bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcium-sensing receptor regulation of PTH-inhibitable proximal tubule phosphate transport
Authors: Ba Jianming , Brown Dennis , Friedman Peter A.
Published: March 2015

Inorganic phosphate (Pi) is absorbed by proximal tubules through a cellular pathway that is inhibited by parathyroid hormone (PTH). The calcium-sensing receptor (CaSR) is expressed on apical membranes of proximal tubules. In the present studies, we determined the effect of luminal and/or basolateral PTH on phosphate absorption and tested the hypothesis that CaSR activation blocks PTH-inhibitable phosphate absorption. Single proximal S3 tubules were dissected from the kidneys of mice and studied by the Burg technique. Tubules were bathed with DMEM culture media supplemented with 6% BSA and perfused with an ultrafiltrate prepared from the bathing solution.33P and FITC-inulin were added to the luminal perfusate to measure phosphate absorption ( JPi) and fluid absorption ( Jv), respectively. JPiaveraged 2.9 pmol·min–1·mm–1under control conditions and decreased by 20% upon addition of serosal PTH. PTH had no effect on Jv. Inclusion of PTH in the luminal perfusate reduced JPito 2.1 pmol · min–1· mm–1. Combined addition of PTH to perfusate and bathing solutions reduced JPito 1.5 pmol · min–1· mm–1without affecting Jv. Indirect immunofluorescence studies revealed abundant PTH receptor (PTH1R) expression on brush-border membranes, with lower amounts on basolateral membranes. CaSRs were localized primarily, but not exclusively, to brush-border membranes. CaSR activation with luminal Gd3+abolished the inhibitory action of PTH on JPi. Addition of Gd3+to the serosal bathing solution had no effect on PTH-sensitive JPi. Gd3+i.e., PTH-independent JPi. Gd3+did not affect basal, had no effect on Jvwhen added to lumen or bath. Dopamine-inhibitable JPiwas not affected by Gd3+. Experiments with proximal-like opossum kidney cells showed that elevated extracellular Ca2+or NPS R467, a type II calcimimetic, inhibited PTH action on Piuptake. In conclusion, PTH1Rs are expressed on apical and basolateral membranes of mouse proximal tubules. Stimulating apical or basolateral PTH1R inhibits phosphate absorption. CaSR activation specifically regulates PTH-suppressible phosphate absorption.
Decreased abundance of collecting duct urea transporters UT-A1 and UT-A3 with ECF volume expansion
Authors: Wang Xiao-Yan , Beutler Kathleen , Nielsen Jakob , Nielsen Søren , Knepper Mark A. , Masilamani Shyama
Published: March 2015

Clinical disorders of extracellular fluid (ECF) volume regulation are often associated with changes in plasma urea concentration. To investigate possible renal causes, we measured the relative abundance of the urea transporters UT-A1, UT-A2, and UT-A3 in renal medulla of rats with aldosterone-induced NaCl retention. ECF volume-expanded rats received aldosterone by osmotic minipump plus a diet containing a high level of NaCl. Control rats received the same infusion of aldosterone plus a virtually NaCl-free diet, which prevented ECF volume expansion. Preliminary measurements demonstrated transient positive Na and water balance, decreased serum urea concentration, and increased urea clearance, but no change in creatinine clearance. Immunoblotting of homogenates from inner medulla showed a marked decrease in the abundance of the collecting duct urea transporters UT-A1 and UT-A3. There were no differences in the abundance of UT-A2, aquaporin (AQP)-2, AQP-3, or AQP-4 in ECF volume-expanded rats vs. controls. Time course experiments demonstrated that changes in UT-A1 abundance paralleled the fall in serum urea concentration after the switch from a low-NaCl to a high-NaCl diet, whereas the fall in UT-A3 abundance was delayed. Candesartan administration markedly decreased the abundance of UT-A1 and UT-A3 in the renal inner medulla, which is consistent with a role for the angiotensin II type 1 receptor in urea transport regulation. The results support the view that ECF-related changes in serum urea concentration are mediated, at least in part, through altered urea transporter abundance.
Renal interstitial hydrostatic pressure and natriuretic responses to volume expansion in pregnant rats
Authors: Khraibi Ali A. , Solhaug Michael J. , Dobrian Anca D. , Berndt Theresa J.
Published: March 2015

During normal pregnancy, a gradual plasma volume expansion (VE) occurs and reaches a maximum level at late term. Pressure natriuresis and renal interstitial hydrostatic pressure (RIHP) responses are attenuated in pregnant rats. Also, basal RIHP is lower in pregnant rats, suggesting an increase in renal interstitial compliance during pregnancy. This adaptation may contribute to the increase in plasma volume that is required for a normal pregnancy, because increases in RIHP have been consistently shown to produce natriuresis and diuresis. Acute saline VE (5% body wt/30 min) has been shown to increase RIHP in normal nonpregnant rats. Therefore, the objective of this study was to determine RIHP, natriuretic, and diuretic responses to VE in nonpregnant ( n = 7), midterm pregnant ( n = 8), and late-term pregnant ( n = 8) Sprague-Dawley rats. Although VE significantly increased RIHP, fractional excretion of sodium (FENa), and urine flow rate (V˙) in all groups, ΔRIHP was highest for nonpregnant (3.0 ± 0.3 mmHg) compared with midterm pregnant (1.6 ± 0.1 mmHg; P < 0.05 vs. nonpregnant) and late-term pregnant rats (1.2 ± 0.1 mmHg; P < 0.05 vs. both midterm pregnant and nonpregnant rats). ΔFENaand ΔV˙ were similar in all groups: 5.8 ± 1.0% and 231 ± 27 μl/min for nonpregnant, 6.8 ± 1.3% and 173 ± 16 μl/min for midterm pregnant, and 7.6 ± 1.2% and 203 ± 10 μl/min for late-term pregnant rats, respectively. In conclusion, basal RIHP and the increase in RIHP during VE were attenuated during pregnancy; however, the natriuretic and diuretic responses to VE remain intact during the course of pregnancy.
Regulation by glucocorticoids and osmolality of expression of ROMK (Kir 1.1), the apical K channel of thick ascending limb
Authors: Gallazzini Morgan , Attmane-Elakeb Amel , Mount David B. , Hebert Steven C. , Bichara Maurice
Published: March 2015

