice that lack all ACE, but in contrast to the complete knockout, ACE.2 mice have normal medullary histology and creatinine clearance. To explore the urine-concentrating defect in ACE.2 mice, renal medullary transport proteins were analyzed using Western blot analysis. In the inner medulla, UT-A1, ClC-K1, and aquaporin-1 (AQP1) were significantly reduced to 28 ± 5, 6 ± 6, and 39 ± 5% of the level in wild-type mice, respectively, whereas AQP2 and UT-B were unchanged. In the outer medulla, Na+-K+-2Cl−cotransporter (NKCC2/BSC1) and AQP1 were significantly reduced to 56 ± 11 and 29 ± 6%, respectively, whereas Na+-K+-ATPase, UT-A2, UT-B, and AQP2 were unchanged, and renal outer medullary potassium channel was significantly increased to 711 ± 187% of the level in wild-type mice. The abnormal expression of these transporters was similar in ACE.2 mice backcrossed onto a C57BL/6 or a Swiss background and was not rescued by ANG II infusion. We conclude that the urine-concentrating defect in ACE.2 mice is associated with, and may result from, downregulation of some or all of these key urea, salt, and water transport proteins.
Contribution of quinolinic acid in the development of anemia in renal insufficiency
Authors: Pawlak Dariusz , Koda Mariusz , Pawlak Sebastian , Wolczynski Slawomir , Buczko Wlodzimierz
Published: March 2015

Quinolinic acid (QA) is a potent endogenous excitotoxin; elevation of its concentration in an organism has been implicated in the pathogenesis of various disorders. The purpose of this study was the assessment of QA impact on the process of erythropoiesis. Marked increase of QA concentration was observed in plasma and peripheral tissues of uremic rats. These changes were proportional to the amount of the removed renal tissue and positively correlated with the concentration of creatinine but negatively correlated with hematological parameters, i.e., hematocrit and Hb red blood cells count. The changes were accompanied by a slight decrease in the concentration of endogenic erythropoietin (EPO) in the plasma of animals with uremia. Chronic treatment with QA diminished the increase in EPO concentration after introduction of cobalt in rats. These changes were associated with the decrease in all hematological parameters after QA administration. The in vitro study in the conditions of hypoxia showed that QA inhibited the EPO release from HepG2 cells to the culture base. Additionally, in HepG2 cells QA had a dose-dependent inhibitory effect on hypoxia- and cobalt-induced EPO gene expression without any cell toxicity. In conclusion, the erythropoiesis in chronic renal failure could be attributed to the influence of QA on EPO synthesis. Thus we propose that QA can be a uremic toxin responsible for anemia in animals or patients with renal failure.
Effect of immunosuppressive agents on glucocorticoid receptor function in A6 cells
Authors: Edinger Robert S. , Watkins Simon C. , Pearce David , Johnson John P.
Published: March 2015

Immunosuppressive agents such as FK-506 and rapamycin inhibit aldosterone- stimulated Na+transport in A6 cells. Concentration dependence is consistent with the known affinities of these agents for immunophilins. The inhibition was also dependent on time, requiring preincubation with FK-506 or rapamycin before inhibition was seen. The present studies were designed to determine whether this inhibition was pretranscriptional and whether it was due to an effect on either receptor translocation or nuclear accumulation. Because transport effects of steroids in A6 cells are mediated by glucocorticoid receptors (GRs), we examined the transcriptional response of GR-regulated reporters transfected into these cells. Preincubation of cells with FK-506 and rapamycin completely blocked reporter gene activation, whereas preincubation with cyclosporin A partially inhibited this activation. A minimum of 8 h of preincubation was required before the effect was seen. Using a transiently transfected green fluorescent protein-GR construct, we examined the effect of FK-506 and rapamycin on GR translocation. GR translocation induced by dexamethasone was extremely rapid (<5 min) and was largely unaffected by FK-506 or rapamycin but was completely blocked by geldanamycin. Digital deconvolutions revealed a punctate nuclear accumulation of GR, which was still seen after preincubation with immunosuppressive agents. These agents clearly inhibit steroid action by blocking GR-stimulated gene transcription, but this effect is not mediated by altered translocation or nuclear accumulation of receptors. Inhibition of steroid-regulated gene transcription by immunosuppressive agents may explain the electrolyte abnormalities seen in patients receiving these drugs.
Immunomorphometric study of rat renal inner medulla
Authors: Mejia Raymond , Wade James B.
Published: March 2015

We utilized immunofluorescent immunolabeling of renal tissue sections to identify and count tubules at specified depths of the rat renal inner medulla. We used primary antibodies to aquaporin-1 (AQP1; labeling thin descending limbs), aquaporin-2 (AQP2; labeling inner medullary collecting ducts), the rat kidney-specific chloride channel (ClC-K1; labeling thin ascending limbs), and von Willebrand factor (labeling descending vasa recta). Secondary antibodies conjugated to different fluorophores were used, giving up to a three-color display. Labeled structures were then identified and counted. At each level sampled in the inner medulla, many more thin limbs were labeled by ClC-K1 than AQP1. In addition, thin limbs were found to label with antibodies to ClC-K1 on both sides of their hairpin turns. We conclude that the descending thin limbs shift from expressing AQP1 to expressing ClC-K1 some distance before the point where they turn and begin to ascend. Mathematical models can use our quantitative data to explore implications for the urine-concentrating mechanism.
Urotensin II is a nitric oxide-dependent vasodilator and natriuretic peptide in the rat kidney
Authors: Zhang Andrew Y. , Chen Ya-Fei , Zhang David X. , Yi Fu-Xian , Qi Jenson , Andrade-Gordon Patricia , de Garavilla Lawrence , Li Pin-Lan , Zou Ai-Ping
Published: March 2015

