bluuebunny/crossref_metadata_2025 huggingface dataset:
Redox regulation of HIF-1α levels and HO-1 expression in renal medullary interstitial cells
Authors: Yang Zhi-Zhang , Zhang Andrew Y. , Yi Fu-Xian , Li Pin-Lan , Zou Ai-Ping
Published: March 2015

The present study hypothesized that superoxide (O[Formula: see text]·) importantly contributes to the regulation of hypoxia-inducible factor (HIF)-1α expression at posttranscriptional levels in renal medullary interstitial cells (RMICs) of rats. By Western blot analysis, it was found that incubation of RMICs with O[Formula: see text]· generators xanthine/xanthine oxidase and menadione significantly inhibited the hypoxia- or CoCl2-induced increase in HIF-1α levels and completely blocked the increase in HIF-1α levels induced by ubiquitin-proteasome inhibition with CBZ-LLL in the nuclear extracts from these cells. Under normoxic conditions, a cell-permeable O[Formula: see text]· dismutase (SOD) mimetic, 4-hydroxyl-tetramethylpiperidin-oxyl (TEMPOL) and PEG-SOD, significantly increased HIF-1α levels in RMICs. Two mechanistically different inhibitors of NAD(P)H oxidase, diphenyleneiodonium and apocynin, were also found to increase HIF-1α levels in these renal cells. Moreover, introduction of an anti-sense oligodeoxynucleotide specific to NAD(P)H oxidase subunit, p22phox, into RMICs markedly increased HIF-1α levels. In contrast, the OH· scavenger tetramethylthiourea had no effect on the accumulation of HIF-1α in these renal cells. By Northern blot analysis, scavenging or dismutation of O[Formula: see text]· by TEMPOL and PEG-SOD was found to increase the mRNA levels of an HIF-1α-targeted gene, heme oxygenase-1. These results indicate that increased intracellular O[Formula: see text]· levels induce HIF-1α degradation independently of H2O2and OH· radicals in RMICs. NAD(P)H oxidase activity may importantly contribute to this posttranscriptional regulation of HIF-1α in these cells under physiological conditions.
Crescentic glomerulonephritis is diminished in fibrinogen-deficient mice
Authors: Drew Angela F. , Tucker Heidi L. , Liu Hong , Witte David P. , Degen Jay L. , Tipping Peter G.
Published: March 2015

First published August 21, 2001; 00.1152/ajprenal.0002.2001.— Crescentic forms of glomerulonephritis are characterized by the accumulation of fibrin and cells in Bowman's space and are associated with a rapid loss of renal function. Accumulation of fibrin in the glomerular tufts is thought to promote macrophage infiltration and glomerular injury. To directly explore the role of fibrin(ogen) in the development of crescentic glomerulonephritis, antiglomerular basement membrane nephritis was induced in fibrinogen-deficient and control mice. Glomeruli from control mice developed severe disease including fibrin deposits, inflammatory cell accumulation, and crescent formation (46.3 ± 7.3% of glomeruli). Fibrinogen-deficient mice developed significantly milder disease with fewer glomerular crescents (24.0 ± 4.7% of glomeruli; P < 0.03). Glomerular macrophage accumulation was diminished in fibrinogen-deficient mice (0.9 ± 0.4 macrophages/glomerular cross section) relative to control mice (3.9 ± 1.4 macrophages/glomerular cross section; P < 0.03). Finally, renal function as assessed by serum creatinine was better maintained in fibrinogen-deficient mice. These results indicate that although fibrin(ogen) is not essential for the development of glomerular crescents, it contributes significantly to the pathogenesis of crescentic glomerulonephritis by promoting glomerular macrophage accumulation and impairing filtration.
Membrane repolarization is delayed in proximal tubules after ischemia-reperfusion: possible role of microtubule-organizing centers
Authors: Wald Flavia A. , Figueroa Yolanda , Oriolo Andrea S. , Salas Pedro J. I.
Published: March 2015