Mechanisms of regulation of ROMK channel mRNA and protein expression in medullary thick ascending limb (MTAL) were assessed in rat MTAL fragments incubated for 7 h. ROMK mRNA was quantified by quantitative RT-PCR and ROMK protein by immunoblotting analysis of crude membranes. Medium hyperosmolality (450 mosmol/kgH2O; NaCl plus urea added to isoosmotic medium) increased ROMK mRNA ( P < 0.04) and protein ( P < 0.006), and 10 nM dexamethasone also increased ROMK mRNA ( P < 0.02). Hyperosmolality and dexamethasone had no additive effects on ROMK mRNA. NaCl alone, but not urea or mannitol, reproduced the hyperosmolality effect on ROMK mRNA. 1-Deamino-(8-d-arginine) vasopressin (1 nM) or 0.5 mM 8-bromo-cAMP had no effect per se on ROMK mRNA and protein. However, 8-bromo-cAMP abolished the stimulatory effect of dexamethasone on ROMK mRNA in the isoosmotic but not in the hyperosmotic medium ( P < 0.004). In in vivo studies, the abundance of ROMK protein and mRNA increased in adrenalectomized (ADX) rats infused with dexamethasone compared with ADX rats ( P < 0.02). These results establish glucocorticoids and medium NaCl concentration as direct regulators of MTAL ROMK mRNA and protein expression, which may be modulated by cAMP-dependent factors.
Effects of renal denervation on tubular sodium handling in rats with CBL-induced liver cirrhosis
Authors: Jonassen Thomas E. N. , Brønd Lone , Torp Malene , Græbe Martin , Nielsen Søren , Skøtt Ole , Marcussen Niels , Christensen Sten
Published: March 2015

This study was designed to examine the effect of bilateral renal denervation (DNX) on thick ascending limb of Henle's loop (TAL) function in rats with liver cirrhosis induced by common bile duct ligation (CBL). The CBL rats had, as previously shown, sodium retention associated with hypertrophy of the inner stripe of the outer medulla (ISOM) and increased natriuretic effect of furosemide in vivo, and semiquantitative immunoblotting showed increased expression of the furosemide-sensitive Na-K-2Cl cotransporter type 2 (NKCC2) in ISOM from CBL rats. DNX significantly attenuated the sodium retention in the CBL rats, which was associated with normalization of the natriuretic effect of furosemide, as well as a significant reduction in the expression of NKCC2 in the ISOM. However, the marked hypertrophy of the ISOM found in CBL rats was not reversed by DNX. Together, these data indicate that increased renal sympathetic nerve activity known to be present in CBL rats plays a significant role in the formation of sodium retention by stimulating sodium reabsorption in the TAL via increased renal abundance of NKCC2.
Cell proliferation is insufficient, but loss of tuberin is necessary, for chemically induced nephrocarcinogenicity
Authors: Yoon Hae-Seong , Monks Terrence J. , Everitt Jeffrey I. , Walker Cheryl L. , Lau Serrine S.
Published: March 2015

Although 2,3,5-tris-(glutathion- S-yl)hydroquinone (TGHQ; 2.5 μmol/kg ip) markedly increased cell proliferation within the outer stripe of the outer medulla (OSOM) of the kidney in both wild-type ( Tsc2+/+ ) and mutant Eker rats ( Tsc2 EK/+), only TGHQ-treated Tsc2 EK/+ rats developed renal tumors, indicating that cell proliferation per se was not sufficient for tumor development. Tuberin expression was initially induced within the OSOM after TGHQ treatment but was lost within TGHQ-induced renal tumors. High extracellular signal-regulated kinase (ERK) activity occurred in the OSOM of Tsc2 EK/+ rats at 4 mo and in TGHQ-induced renal tumors. Cyclin D1 was also highly expressed in TGHQ-induced renal tumors. Reexpression of Tsc2 in tuberin-negative cells decreased ERK activity, consistent with the growth-suppressive effects of this tumor suppressor gene. Thus 1) stimulation of cell proliferation after toxicant insult is insufficient for tumor formation; 2) tuberin induction after acute tissue injury suggests that Tsc2 is an acute-phase response gene, limiting the proliferative response after injury; and 3) loss of Tsc2 gene function is associated with cell cycle deregulation.
Melatonin reduces renal interstitial inflammation and improves hypertension in spontaneously hypertensive rats
Authors: Nava Mayerly , Quiroz Yasmir , Vaziri Nosratola , Rodríguez-Iturbe Bernardo
Published: March 2015