Recent studies have indicated that urotensin II (UII), a cyclic peptide, is vasoactive and may be involved in cardiovascular dysfunctions. It remains unknown, however, whether UII plays a role in the control of renal vascular tone and tubular function. In the present study, a continuous infusion of synthetic human UII (hUII) into the renal artery (RA) in anesthetized rats was found to increase renal blood flow (RBF) and urinary water and sodium excretion (UV and UNaV) in a dose-dependent manner. At a dose of 20 ng · kg–1 · min–1, it increased RBF by 20% and UV and UNaV by 94 and 109%, respectively. Nitric oxide (NO) synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME) completely abolished hUII-induced increases in RBF and water/sodium excretion. In isolated, pressurized, and phenylephrine-precontracted small RA with internal diameter of ∼200 μm, hUII produced a concentration-dependent vasodilation with a maximal response of 55% at 1.5 μM. l-NAME significantly blocked this hUII-induced vasodilation by 60%. In denuded RA, hUII had neither vasodilator nor vasoconstrictor effect. With the use of 4,5-diaminofluorescein diacetate-based fluorescence imaging analysis of NO levels, hUII (1 μM) was shown to double the NO levels within the endothelium of freshly dissected small RA, and l-NAME blocked this UII-induced production of endothelial NO. These results indicate that UII produces vasodilator and natriuretic effects in the kidney and that UII-induced vasodilation is associated with increased endothelial NO in the RA.
Contribution of prostaglandin EP2receptors to renal microvascular reactivity in mice
Authors: Imig John D. , Breyer Matthew D. , Breyer Richard M.
Published: March 2015

The present studies were performed to determine the contribution of EP2receptors to renal hemodynamics by examining afferent arteriolar responses to PGE2, butaprost, sulprostone, and endothelin-1 in EP2receptor-deficient male mice (EP2−/−). Afferent arteriolar diameters averaged 17.8 ± 0.8 μm in wild-type (EP2+/+) mice and 16.7 ± 0.7 μm in EP2−/− mice at a renal perfusion pressure of 100 mmHg. Vessels from both groups of mice responded to norepinephrine (0.5 μM) with similar 17–19% decreases in diameter. Diameters of norepinephrine-preconstricted afferent arterioles increased by 7 ± 2 and 20 ± 6% in EP2+/+ mice in response to 1 μM PGE2and 1 μM butaprost, respectively. In contrast, afferent arteriolar diameter of EP2−/− mice decreased by 13 ± 3 and 16 ± 6% in response to PGE2and butaprost. The afferent arteriolar vasoconstriction to butaprost in EP2−/− mice was eliminated by angiotensin-converting enzyme inhibition. Sulprostone, an EP1and EP3receptor ligand, decreased afferent arteriolar diameter in both groups; however, the vasoconstriction in the EP2−/− mice was greater than in the EP2+/+ mice. Endothelin-1-mediated afferent arteriolar diameter responses were enhanced in EP2−/− mice. Afferent arteriolar diameter decreased by 29 ± 7% in EP2−/− and 12 ± 7% in EP2+/+ mice after administration of 1 nM endothelin-1. These results demonstrate that the EP2receptor mediates a portion of the PGE2afferent arteriolar vasodilation and buffers the renal vasoconstrictor responses elicited by EP1and EP3receptor activation as well as endothelin-1.
Low-calcium diet in hypercalciuric enuretic children restores AQP2 excretion and improves clinical symptoms
Authors: Valenti Giovanna , Laera Antonia , Gouraud Sabine , Pace Giuseppe , Aceto Gabriella , Penza Rosa , Selvaggi Francesco P. , Svelto Maria
Published: March 2015

In this study, we analyzed the effect of a therapeutic intervention in 46 enuretic children, 26 (57%) of whom were hypercalciuric. All the patients ( n = 46) were treated with DDAVP for 3–6 mo. The hypercalciuric patients ( n = 26) received a low-calcium diet (∼500 mg/day) for the same period. After the therapy, the bed-wetting episodes stopped in 80% of the 46 patients tested. In those patients having low-AVP levels before the therapy, circulating AVP concentration returned to normal (>4 pg/ml), and the hypercalciuria was resolved in the hypercalciuric patients (calcium/creatinine ratio <0.2). Urinary aquaporin-2 (AQP2) levels were semiquantified by densitometric scanning and reported as a ratio between the intensity of the signal in the day vs. the night urine samples (day/night AQP2 ratio). In the hypercalciuric patients, the day/night AQP2 ratio returned to values close to those found in the healthy children (from 1.19 ± 0.20 before to 0.69 ± 0.10 after the treatment, n = 26, P = 0.03). In contrast, in the normocalciuric children we saw no significant modulation of AQP2 excretion (from 1.07 ± 0.14 before to 0.99 ± 0.14 after the treatment, n = 20). This study clearly demonstrates that urinary calcium levels modulate AQP2 excretion and is likely to be useful for treatment of children with enuresis.
Glucocorticoid regulation of the murine PHEX gene
Authors: Hines Eric R. , Collins James F. , Jones Marci D. , Serey Samantha H. , Ghishan Fayez K.
Published: March 2015

The phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) is a member of the neutral endopeptidase family, which is expressed predominantly on the plasma membranes of mature osteoblasts and osteocytes. Although it is known that the loss of PHEX function results in X-linked hypophosphatemic rickets, characterized by abnormal bone matrix mineralization and renal phosphate wasting, little is known about how PHEX is regulated. We therefore sought to determine whether the murine PHEX gene is regulated by glucocorticoids (GCs), which are known to influence phosphate homeostasis and bone metabolism. Northern blot analysis revealed increased PHEX mRNA expression in GC-treated suckling mice (1.5-fold) and in rat osteogenic sarcoma (UMR-106) cells (2.5-fold). An increase was also seen in PHEX promoter activity in transiently transfected UMR-106 cells with GC treatment. Analysis of nested promoter deletions revealed that an atypical GC response element was located between −337 and −315 bp. Mutational analysis and electrophoretic mobility shift assays further identified −326 to −321 bp as a site involved in GC regulation. Supershift analyses and electrophoretic mobility shift assay competition studies indicated that the core binding factor α1-subunit transcription factor is able to bind to this region and may therefore play a role in the GC response of the murine PHEX gene.
Anomalous fractional clearance of negatively charged Ficoll relative to uncharged Ficoll
Authors: Guimarães Marco A. M. , Nikolovski Julijana , Pratt Lynette M. , Greive Kerryn , Comper Wayne D.
Published: March 2015

Recent studies, using low-temperature perfusion of rat kidneys, have claimed the existence of renal charge selectivity simply on the basis of the differential excretion rates of uncharged Ficoll and charged proteins. To test for the existence of charge selectivity in vivo, we examined the clearance of negatively charged Ficoll compared with uncharged Ficoll. A short-term approach to steady state was used to study the fractional clearances. Relative clearances were also examined using an osmotic pump technique where the tracers reach a steady-state value in conscious rats after 7 days. Carboxymethyl Ficoll was stable during filtration and renal passage, was not taken up by the kidneys, and did not bind to plasma proteins. There was no significant difference in the fractional clearance of molecules with radius of 36 Å for Ficoll (fractional clearance = 0.048 ± 0.038, n = 5) and negatively charged carboxymethyl Ficoll (fractional clearance = 0.028 ± 0.019, n = 5). For molecules with radii greater than 36 Å, carboxymethyl Ficoll had facilitated clearance with respect to uncharged Ficoll [for example, at a radius of 60 Å fractional clearance for Ficoll = 0.0012 ± 0.0005 ( n = 5), whereas that for carboxymethyl Ficoll = 0.015 ± 0.005 ( n = 5)]. Renal function was not compromised by carboxymethyl Ficoll as uncharged Ficoll in urine exhibited similar hydrodynamic size profiles when studied in the presence of excess unlabeled carboxymethyl Ficoll. The facilitated clearance of negatively charged Ficoll with respect to uncharged Ficoll reveals a property of the capillary wall, which has been previously observed with other nonproteinaceous polyanions. This study demonstrates that the glomerular capillary wall is not charge selective in the form of excluding negatively charged Ficoll. However, the charge properties of the capillary wall may influence the facilitated transport of charged Ficoll compared with uncharged Ficoll.
Targeted degradation of ENaC in response to PKC activation of the ERK1/2 cascade
Authors: Booth Rachell E. , Stockand James D.
Published: March 2015

Renal A6 epithelial cells were used to determine the mechanism by which protein kinase C (PKC) decreases epithelial Na+channel (ENaC) activity. Activation of PKC reduced relative Na+reabsorption to <20% within 60 min. This decrease was sustained over the next 24–48 h. In response to PKC signaling, α-, β-, and γ-ENaC levels were 0.97, 0.36, and 0.39, respectively, after 24 h, with the levels of the latter two subunits being significantly decreased. The PKC-mediated decreases in β- and γ-ENaC were significantly reversed by simultaneous addition of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase-1/2 inhibitors U-0126 and PD-98059. These inhibitors, in addition, protected Na+reabsorption from PKC, demonstrating that the MAPK1/2 cascade, in some instances, plays a central role in downregulation of ENaC activity. The effects of PKC on β- and γ-ENaC levels were additive with those of inhibitors of transcription (actinomycin D) and translation (emetine and cycloheximide), suggesting that PKC promotes subunit degradation and does not affect subunit synthesis. The bulk of whole cell γ-ENaC was degraded within 1 h after treatment with inhibitors of synthesis; however, a significant pool was “protected” from inhibitors for up to 12 h. PKC affected this protected pool of γ-ENaC. Moreover, proteosome inhibitors (MG-132 and lactacystin) reversed PKC effects on this protected pool of γ-ENaC. Thus PKC signaling via MAPK1/2 cascade activation in A6 cells promotes degradation of γ-ENaC.
P2 receptor-mediated afferent arteriolar vasoconstriction during calcium blockade
Authors: Inscho Edward W. , Cook Anthony K.
Published: March 2015

Experiments were performed to determine the role of L-type calcium channels on the afferent arteriolar vasoconstrictor response to ATP and UTP. With the use of the blood-perfused juxtamedullary nephron technique, kidneys were perfused at 110 mmHg and the responses of arterioles to α,β-methylene ATP, ATP, and UTP were determined before and during calcium channel blockade with diltiazem. α,β-Methylene ATP (1.0 μM) decreased arteriolar diameter by 8 ± 1% under control conditions. This response was abolished during calcium channel blockade. In contrast, 10 μM UTP reduced afferent arteriolar diameter to a similar degree before (20 ± 4%) and during (14 ± 4%) diltiazem treatment. Additionally, diltiazem completely prevented the vasoconstriction normally observed with ATP concentrations below 10 μM and attenuated the response obtained with 10 μM ATP. These data demonstrate that L-type calcium channels play a significant role in the vasoconstrictor influences of α,β-methylene ATP and ATP but not UTP. The data also suggest that other calcium influx pathways may participate in the vasoconstrictor response evoked by P2 receptor activation. These observations support previous findings that UTP-mediated elevation of intracellular calcium concentration in preglomerular vascular smooth muscle cells relies primarily on calcium release from intracellular pools, whereas ATP-mediated responses involve both voltage-dependent calcium influx, through L-type calcium channels, and the release of calcium from intracellular stores. These results support the argument that P2X and P2Y receptors influence the diameter of afferent arterioles through activation of disparate signal transduction mechanisms.
Sustained activation of MAPK/ERKs signaling pathway in cystic kidneys from bcl-2 −/− mice
Authors: Sorenson Christine M. , Sheibani Nader
Published: March 2015

Cell proliferation, survival, and differentiation are carefully orchestrated processes during nephrogenesis that become aberrant during renal cyst formation. Signaling through focal adhesion kinase (FAK) impacts these processes, although its role during nephrogenesis requires further delineation. We previously demonstrated that phosphorylation of FAK and paxillin is not downregulated in cystic kidneys from B cell lymphoma/leukemia-2 (bcl-2) −/− mice. Here we examine whether FAK downstream signaling pathways are affected in these cystic kidneys. Cystic kidneys from bcl-2 −/− mice exhibited sustained phosphorylation of Src and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK, ERK1). However, similar levels of expression were noted for phosphorylated c-Jun NH2-terminal kinase, phosphatidylinositol-3-kinase, and its target protein kinase B/ATP-dependent tyrosine kinase in kidneys from postnatal day 20 bcl-2 +/+ and bcl-2 −/− mice. We also examined expression of the adapter protein Shc, implicated in growth and apoptosis. Expression of p66Shcdecreases to low levels in postnatal kidneys, whereas p52/p46Shcwas constitutively expressed during nephrogenesis. Shc expression was similar in normal and cystic kidneys. Therefore, sustained activation of MAPK/ERKs through the Src/FAK pathway may contribute to the hyperproliferation observed in cystic kidneys from bcl-2 −/− mice.
BMP7 antagonizes TGF-β-dependent fibrogenesis in mesangial cells
Authors: Wang Shinong , Hirschberg Raimund
Published: March 2015

Exogenous administration of recombinant human bone morphogenetic protein (BMP)-7 was recently shown to ameliorate renal glomerular and interstitial fibrosis in rodents with experimental renal diseases. We tested the hypothesis that BMP7 functions by antagonizing profibrogenic events that are induced by transforming growth factor (TGF)-β in cultured mesangial cells. Incubation of murine mesangial cells with TGF-β (50–200 pM) increased cell-associated collagen type IV and fibronectin, soluble collagen type IV, thrombospondin, and connective tissue growth factor (CTGF). Coincubation with recombinant human BMP7 (200 pM) reduced the increase of these ECM proteins and CTGF. The changes in collagen type IV and fibronectin proteins occurred without concomitant changes in collagen type α1IV and fibronectin mRNA levels, suggesting that TGF-β and BMP7 act primarily by affecting ECM protein degradation. Indeed, TGF-β decreases the levels and activity of matrix metalloprotease (MMP)-2, the major metalloprotease that is secreted by mesangial cells. Moreover, BMP7 inhibits TGF-β-induced activation of MMP2. Because TGF-β reduces the activity of MMPs through increasing plasminogen activator inhibitor (PAI)-1, we tested whether BMP7 interferes with this TGF-β effect. BMP7 reduces, by about two-thirds, the activation of a PAI-1 promoter/luciferase reporter in cells stably transfected with this construct. The findings from these studies indicate that BMP7 reduces TGF-β-induced ECM protein accumulation in cultured mesangial cells primarily by maintaining levels and activity of MMP2 partially through prevention of TGF-β-dependent upregulation of PAI-1.
Proinflammatory effects of oxidative stress in chronic kidney disease: role of additional angiotensin II blockade
Authors: Agarwal Rajiv ,   (With the Technical Assistance of Shawn D. Chase)
Published: March 2015