Several studies have demonstrated that treatment with antioxidants improves hypertension in spontaneously hypertensive rats (SHR). Because our laboratory has shown that renal infiltration of immune cells plays a role in the development of hypertension (Rodriguez-Iturbe B, Quiroz Y, Nava M, Bonet L, Chavez M, Herrera-Acosta J, Johnson RJ, and Pons HA. Am J Physiol Renal Physiol 282: F191–F201, 2002), we did the present studies to define whether the antihypertensive effect of antioxidants was associated with an improvement in renal inflammation. Melatonin was administered as an antioxidant. For 6 wk, melatonin was added to the drinking water (10 mg/100 ml) given to a group of SHR (SHR-Mel; n = 10), and we compared them with groups of untreated SHR ( n = 10) and Wistar-Kyoto (WKY) control rats ( n = 10). Hypertension became increasingly severe in the SHR group [195 ± 14.3 (SD) mmHg at the end of the experiment] and improved in the SHR-Mel group (149 ± 20.4 mmHg, P < 0.001) in association with a 40–60% reduction in the renal infiltration of lymphocytes, macrophages, and angiotensin II-positive cells. Intracellular superoxide and renal malondialdehyde content were reduced by melatonin treatment as was the immunohistological expression of the 65-kDA DNA-binding subunit of NF-κB. We conclude that melatonin treatment ameliorates hypertension in SHR in association with a reduction in interstitial renal inflammation. Decreased activation of NF-κB, likely resulting from a reduction in local oxidative stress, may play a role in the suppression of renal immune infiltration and, thereby, in the antihypertensive effects of melatonin.
Glucose-induced changes in integrins and matrix-related functions in cultured human glomerular epithelial cells
Authors: Kitsiou Paraskevi V. , Tzinia Athina K. , Stetler-Stevenson William G. , Michael Alfred F. , Fan Wei-Wei , Zhou Bing , Tsilibary Effie C.
Published: March 2015

In cultured human glomerular epithelial cells (HGEC), 25 mM glucose resulted in decreased expression of α3-, α2-, and β1-integrins and increased expression of α5- and αvβ3-integrins. This change was accompanied by decreased binding of HGEC to type IV collagen. In the presence of normal (5 mM) glucose concentration, cell binding to type IV collagen was primarily mediated by α2β1- and α5β1-integrins, as indicated by experiments in which cell adhesion to type IV collagen was competed by specific anti-integrin monoclonal antibodies. In the presence of high (25 mM) glucose, the upregulated α5- and αvβ3-integrins were mainly involved in cell binding to type IV collagen. Furthermore, high glucose decreased expression of matrix metalloproteinase-2 (MMP-2), a collagenase regulated in part by α3β1-integrin, as suggested by the use of ligand-mimicking antibodies against these integrins, which resulted in release of increased amounts of MMP-2 in the culture medium. Finally, tissue inhibitor of metalloproteinase-2, the specific inhibitor of MMP-2, was upregulated in high glucose and could contribute to matrix accumulation. These changes could help explain basement membrane thickening in diabetes.
Interaction with grp58 increases activity of the thiazide-sensitive Na-Cl cotransporter
Authors: Wyse Bruce , Ali Nawb , Ellison David H.
Published: March 2015

The thiazide-sensitive sodium-chloride cotransporter (NCC) is expressed by distal convoluted tubule cells of the mammalian kidney. We used yeast two-hybrid screening to identify that glucose-regulated protein 58 (grp58), a protein induced by glucose deprivation, binds to the COOH terminus of the NCC. Immunoprecipitation of rat kidney cortex homogenates using a guinea pig anti-NCC antibody confirmed that grp58 associates with the NCC in vivo. Northern blots indicated that grp58 is highly expressed in rat kidney cortex. Immunofluorescence showed that grp58 protein abundance in kidney is highest in epithelial cells of the distal nephron, where it colocalizes with NCC near the apical membrane. To determine whether this interaction has a functional significance, NCC and grp58 cRNA were coexpressed in Xenopus laevis oocytes. In oocytes overexpressing grp58, sodium uptake was increased compared with control. Because oocytes express endogenous grp58, antisense experiments were performed to evaluate whether endogenous grp58 affected NCC activity in oocytes. Sodium uptake was lower in oocytes injected with both antisense grp58 cRNA and sense NCC compared with sense NCC oocytes. Western blot analysis did not show any effect of grp58 expression on processing of the NCC. These data indicate a novel, functionally important interaction between grp58 and the NCC in rat kidney cortex.
Hyperuricemia induces a primary renal arteriolopathy in rats by a blood pressure-independent mechanism
Authors: Mazzali Marilda , Kanellis John , Han Lin , Feng Lili , Xia Yi-Yang , Chen Qiang , Kang Duk-Hee , Gordon Katherine L. , Watanabe Susumu , Nakagawa Takahiko , Lan Hui Y. , Johnson Richard J.
Published: March 2015

Hyperuricemia is associated with hypertension and vascular disease, but whether this represents a causal relationship or an epiphenomenon remains unknown. We recently reported a model of mild hyperuricemia in rats that results in increased blood pressure and mild renal fibrosis. In this study, we examined the effect of hyperuricemia on the renal vasculature. Rats fed 2% oxonic acid and a low-salt diet for 7 wk developed mild hyperuricemia (1.8 vs. 1.4 mg/dl, P < 0.05), hypertension [147 vs. 127 mmHg systolic blood pressure (SBP), P < 0.05], and afferent arteriolar thickening, with a 35% increase in medial area ( P < 0.05). Allopurinol or benziodarone prevented the hyperuricemia, hypertension, and arteriolopathy. Hydrochlorothiazide treatment did not prevent the hyperuricemia or arteriolopathy despite controlling blood pressure. In contrast, the arteriolopathy and hypertension were prevented by both enalapril and losartan. Uric acid also directly stimulated vascular smooth muscle cell proliferation in vitro, and this was partially inhibited by losartan. Thus hyperuricemia induces a renal arteriolopathy in rats that is blood pressure independent and involves the renin-angiotensin system.
Cell death induced by acute renal injury: a perspective on the contributions of apoptosis and necrosis
Authors: Padanilam Babu J.
Published: March 2015