Oxidative stress plays an important role in causing progressive chronic kidney disease (CKD). We examined the influence of add-on ANG II receptor blockade administered as losartan (50 mg/day for 1 mo) on oxidative stress and proinflammatory state of the kidney in patients with CKD. All subjects were taking an angiotensin-converting enzyme inhibitor plus other antihypertensive agents. Oxidative stress to lipids and proteins was measured by an HPLC assay for malondialdehyde (MDA) and carbonyl concentration, respectively. Urinary inflammation was measured by monocyte chemotactic protein-1 (MCP-1) excretion rate. The etiology of CKD was type 2 diabetes mellitus in 12 and glomerulonephritis in 4 patients. There was no change in proteinuria or 24-h ambulatory blood pressure (BP) with add-on ANG II receptor blockade with losartan therapy. Before losartan therapy, urinary protein and albumin oxidation were 99 and 71% higher, respectively, compared with in plasma ( P < 0.05). There was a 35% reduction in urinary oxidized albumin with add-on losartan therapy ( P = 0.036). Urinary and plasma MDA were elevated compared with age-matched controls. Urinary MDA was significantly reduced from 4.75 ± 3.23 to 3.39 ± 2.17 μmol/g creatinine with add-on losartan therapy. However, plasma MDA or oxidized proteins did not change in response to additional ANG II blockade. A good correlation was seen between the change in urinary oxidized albumin and MCP-1 levels ( r= 0.61, P = 0.012). These data demonstrate that oxidative damage to urinary protein and lipids can be reduced with additional ANG II receptor blockade, independently of reductions in proteinuria or BP. Urinary measurements of markers of oxidative damage to lipids and proteins appear to be more sensitive than plasma measurements in patients with CKD. The significant association of the change in urinary MCP-1 with a reduction in oxidative stress supports the role of the redox state in the kidney with renal fibrosis and progressive kidney damage.
Cloning and localization of KCC4 in rabbit kidney: expression in distal convoluted tubule
Authors: Velázquez Heino , Silva Teresa
Published: March 2015

Cl-dependent K secretion is a feature of renal distal tubules and collecting ducts. Recent cloning and identification of K-Cl cotransporter proteins led us to search for additional novel KCC isoforms expressed in the renal distal nephron. A human expressed sequence tag (EST) with high homology to KCC1 was identified. The rabbit isoform was cloned by homology using degenerate primers and rapid amplification of cDNA ends (RACE). Our isoform is the rabbit homologue of mouse and human KCC4 published previously. The 4.35-kb rabbit KCC4 cDNA encodes a protein of 1,106 amino acids. Antibodies were generated to both NH2-terminal and COOH-terminal fusion proteins. Northern and Western blot analyses showed widespread mRNA and protein expression in many rabbit organs, in renal cortex, outer medulla, and inner medulla but not in skeletal muscle. Immunohistochemical localization of KCC4 showed expression exclusively along the basolateral membrane in many nephron segments. The distal convoluted tubule and connecting tubule exhibited the highest level of KCC4 immunoreactivity, followed by the medullary thick ascending limb. A low level of immunoreactivity was detected in the proximal tubule and collecting ducts. We postulate that KCC4 mediates potassium and chloride exit from the cell and may play an important role in salt absorption by the distal convoluted tubule.
Renal arginine and protein synthesis are increased during early endotoxemia in mice
Authors: Hallemeesch Marcella M. , Soeters Peter B. , Deutz Nicolaas E. P.
Published: March 2015

The kidney has an important function in arginine metabolism, because the kidney is the main endogenous source for de novo arginine production from circulating citrulline. In conditions such as sepsis, nitric oxide (NO) production is increased and is dependent on extracellular arginine availability. To elucidate the adaptive role of renal de novo arginine synthesis in a condition of increased NO production, we studied renal arginine metabolism in a mouse model of endotoxemia. Because arginine flux is largely dependent on protein flux, we also measured protein metabolism in mice. Female mice were injected intraperitoneally with lipopolysaccharide; control mice received 0.9% NaCl. Six hours later, renal blood flow was measured with the use of para-aminohippuric acid. Arginine and protein metabolism were studied using organ-balance, stable-isotope techniques. Systemic NO production was increased in the endotoxin-treated mice. In addition, renal protein synthesis and de novo arginine production from citrulline were increased. However, no effect on renal NO production was observed. In conclusion, increased renal de novo arginine production may serve to sustain systemic NO production. To our knowledge, it was shown for the first time that renal protein synthesis is enhanced in the early response to endotoxemia.
Bidirectional regulation of renal hemodynamics by activation of PAR1and PAR2in isolated perfused rat kidney
Authors: Gui Yu , Loutzenhiser Rodger , Hollenberg Morley D.
Published: March 2015

Proteinase-activated receptors (PARs) are activated by either serine proteinases or synthetic peptides corresponding to the NH2-terminal tethered ligand sequences that are unmasked by proteolytic cleavage. Although PARs are highly expressed in the kidney, their roles in regulating renal function are not known. In the present study, we evaluated the impact of PAR activation on renal hemodynamics using PAR1- and PAR2-activating peptides (TFLLR-NH2and SLIGRL-NH2) and proteinases (thrombin and trypsin) as PAR agonists in the isolated perfused rat kidney preparation. PAR1activation resulted in renal vasoconstriction and a marked reduction in the glomerular filtration rate (GFR). In contrast, PAR2activation caused vasodilation, partially reversing the vasoconstriction induced by TFLLR-NH2and ANG II and increasing GFR that had been prereduced by ANG II. The vasoconstrictor actions of PAR1activation were abolished by protein kinase C inhibition. The PAR2-induced vasodilation was only partially blocked by NG-nitro-l-arginine methyl ester, suggesting both nitric oxide-dependent and -independent mechanisms. Although PAR4mRNA was detected in renal parenchyma, the PAR4-activating peptide AYPGKF-NH2had no effect on renal perfusion flow rate. We conclude that PAR1and PAR2play bidirectional roles in the regulation of renal hemodynamics.
Angiotensin blocks substance P release from renal sensory nerves by inhibiting PGE2-mediated activation of cAMP
Authors: Kopp Ulla C. , Cicha Michael Z. , Smith Lori A.
Published: March 2015