In humans and experimental models of renal ischemia, tubular cells in various nephron segments undergo necrotic and/or apoptotic cell death. Various factors, including nucleotide depletion, electrolyte imbalance, reactive oxygen species, endonucleases, disruption of mitochondrial integrity, and activation of various components of the apoptotic machinery, have been implicated in renal cell vulnerability. Several approaches to limit the injury and augment the regeneration process, including nucleotide repletion, administration of growth factors, reactive oxygen species scavengers, and inhibition of inducers and executioners of cell death, proved to be effective in animal models. Nevertheless, an effective approach to limit or prevent ischemic renal injury in humans remains elusive, primarily because of an incomplete understanding of the mechanisms of cellular injury. Elucidation of cell death pathways in animal models in the setting of renal injury and extrapolation of the findings to humans will aid in the design of potential therapeutic strategies. This review evaluates our understanding of the molecular signaling events in apoptotic and necrotic cell death and the contribution of various molecular components of these pathways to renal injury.
Expression of myosin VI within the early endocytic pathway in adult and developing proximal tubules
Authors: Biemesderfer Daniel , Mentone Sue Ann , Mooseker Mark , Hasson Tama
Published: March 2015

Myosin VI is a reverse-direction molecular motor implicated in membrane transport events. Because myosin VI is most highly expressed in the kidney, we investigated its renal localization by using high-resolution immunocytochemical and biochemical methods. Indirect immunofluorescence microscopy revealed myosin VI at the base of the brush border in proximal tubule cells. Horseradish peroxidase uptake studies, which labeled endosomes, and double staining for clathrin adapter protein-2 showed that myosin VI was closely associated with the intermicrovillar (IMV) coated-pit region of the brush border. Localization of myosin VI to the IMV region was confirmed at the electron microscopic level by colloidal gold labeling of ultrathin cryosections. In addition, antigen retrieval demonstrated a small but significant pool of myosin VI on the microvilli. To confirm the association of myosin VI with the IMV compartment, these membranes were separated from other membrane compartments by using 15–25% OptiPrep density gradients. Immunoblotting of the gradient fractions confirmed that myosin VI was enriched with markers for the IMV microdomain of the brush border, suggesting that myosin VI associates with proteins in this compartment. Finally, we examined the expression of myosin VI during nephron development. We found myosin VI present in a diffuse cytoplasmic pattern at stage II (S-shaped body phase) and that it was only redistributed fully to the brush border in the stage IV nephron. These studies support a model for myosin VI function in the endocytic process of the proximal tubule.
Podocyte slit-diaphragm protein nephrin is linked to the actin cytoskeleton
Authors: Yuan Huaiping , Takeuchi Emiko , Salant David J.
Published: March 2015

Nephrin is an Ig-like transmembrane protein. It is a major component of the podocyte slit diaphragm and is essential for maintaining normal glomerular permeability. CD2-associated protein (CD2AP) is also necessary for normal glomerular permeability and is a putative nephrin adapter molecule. Here, we document that nephrin and CD2AP are linked to the actin cytoskeleton. As detected by Western blot analysis, nephrin and CD2AP were both insoluble when cell membranes from normal rat glomeruli were extracted with 0.5% Triton X-100 (TX-100) at 4°C in the presence of divalent cations, but they were solubilized when the extraction included potassium iodide (KI) to depolymerize F-actin. In addition, a small fraction of the solubilized nephrin and CD2AP was recovered in the low-density fractions of OptiPrep flotation gradients, which indicates that a portion of nephrin, possibly associated with CD2AP, resides in a cholesterol- or sphingolipid-rich region of the plasma membrane. Immunofluorescent staining of unfixed sections of normal rat kidney for nephrin, CD2AP, and F-actin was unaltered by treatment with TX-100 but was greatly diminished by addition of KI. Nephrin staining was slightly reduced by cholesterol depletion with methyl-β-cyclodextrin in the presence of TX-100 but was nearly absent after addition of KI. These results document that nephrin anchors the slit diaphragm to the actin cytoskeleton, possibly by linkage to CD2AP, and that nephrin traverses a relatively cholesterol-poor region of the podocyte plasma membrane. In addition, a small pool of actin-associated nephrin and CD2AP resides in lipid rafts, possibly in the cholesterol-rich apical region of the podocyte-foot processes.
Rottlerin inhibits tonicity-dependent expression and action of TonEBP in a PKCδ-independent fashion
Authors: Zhao Hongyu , Tian Wei , Cohen David M.
Published: March 2015

Novel protein kinase C (PKC) isoforms PKCδ and PKCε have recently been implicated in signaling by hypertonic stress. We investigated the role of the putative PKCδ inhibitor rottlerin on tonicity-dependent gene regulation. In the renal medullary mIMCD3 cell line, rottlerin blocked tonicity-dependent transcription of a tonicity enhancer (TonE)-driven luciferase reporter gene, as well as tonicity-dependent transcription of the physiological tonicity effector gene aldose reductase, but not urea-dependent transcription. Consistent with these data, rottlerin inhibited tonicity-dependent expression of TonE binding protein (TonEBP) at the mRNA and protein levels. Another inhibitor of both novel and conventional PKC isoforms, GF-109203X, suppressed TonEBP-dependent transcription but failed to influence tonicity-inducible TonEBP expression. Global PKC downregulation with protracted phorbol ester treatment, however, failed to influence tonicity-dependent signaling, arguing against a PKCδ-dependent mechanism of rottlerin action in this model. In addition, hypertonic stress failed to induce phosphorylation of PKCδ. Furthermore, in a PC-12 cell model with a comparable degree of tonicity-dependent transcription, constitutive overexpression of dominant negative-acting PKCδ or PKCε effectively decreased tonicity signaling to extracellular signal-regulated kinase activation, as expected, but failed to influence TonE-dependent transcription. TonE-dependent transcription, however, remained rottlerin sensitive in this PC-12 cell model. In the aggregate, these data indicate that rottlerin dramatically inhibits tonicity-dependent TonEBP expression and TonE-dependent transcription but, despite its reputed mode of action, does so through a PKCδ-independent pathway.
Biphasic regulation of plasminogen activator/inhibitor by LDL in mesangial cells
Authors: Song Chi Young , Kim Bong Cho , Hong Hye Kyoung , Kim Byoung Kwon , Kim Young Sook , Lee Hyun Soon
Published: March 2015

Lipid abnormalities and dysregulation of the plasminogen activator (PA)/plasmin system may be involved in the development of glomerulosclerosis. We investigated the effects of low-density lipoprotein (LDL) on PA inhibitor-1 (PAI-1), urokinase-type PA (uPA), and tissue-type PA (tPA) in relationship to protein kinase C (PKC) in cultured human mesangial cells (HMC). LDL (200 μg/ml) induced two peaks of PKC activation at hours 0.25 and 6, with translocation of PKC-α, -β1, and -δ from cytosol to the membrane. The second increase in PKC activity gradually decreased to the control value by hour 18. LDL downregulated 2.4-kb PAI-1, uPA, and tPA mRNA expression within 6 h of incubation with HMC. On the other hand, after 12–48 h, LDL-treated cells showed a significant increase in PAI-1, tPA, and uPA mRNA levels. LDL induced up to a twofold increase in PAI-1 antigen levels in the extracellular matrix of HMC after 24–48 h as well as increased PA inhibitory activity in the culture medium. Analysis of the adhesion plaques from cells incubated with LDL for 48 h by zymography showed increased intensity of lysis near molecular weights of ∼55,000 and 100,000. LDL slightly increased tPA release at hours 24 and 48 but did not increase PA activity in culture medium. The stimulatory effects of LDL on PAI-1, tPA, and uPA gene regulation in HMC were blocked by the inhibition of PKC using GF-109203X 12 h after treatment with LDL or downregulation of PKC using phorbol myristate acetate. In summary, LDL regulates PAI-1, uPA, and tPA in biphasic patterns in HMC, and the upregulation of PAI-1, uPA, and tPA after long-term LDL exposure seems to be mediated by a delayed PKC activation associated with an increased PA inhibitory activity. These results suggest that LDL, after prolonged incubations with HMC, causes a PA/inhibitor imbalance favoring accumulation of matrix.
Antioxidant ameliorates cisplatin-induced renal tubular cell death through inhibition of death receptor-mediated pathways
Authors: Tsuruya Kazuhiko , Tokumoto Masanori , Ninomiya Toshiharu , Hirakawa Makoto , Masutani Kohsuke , Taniguchi Masatomo , Fukuda Kyoichi , Kanai Hidetoshi , Hirakata Hideki , Iida Mitsuo
Published: March 2015

We have recently demonstrated the direct involvement of the death receptor-mediated apoptotic pathways in cisplatin-induced renal tubular cell (RTC) death. Reactive oxygen species are thought to be a major cause of cellular damage in such injury. The aim of this study was to examine the mechanism through which antioxidants ameliorate cisplatin-induced RTC death, with special emphasis on death receptor-mediated apoptotic pathways. Cisplatin was added to cultures of normal rat kidney (NRK52E) cells or injected in rats. NRK52E cells and rats were also treated with dimethylthiourea (DMTU), a hydroxyl radical scavenger. We then examined the mRNA levels of death ligands and receptors, caspase-8 activity, cell viability, cell death, renal function, and histological alterations. RT-PCR indicated cisplatin-induced upregulation of Fas, Fas ligand, and TNF-α mRNAs and complete inhibition by DMTU in vitro and in vivo. Cisplatin increased caspase-8 activity of NRK52E cells, and DMTU prevented such activation. Exposure to cisplatin reduced viability of NRK52E cells, examined by WST-1 assay, and increased apoptosis and necrosis of the cells, examined by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and fluorescence-activated cell sorter analysis. DMTU abrogated cisplatin-induced changes in cell viability and apoptosis and/or necrosis. Cisplatin-induced renal dysfunction and histological damage were also prevented by DMTU. DMTU did not hinder cisplatin incorporation into RTCs. Our results suggest that antioxidants can ameliorate cisplatin-induced acute renal failure through inactivation of the death receptor-mediated apoptotic pathways.
Reduced osmotically inactive Na storage capacity and hypertension in the Dahl model
Authors: Titze Jens , Krause Holger , Hecht Hermann , Dietsch Peter , Rittweger Jörn , Lang Rainer , Kirsch Karl A. , Hilgers Karl F.
Published: March 2015

Recent evidence suggested that Na can be stored in an osmotically inactive form. We investigated whether osmotically inactive Na storage is reduced in a rat model of salt-sensitive (SS) hypertension. SS and salt-resistant (SR) Dahl-Rapp rats as well as Sprague-Dawley (SD) rats were fed a high (8%)- or low (0.1%)-NaCl diet for 4 wk ( n = 10/group). Mean arterial pressure (MAP) was measured at the end of the experiment. Wet and dry weights, water content, total body Na (TBS), and bone Na content were measured by dessication and dry ashing. MAP was higher in both Dahl strains than in SD rats. In SS rats, 8% NaCl led to Na accumulation, water retention, and hypertension due to impaired renal Na excretion. There was no dietary-induced Na retention in SR and SD rats. TBS was variable; nevertheless, TBS was significantly correlated with body water and MAP in all strains. However, the extent of Na-associated volume and MAP increases was strain specific. Osmotically inactive Na in SD rats was threefold higher than in SS and SR rats. Both SS and SR Dahl rat strains displayed reduced osmotically inactive Na storage capacity compared with SD controls. A predisposition to fluid accumulation and high blood pressure results from this alteration. Additional factors, including impaired renal Na excretion, probably contribute to hypertension in SS rats. Our results draw attention to the role of osmotically inactive Na storage.
General inhibition of renocortical cyclooxygenase-2 expression by the renin-angiotensin system
Authors: Castrop Hayo , Klar Jürgen , Wagner Charlotte , Höcherl Klaus , Kurtz Armin
Published: March 2015