Activation of renal sensory nerves involves PGE2-mediated release of substance P (SP) via activation of the cAMP-PKA pathway. The PGE2-mediated SP release is suppressed by a low- and enhanced by a high-sodium (Na+) diet, suggesting an inhibitory effect of ANG. We now examined whether ANG II is present in the pelvic wall and inhibits PGE2-mediated SP release by blocking PGE2-mediated increases in cAMP. ANG II levels in renal pelvic tissue were 710 ± 95 and 260 ± 30 fmol/g tissue in rats fed a low- and high-Na+diet, respectively. In a renal pelvic preparation from high-Na+-diet rats, 0.14 μM PGE2produced an increase in SP release from 7 ± 1 to 19 ± 3 pg/min that was blocked by 15 nM ANG II. Treating pelvises with pertussis toxin (PTX) abolished the effects of ANG II. In pelvises from low-Na+rats, neither basal nor bradykinin-mediated SP release was altered by PGE2. However, the bradykinin-mediated release of SP was enhanced by the permeable cAMP analog CPT-cAMP, from 4 ± 1 to 11 ± 2 pg/min, a response similar to that in normal-Na+-diet rats. In vivo, renal pelvic administration of PGE2enhanced the afferent renal nerve activity (ARNA) response to bradykinin in normal- but not in low-Na+diet rats. CPT-cAMP produced similar enhancement of the ARNA responses to bradykinin in normal- and low-Na+-diet rats, 1,670 ± 490 and 1,760 ± 400%·s (area under the curve of ARNA vs. time). Similarly, the ARNA responses to increases in renal pelvic pressure were similarly enhanced by CPT-cAMP in normal- and low-Na+-diet rats. In conclusion, renal pelvic ANG II modulates the responsiveness of renal sensory nerves by suppressing PGE2-mediated activation of adenylyl cyclase via a PTX-sensitive mechanism.
ANG II reduces net acid secretion in rat outer medullary collecting duct
Authors: Wall Susan M. , Fischer Michael P. , Glapion Dawn M. , De La Calzada Mae
Published: March 2015

In rat outer medullary collecting duct (OMCD), the mechanism(s) and regulation of H+secretion are not understood fully. The effect of changes in acid-base balance and the renin-angiotensin system on net H+secretion was explored. Rats received NaCl, NaHCO3, NH4Cl, or nothing in their drinking water for 7 days. Total ammonia and total CO2( JtCO2) fluxes were measured in OMCD tubules perfused in vitro from rats in each treatment group. JtCO2was reduced in tubules from rats drinking NH4Cl relative to those drinking NaHCO3. Because NH4Cl intake increases plasma renin and aldosterone, we asked if upregulation of the renin-angiotensin system reduces net H+secretion. Deoxycorticosterone pivalate administered in vivo did not affect JtCO2. However, ANG II given in vivo at 0.1 ng/min reduced JtCO2by 35%. To determine if ANG II has a direct effect on acid secretion, JtCO2was measured with ANG II applied in vitro. ANG II (10-8M) present in the bath solution reduced JtCO2by 35%. This ANG II effect was not observed in the presence of the AT1receptor blocker candesartan. In conclusion, in rat OMCD, JtCO2is paradoxically reduced with NH4Cl ingestion. Increased circulating ANG II, as occurs during metabolic acidosis, reduces JtCO2.
Urinary excretion of viable podocytes in health and renal disease
Authors: Vogelmann Stefanie U. , Nelson W. James , Myers Bryan D. , Lemley Kevin V.
Published: March 2015

The loss of glomerular visceral epithelial cells (podocytes) has been associated with the development of glomerular sclerosis and loss of renal function. Viability of podocytes recovered from urine of subjects with glomerular disease and of healthy controls was investigated by propidium iodide exclusion and TUNEL staining. Podocyte loss was quantified by cytospin. The growth behavior in culture of urinary cells and their expression of specific markers were examined. The majority of urinary podocytes are viable, although apoptosis occurs in about one-half of the cells. Patients with active glomerular disease excreted up to 388 podocytes/mg creatinine, whereas healthy controls and patients with quiescent disease generally excreted <0.5 podocytes/mg creatinine. The identity of cultured cells was confirmed by their morphology, growth behavior, and expression of podocyte-specific markers. The difference in growth behavior between healthy controls and subjects with active glomerular disease suggests that in active disease viable podocytes detach from the glomerular tuft due to local environmental factors rather than defects in the podocytes per se, whereas in healthy individuals mostly senescent podocytes are shed.
Influence of testosterone on regulation of ODC, antizyme, andN1-SSAT gene expression in mouse kidney
Authors: Levillain Olivier , Greco Anna , Diaz Jean-Jacques , Augier Roger , Didier Anne , Kindbeiter Karine , Catez Frédéric , Cayre Myriam
Published: March 2015