Because across-the-board data indicate that renin and cyclooxygenase-2 (COX-2) expression in the kidney cortex are regulated in parallel and because ANG II can inhibit COX-2 expression, the purpose of our study was to characterize a potential general inhibitory feedback of the renin-angiotensin system on renocortical COX-2 expression in vivo. Rats were fed a high-, normal-, or low-salt diet or were chronically infused with furosemide (60 mg · kg−1 · day−1) or the left renal artery was clipped, and the animals were treated in addition to or without the angiotensin-converting enzyme inhibitor ramipril (10 mg · kg−1 · day−1). A high-salt diet reduced expression of COX-2, whereas a low-salt diet, furosemide infusion, and renal artery stenosis stimulated COX-2 expression. Additional angiotensin-converting enzyme inhibition led to further increases in renocortical COX-2 expression by 62, 136, 300, 50, and 70% for a high-, normal-, and low-salt diet, furosemide infusion, and renal artery stenosis, respectively. Thus our data suggest a general inhibitory effect of the renin-angiotensin system on renocortical COX-2 expression.
Expression and function of sodium transporters in two opossum kidney cell clonal sublines
Authors: Gomes Pedro , Xu Jing , Serrão Paula , Dória Sofia , Jose Pedro A. , Soares-da-Silva Patrício
Published: March 2015

The present study describes characteristic features of two clonal subpopulations of opossum kidney (OK) cells (OKLCand OKHC) that are functionally different but morphologically identical. The most impressive differences between OKHCand OKLCcells are the overexpression of Na+-K+-ATPase and type 3 Na+/H+exchanger by the former, accompanied by an increased Na+-K+-ATPase activity (57.6 ± 5.6 vs. 30.0 ± 0.1 nmol Pi· mg protein−1· min−1); the increased ability to translocate Na+from the apical to the basolateral surface; and the increased Na+-dependent pHirecovery (0.254 ± 0.016 vs. 0.094 ± 0.011 pH units/s). Vmaxvalues (in pH units/s) for Na+-dependent pHirecovery in OKHCcells (0.00521 ± 0.0004) were twice ( P < 0.05) those in OKLC(0.00202 ± 0.0001), with similar Kmvalues (in mM) for Na+(OKLC, 21.0 ± 5.5; OKHC, 14.0 ± 5.6). In addition, we measured the activities of transporters (organic ions, α-methyl-d-glucoside, l-type amino acids, and Na+) and enzymes (adenylyl cyclase, aromaticl-amino acid decarboxylase, and catechol- O-methyltransferase). The cells were also characterized morphologically by optical and scanning electron microscopy and karyotyped. It is suggested that OKLCand OKHCcells constitute an interesting cell model for the study of renal epithelial physiology and pathophysiology, namely, hypertension.
Water transport in neonatal and adult rabbit proximal tubules
Authors: Quigley Raymond , Baum Michel
Published: March 2015

We have recently demonstrated that although the osmotic water permeability ( Pf) of neonatal proximal tubules is higher than that of adult tubules, the Pfof brush-border and basolateral membrane vesicles from neonatal rabbits is lower than that of adults. The present study examined developmental changes in the water transport characteristics of proximal convoluted tubules (PCTs) in neonatal (9–16 days old) and adult rabbits to determine whether the intracellular compartment or paracellular pathway is responsible for the maturational difference in transepithelial water transport. The permeability of n-butanol was higher in the neonatal PCT than the adult PCT at all temperatures examined, whereas the diffusional water permeability was identical. Increasing the osmotic gradient increased volume absorption in both the neonatal and the adult PCT to the same degree. The Pfwas not different between the neonatal and the adult PCT at any osmotic gradient studied. To assess solvent drag as a measure of the paracellular transport of water, the effect of the osmotic gradient on mannitol and chloride transport were measured. There was no change in chloride or mannitol transport with the increased osmotic gradient in either group, indicating that there was no detectable paracellular water movement. In addition, the mannitol permeability of the neonatal PCT was found to be lower than that of the adult PCT with the isotonic bath (8.97 ± 4.01 vs. 40.49 ± 13.89 μm/s, P < 0.05). Thus the intracellular compartment of the neonatal PCT has a lower resistance for water transport than the adult PCT and is responsible for the higher than expected Pfin the neonatal PCT.
Role of prostanoids in regulation of the renin-angiotensin-aldosterone system by salt intake
Authors: Höcherl Klaus , Kammerl Martin C. , Schumacher Karl , Endemann Dirk , Grobecker Horst F. , Kurtz Armin
Published: March 2015

We investigated the effect of cyclooxygenase (COX) activity on the regulation of the renin-angiotensin-aldosterone system by salt intake. Therefore, Sprague-Dawley rats were subjected to different salt diets [0.02, 0.6, and 8% NaCl (wt/wt)] and treated with the selective COX-2 inhibitor rofecoxib (10 mg · kg body wt−1· day−1) or with ketorolac at a dose selective for COX-1 inhibition (2 mg · kg body wt−1· day−1) for 3, 7, 14, and 21 days. Rofecoxib and ketorolac caused a similar reduction of renocortical PGE2formation with a low-salt diet. Rofecoxib did not change plasma renin activity or renocortical renin mRNA abundance with any of the diets but clearly lowered plasma aldosterone concentration. In contrast, ketorolac delayed the increase in plasma renin activity and of renin mRNA in response to low salt intake but did not change plasma aldosterone concentration. Prolonged treatment with rofecoxib but not with ketorolac caused an upregulation of COX-2 expression while COX-1 mRNA abundance remained unchanged. These findings suggest that COX-1-derived, but not COX-2-derived, prostanoids are of relevance for the regulation of the renin system by salt intake.
Role of MAPK pathways in light chain-induced cytokine production in human proximal tubule cells
Authors: Sengul Sule , Zwizinski Craig , Batuman Vecihi
Published: March 2015