Polyamines are involved in the control of the cell cycle and cell growth. In murine kidney, testosterone enhances gene expression of ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis. In this study, we document the time course effect of testosterone on 1) gene expression of ODC, antizyme 1 (AZ1), and spermidine/spermine- N1-acetyltransferase ( N1-SSAT); 2) ODC activity in proximal convoluted tubules (PCT) and cortical proximal straight tubules (CPST); and 3) renal polyamine levels. Female mice were treated with testosterone for a period of 1, 2, 3, and 5 consecutive days. ODC gene expression was extremely low in kidneys of untreated female mice compared with that of males. Consequently, the renal putrescine level was sevenfold lower in females than in males, whereas spermidine and spermine levels did not differ between sexes. In female kidneys, testosterone treatment sharply increased ODC mRNA and protein levels as well as ODC activity. Testosterone increased the expression of ODC in PCT and CPST over different time courses, which suggests that ODC activity is differentially regulated in distinct tubules. The expression of AZ1 and N1-SSAT mRNA was similar in male and female mouse kidneys. Testosterone treatment enhanced AZ1 and N1-SSAT mRNA levels in a time-dependent manner by unknown molecular mechanisms. Putrescine and spermidine levels increased after testosterone treatment in female kidneys. Surprisingly, although ODC protein and activity were undetectable in female kidneys, the levels of AZ1 mRNA and protein were similar to those in males. Therefore, one may propose that ODC protein could be continuously degraded by AZ1 in female kidneys.
Nox4 mediates angiotensin II-induced activation of Akt/protein kinase B in mesangial cells
Authors: Gorin Yves , Ricono Jill M. , Kim Nam-Ho , Bhandari Basant , Choudhury Goutam Ghosh , Abboud Hanna E.
Published: March 2015

ANG II induces protein synthesis through the serine-threonine kinase Akt/protein kinase B (PKB) in mesangial cells (MCs). The mechanism(s) of activation of Akt/PKB particularly by G protein-coupled receptors, however, is not well characterized. We explored the role of the small GTPase Rac1, a component of the phagocyte NADPH oxidase, and the gp91phoxhomologue Nox4/Renox in this signaling pathway. ANG II causes rapid activation of Rac1, an effect abrogated by phospholipase A2inhibition and mimicked by arachidonic acid (AA). Northern blot analysis revealed high levels of Nox4 transcript in MCs and transfection with antisense (AS) oligonucleotides for Nox4 markedly decreased NADPH-dependent reactive oxygen species (ROS)-producing activity. Dominant negative Rac1 (N17Rac1) as well as AS Nox4 inhibited ROS generation in response to ANG II and AA, whereas constitutively active Rac1 stimulated ROS formation. Moreover, N17Rac1 blocked stimulation of NADPH oxidase activity by AA. N17Rac1 or AS Nox4 abolished ANG II- or AA-induced activation of the hypertrophic kinase Akt/PKB. In addition, AS Nox4 inhibited ANG II-induced protein synthesis. These data provide the first evidence that activation by AA of a Rac1-regulated, Nox4-based NAD(P)H oxidase and subsequent generation of ROS mediate the effect of ANG II on Akt/PKB activation and protein synthesis in MCs.
Renal function in NHE3-deficient mice with transgenic rescue of small intestinal absorptive defect
Authors: Woo Alison L. , Noonan William T. , Schultheis Patrick J. , Neumann Jonathan C. , Manning Patrice A. , Lorenz John N. , Shull Gary E.
Published: March 2015

The degree to which loss of the NHE3 Na+/H+ exchanger in the kidney contributes to impaired Na+-fluid volume homeostasis in NHE3-deficient ( Nhe3 −/−) mice is unclear because of the coexisting intestinal absorptive defect. To more accurately assess the renal effects of NHE3 ablation, we developed a mouse with transgenic expression of rat NHE3 in the intestine and crossed it with Nhe3 −/− mice. Transgenic Nhe3 −/− (tg Nhe3 −/−) mice tolerated dietary NaCl depletion better than nontransgenic knockouts and showed no evidence of renal salt wasting. Unlike nontransgenic Nhe3 −/− mice, tg Nhe3 −/− mice tolerated a 5% NaCl diet. When fed a 5% NaCl diet, tg Nhe3 −/− mice had lower serum aldosterone than tg Nhe3 −/− mice on a 1% NaCl diet, indicating improved extracellular fluid volume status. Na+-loaded tg Nhe3 −/− mice had sharply increased urinary Na+ excretion, reflective of increased absorption of Na+ in the small intestine; nevertheless, they remained hypotensive, and renal studies showed a reduction in glomerular filtration rate (GFR) similar to that observed in nontransgenic Nhe3 −/− mice. These data show that reduced GFR, rather than being secondary to systemic hypovolemia, is a major renal compensatory mechanism for the loss of NHE3 and indicate that loss of NHE3 in the kidney alters the set point for Na+-fluid volume homeostasis.
Increased expression of cyclooxygenase-1 and -2 in the diabetic rat renal medulla
Authors: Nasrallah Rania , Landry Anne , Singh Sonia , Sklepowicz Monika , Hébert Richard L.
Published: March 2015