We previously demonstrated that light chain (LC) endocytosis by human proximal tubule cells (PTCs) leads to production of cytokines through activation of NF-κB. Here, we examined the role of MAPK pathways in these responses using four species of myeloma LCs (κ1, κ2, κ3, and λ1) previously shown to induce cytokine production by PTCs. Among these, κ1-LC, which yielded the strongest cytokine responses, was selected for detailed studies. Activation of MAPKs was probed by Western blot analysis for the active kinases, ERK 1/2, JNK 1/2, and p38 in κ1-LC-exposed human PTCs. To evaluate the functional role of MAPKs in LC-induced cytokine responses, we tested the effects of U-0126, an ERK inhibitor; SP-600125, an inhibitor of JNK; SB-203580, a p38 inhibitor; and curcumin, a JNK-AP-1 inhibitor, all added to media before 4-h exposure to 1.5 mg/ml κ1-LC. IL-6 and monocyte chemotactic protein-1 (MCP-1) were determined by ELISA. Both LC and human serum albumin (HSA) activated ERK, although the HSA effect was weaker. κ1-LC stimulated all three MAPKs, although phosphorylation of ERK was more pronounced and sustained than others. Inhibitors of ERK, JNK, and p38 reduced LC-induced IL-6 and MCP-1 production. These findings suggest that activation of MAPKs plays a role in LC-induced cytokine responses in PTCs. Activation of MAPKs may be involved in cytokine responses induced by other proteins as well as LCs and may be pivotal in the pathophysiology of tubulointerstitial injury in proteinuric diseases.
Urea transporters are distributed in endothelial cells and mediate inhibition ofl-arginine transport
Authors: Wagner Laszlo , Klein Janet D. , Sands Jeff M. , Baylis Chris
Published: March 2015

Our laboratory previously reported that uremic levels of urea inhibitl-arginine (l-Arg) transport into endothelial cells. The present study further investigated this effect. We measuredl-Arg transport in cultured bovine aortic endothelial cells with normal or high urea (25 mM). The urea transport inhibitor phloretin abolished the inhibitory effect of urea on l-Arg transport, suggesting a role for urea transporters (UTs). We screened bovine aortic endothelial cells and several other endothelial cell types for the presence of UTs by using Western blot analysis. UT-B was present in all endothelial cells, irrespective of species or location of derivation, whereas UT-A distribution was variable and sparse. UT-B was also abundant in rat aorta, mesenteric blood vessels, and spinotrapezius muscle, whereas UT-A distribution was, again, variable and sparse. Chronic elevation of urea had variable, inconsistent effects on UT abundance. This study showed that urea must enter endothelial cells, probably by UT-B, to inhibit l-Arg transport. In view of the wide distribution of UT-B in rat vasculature, elevated blood urea nitrogen may lead to endothelial l-Arg deficiency in vivo.
Nystatin and valinomycin induce tubuloglomerular feedback
Authors: Ren Yilin , Yu Hong , Wang Hong , Carretero Oscar A. , Garvin Jeffrey L.
Published: March 2015

First published September 21, 2001; 10.1152/ajprenal.00357.2001.—The macula densa expresses a luminal Na+-K+-2Cl−cotransporter and a basolateral Cl−conductance. Although it is known that cotransport of Na+, K+, and Cl−is the first step in tubuloglomerular feedback (TGF), subsequent steps are unclear. We hypothesized that Na+-K+-2Cl−entry via the luminal Na+-K+-2Cl−cotransporter elevates intracellular Cl−, increases electrogenic Cl−efflux across the basolateral membrane, and depolarizes the macula densa, initiating TGF. We perfused afferent arterioles with macula densa attached. The macula densa was perfused with solutions containing either 5 mM Na+and 3 mM Cl−(low NaCl) or 80 mM Na+and 77 mM Cl−(high NaCl). When the macula densa perfusate was changed from low to high NaCl, afferent arteriole diameter decreased from 15.8 ± 0.8 to 13.1 ± 0.7 mm ( P < 0.05). Adding 10 μM furosemide to the macula densa lumen blocked TGF. When nystatin, a group I cation ionophore, was added to the macula densa lumen together with furosemide in the presence of low NaCl, it induced TGF (from 18.0 ± 1.5 to 15.6 ± 1.6 mm; P = 0.003). When valinomycin, a K+-selective ionophore, was added to the macula densa lumen together with furosemide in the presence of low NaCl containing 5 mM K+, it did not induce TGF. Subsequent addition of 50 mM KCl to the macula densa perfusate induced TGF (from 21.7 ± 0.8 to 17.5 ± 1.3 mm; P = 0.0047; n = 6). Adding 50 mM KCl without valinomycin did not induce TGF. When 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 1 μM), a Cl−channel blocker, was added to the bath, it blocked TGF induced by high NaCl, but did not block TGF induced by valinomycin plus 50 mM KCl. NPPB did not alter afferent arteriole constriction induced by norepinephrine. We concluded that increased NaCl in the lumen of the macula densa leads to influx of Cl−via the Na+-K+-2Cl−cotransporter. The accelerated transport increases intracellular Cl−. The subsequent exit of Cl−across the basolateral membrane via Cl−channels in turn leads to depolarization of the macula densa and thereby induces TGF.
Higher basal serine phosphorylation of D1Areceptors in proximal tubules of old Fischer 344 rats
Authors: Asghar Mohammad , Hussain Tahir , Lokhandwala Mustafa F.
Published: March 2015