Alterations in renal prostaglandins (PGs) may contribute to some of the renal manifestations in diabetes leading to nephropathy. PG production is dependent on the activity of cyclooxygenases (COX-1 AND -2) and PG synthases. Our present study investigated levels of these enzymes in streptozotocin-diabetic rats at 2, 4, 6, and 8 wk of diabetes. Immunohistochemical analysis revealed an increase in COX signal in the inner and outer medulla of diabetic rats. This was confirmed by Western blotting, showing up to a fourfold increase in both COX isoforms at 4–6 wk of diabetes. Also, Western blot analysis revealed a sixfold increase in PGE2synthase expression in the outer medullary region of 6-wk diabetic rats but no difference in the inner medulla. In cultured rat inner medullary collecting duct (IMCD), levels of COX were increased two- to threefold in cells exposed for 4 days to 37.5 mM glucose compared with control of 17.5 mM. While no change in PGE2synthase levels was noted, PGE2synthesis was increased. Furthermore, levels of EP1and EP4mRNA were increased, as well as a twofold increase in EP4protein levels. Future studies will determine which COX isoform is contributing to the majority of PGE2produced in the diabetic IMCD and the significance of these findings to disturbances in IMCD function and to the progression of diabetic nephropathy.
Nitric oxide synthesis influences the renal vascular response to heme oxygenase inhibition
Authors: Rodriguez Francisca , Zhang Fan , Dinocca Sandra , Nasjletti Alberto
Published: March 2015

We studied the effects of the heme oxygenase (HO) inhibitor stannous mesoporphyrin (SnMP; 40 μmol/kg iv) on renal hemodynamics in anesthetized rats with and without 48-h pretreatment with N G-nitro-l-arginine methyl ester (l-NAME), an inhibitor of nitric oxide (NO) synthesis. SnMP decreased renal blood flow (RBF) and increased renal vascular resistance (RVR) in both groups. The SnMP-induced reduction of RBF inl-NAME-pretreated rats was more prominent than in rats without pretreatment (43 ± 7 vs. 13 ± 3%) as was the SnMP-induced elevation of RVR (87 ± 31 vs. 14 ± 5%). The renal vasoconstrictor effect of SnMP is linked, in part, to amplification of prevailing neurohormonal constrictor mechanisms, since in l-NAME-pretreated rats it was prevented by concurrent administration of prazosin or losartan. However, SnMP (15 μmol/l) also elicits vasoconstriction in isolated, pressurized renal interlobular arteries and the response is more intense in vessels obtained from l-NAME-pretreated rats than from rats without pretreatment. These data indicate that the status of NO synthesis conditions the vascular response to HO inhibition in the rat kidney.
Vasopressin rapidly increases phosphorylation of UT-A1 urea transporter in rat IMCDs through PKA
Authors: Zhang Chi , Sands Jeff M. , Klein Janet D.
Published: March 2015

The UT-A1 urea transporter plays an important role in maintaining the hyperosmolar milieu of the inner medulla. Vasopressin increases urea permeability in rat terminal inner medullary collecting ducts (IMCDs) within 5–10 min. To elucidate the mechanism, IMCD suspensions were radiolabeled with [32P]orthophosphate. UT-A1 was immunoprecipitated and analyzed by autoradiogram and Western blot. Both the 97- and 117-kDa UT-A1 proteins were phosphorylated. Vasopressin treatment increased the phosphorylation of both UT-A1 proteins at 2 min, which peaked at 5–10 min and remained elevated for up to 30 min. There was a discernable increase in UT-A1 phosphorylation with 10 pM and a 50% increase with 10–100 nM vasopressin. 1-Desamino-8-d-arginine vasopressin (dDAVP) or 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) also increased UT-A1 phosphorylation. The vasopressin-stimulated increase in UT-A1 phosphorylation was blocked by H-89 or a specific peptide inhibitor of protein kinase A. Phosphatase inhibitors (okadaic acid, calyculin) increased UT-A1 phosphorylation. We conclude that vasopressin increases UT-A1 phosphorylation via protein kinase A within 2–5 min in rat IMCDs. This suggests that phosphorylation of UT-A1 may be the mechanism by which vasopressin rapidly increases urea permeability in vivo.
Interleukin-4 and -13 promote basolateral secretion of H+and cathepsin L by glomerular epithelial cells
Authors: Van Den Berg José G. , Aten Jan , Annink Carla , Ravesloot Jan H. , Weber Ekkehard , Weening Jan J.
Published: March 2015

Minimal change nephrosis (MCN) is characterized by massive proteinuria and ultrastructural alterations of glomerular visceral epithelial cells (GVEC). MCN has been associated with elevated production of interleukin (IL)-13 by circulating T lymphocytes and with T helper 2 lymphocyte-dependent conditions. We recently showed that GVEC express IL-4 and IL-13 receptors and that IL-4 and IL-13 increase transcellular ion transport over GVEC monolayers. We therefore hypothesized that IL-13 may directly injure GVEC. Here we demonstrate that IL-4 and IL-13 induce bafilomycin A1-sensitive basolateral proton secretion by cultured GVEC, indicating involvement of vacuolar H+-ATPase. The effects of IL-4 and IL-13 were accompanied by redistribution of the small GTPases Rab5b and Rab7, as shown by confocal immunofluorescence studies. Furthermore, Western blot analysis and assays for cysteine proteinase activity revealed basolateral secretion of the lysosomal proteinase procathepsin L by cultured GVEC, stimulated by IL-4 and IL-13. We speculate that IL-4 and IL-13 influence intracellular trafficking of proteins and promote proteolysis at the basolateral surface of GVEC, which may play a pathogenic role in altered glomerular permeability.