Dopamine (DA) and D1-like receptor agonists promote an increase in Na excretion by means of activation of the D1-like receptor signaling cascade and subsequent inhibition of the Na/H exchanger and Na-K-ATPase in renal proximal tubules. Recently, our laboratory reported that DA and the D1-like receptor agonist failed to inhibit Na-K-ATPase activity in old Fischer 344 rats because of uncoupling of D1Areceptors from G proteins and that this abnormality led to a diminished natriuretic response to DA in old Fischer 344 rats. In this study, we have tested the hypothesis that the mechanism of this uncoupling may be an altered phosphorylation of D1Areceptors in old rats. In experiments performed in renal cortical slices, both DA and SKF-38393, a D1-like receptor agonist, increased the serine phosphorylation of D1Areceptors in adult (6 mo) but not old (24 mo) rats. Interestingly, the basal serine phosphorylation of D1Areceptors was higher in old than in adult rats. Competition ligand binding ([3H]SCH-23390) experiments on the D1-like receptor in adult and old rats with fenoldopam, a D1-like receptor agonist, revealed the presence of two affinity states of the receptors. There was a rightward shift in the agonist displacement of the ligand in old compared with adult rats, as reflected in the IC50values (adult vs. old, 7.46 × 10−9± 2.26 vs. 7.93 × 10−7± 1.33 M). Also, there was a reduction in agonist affinity in the low-affinity receptors in old compared with adult rats (IC50, adult vs. old, 5.67 × 10−5± 1.33 vs. 12.60 × 10−5± 6.50 M). Moreover, the abundance of D1Areceptor proteins was ∼47% lower in the membranes of old compared with adult rats. We speculate that higher basal serine phosphorylation of D1Areceptors may have rendered the D1Areceptor uncoupled from G protein, leading to a reduced agonist affinity state and thus diminished natriuretic response to DA in old rats.
Prospect of a stanniocalcin endocrine/paracrine system in mammals
Authors: Ishibashi Kenichi , Imai Masashi
Published: March 2015

Stanniocalcin (STC) is a calcium- and phosphate-regulating hormone produced in bony fish by the corpuscles of Stannius, which are located close to the kidney. It is a major antihypercalcemic hormone in fish. As the corpuscles of Stannius are absent, and antihypercalcemic hormones are basically not necessary, in mammals, the discovery of a mammalian homolog, STC1, was surprising and intriguing. STC1 displays a relatively high amino acid sequence identity (∼50%) with fish STC. In contrast to fish STC, STC1 is expressed in many tissues, including kidney. More recently, a human gene encoding the second stanniocalcin-like protein, STC2, was identified. STC2 has a lower identity (∼35%) with STC1 and fish STC. Similar to STC1, STC2 is also expressed in a variety of tissues. Research into the functions of STCs in mammals is still at an early stage, and the ultimate physiological and pathological roles of STCs have not yet been established. A few studies indicate that STC1, similar to fish STC, stimulates phosphate absorption in the kidney and intestine, but the function of STC2 is still unknown. However, several interesting findings have been reported on their cellular localization, gene structure, and expression in different physiological and pathological conditions, which will be clues in elucidating the functions of STCs in mammals. STC1 expression is enhanced by hypertonicity in a kidney cell line or by ischemic injuries and neural differentiation in the brain. STC1 expression in the ovary is also enhanced during pregnancy and lactation. Calcitriol upregulates STC1 and downregulates STC2 expression in the kidney. Interestingly, STC1 and STC2 are expressed in many tumor cell lines, and the expression of STC2 is enhanced by estradiol in breast cancer cells. STC2 is also expressed in pancreatic islets. These results suggest that the biological repertoires of STCs in mammals will be considerably larger than in fish and may not be limited to mineral metabolism. This brief review describes recent progress in mammalian STC research.
Generation and phenotypic analysis of CHIF knockout mice
Authors: Aizman Roman , Asher Carol , Füzesi Maria , Latter Hedva , Lonai Peter , Karlish Steven J. D. , Garty Haim
Published: March 2015

Corticosteroid hormone-induced factor (CHIF) is a short epithelial-specific protein that is independently induced by aldosterone and a high-K+diet. It is a member of the FXYD family of single-span transmembrane proteins that include phospholemman, Mat-8, and the γ-subunit of Na+-K+-ATPase. A number of studies have suggested that these proteins are involved in the regulation of ion transport and, in particular, functionally interact with the Na+-K+-ATPase. The present study describes the characterization, targeted disruption, and phenotypic analysis of the mouse CHIF gene. The CHIF knockout mice are viable and not distinguishable from wild-type littermates under normal conditions. Under K+loading, they have a twofold higher urine volume and an increased glomerular filtration rate. Similar but smaller effects are observed in mice fed a low-Na+diet. Treating K+-loaded mice for 10 days with furosemide resulted in lethality in the knockout mice (17 of 39) but not in the wild-type group (1 of 39). The data are consistent with an effect of CHIF on the Na+-K+-ATPase that is specific to the outer and inner medullary duct, its major expression site.
Relationships between caveolae and eNOS: everything in proximity and the proximity of everything
Authors: Goligorsky Michael S. , Li Hong , Brodsky Sergey , Chen Jun
Published: March 2015

Caveolae, flask-shaped invaginations of the plasma membrane occupying up to 30% of cell surface in capillaries, represent a predominant location of endothelial nitric oxide synthase (eNOS) in endothelial cells. The caveolar coat protein caveolin forms high-molecular-weight, Triton-insoluble complexes through oligomerization mediated by interactions between NH2-terminal residues 61–101. eNOS is targeted to caveolae by cotranslational N-myristoylation and posttranslational palmitoylation. Caveolin-1 coimmunoprecipitates with eNOS; interaction with eNOS occurs via the caveolin-1 scaffolding domain and appears to result in the inhibition of NOS activity. The inhibitory conformation of eNOS is reversed by the addition of excess Ca2+/calmodulin and by Akt-induced phosphorylation of eNOS. Here, we shall dissect the system using the classic paradigm of a reflex loop: 1) the action of afferent elements, such as fluid shear stress and its putative caveolar sensor, on caveolae; 2) the ways in which afferent signals may affect the central element, the activation of the eNOS-nitric oxide system; and 3) several resultant well-established and novel physiologically important effector mechanisms, i.e., vasorelaxation, angiogenesis, membrane fluidity, endothelial permeability, deterrance of inflammatory cells, and prevention of platelet aggregation.