We have previously shown that microtubule-organizing centers (MTOCs) attach to the apical network of intermediate filaments (IFs) in epithelial cells in culture and in epithelia in vivo. Because that attachment is important for the architecture of microtubules (MTs) in epithelia, we analyzed whether chemical anoxia in LLC-PK1and CACO-2 cells or unilateral ischemia-reperfusion in rat kidney (performed under fluorane anesthesia) had an effect on the binding and distribution of MTOCs. In culture, we found that chemical anoxia induces MTOC detachment from IFs by morphological and biochemical criteria. In reperfused rat proximal tubules, noncentrosomal MTOCs were fully detached from the cytoskeleton and scattered throughout the cytoplasm at 3 days after reperfusion, when brush borders were mostly reassembled. At that time, MTs were also fully reassembled but, as expected, lacked their normal apicobasal orientation. Two apical membrane markers expressed in S2 and S3 segments were depolarized at the same stage. At 8 days after reperfusion, membrane polarity, MTOCs, and MTs were back to normal. Na+-K+-ATPase was also found redistributed, not to the apical domain but rather to an intracellular compartment, as described by others (Alejandro VS, Nelson W, Huie P, Sibley RK, Dafoe D, Kuo P, Scandling JD Jr., and Myers BD. Kidney Int 48: 1308–1315, 1995). The prolonged depolarization of the apical membrane may have implications in the pathophysiology of acute renal failure.
Acute renal failure after whole body ischemia is characterized by inflammation and T cell-mediated injury
Authors: Burne-Taney Melissa J. , Kofler Julia , Yokota Naoko , Weisfeldt Myron , Traystman Richard J. , Rabb Hamid
Published: March 2015

Acute renal failure (ARF) commonly occurs after whole body ischemia. Most experimental models of ARF have relied on the isolated renal artery clamping model; however, there is a pressing need to develop and understand the pathogenesis of new models with more “clinical relevance.” We evaluated a new murine model of ARF after whole body ischemia reperfusion injury (WBIRI). WBIRI was induced by an infusion of potassium chloride and a cardiac arrest period of 10 min. Resuscitation was achieved by cardiac compressions, ventilation, epinephrine, and fluids. WBIRI leads to a significant increase in serum creatinine (SCr) and renal tubular injury by 24 h. Renal myeloperoxidase (MPO) levels increased at 24 h after WBIRI. Increased expression of the proinflammatory genes, ICAM-1 and IL-6, was also observed in the kidney following WBIRI. On the basis of recent data that T cells are important mediators of isolated renal IRI, WBIRI was evaluated in T cell-deficient nu/nu mice. T cell-deficient mice had a significantly reduced rise in SCr and decreased tubular injury compared with wild-type mice. T cell-deficient mice had a decrease in ICAM-1 expression after WBIRI, but no decrease in renal MPO. This study describes a new, clinically relevant, model of ARF after WBIRI in mice and identifies the T cell as an important mediator of renal injury following WBIRI. Reduced ICAM-1 expression may provide a mechanism for this involvement.
Ammonium transport and pH regulation by K+-Cl-cotransporters
Authors: Bergeron Marc J. , Gagnon Édith , Wallendorff Bernadette , Lapointe Jean-Yves , Isenring Paul
Published: March 2015

The Na+-K+-Cl-cotransporters (NKCCs), which belong to the cation-Cl-cotransporter (CCC) family, are able to translocate [Formula: see text] across cell membranes. In this study, we have used the oocyte expression system to determine whether the K+-Cl-cotransporters (KCCs) can also transport [Formula: see text] and whether they play a role in pH regulation. Our results demonstrate that all of the CCCs examined (NKCC1, NKCC2, KCC1, KCC3, and KCC4) can promote [Formula: see text] translocation, presumably through binding of the ion at the K+site. Moreover, kinetic studies for both NKCCs and KCCs suggest that [Formula: see text] is an excellent surrogate of Rb+or K+and that [Formula: see text] transport and cellular acidification resulting from CCC activity are relevant physiologically. In this study, we have also found that CCCs are strongly and differentially affected by changes in intracellular pH (independently of intracellular [[Formula: see text]]). Indeed, NKCC2, KCC1, KCC2, and KCC3 are inhibited at intracellular pH <7.5, whereas KCC4 is activated. These results indicate that certain CCC isoforms may be specialized to operate in acidic environments. CCC-mediated [Formula: see text] transport could bear great physiological implication given the ubiquitous distribution of these carriers.
Vasoconstrictor and vasodilator effects of adenosine in the kidney
Authors: Hansen Pernille B. , Schnermann Jurgen
Published: March 2015

Adenosine is an ATP breakdown product that in most vessels causes vasodilatation and that contributes to the metabolic control of organ perfusion, i.e., to the match between oxygen demand and oxygen delivery. In the renal vasculature, in contrast, adenosine can produce vasoconstriction, a response that has been suggested to be an organ-specific version of metabolic control designed to restrict organ perfusion when transport work increases. However, the vasoconstriction elicited by an intravenous infusion of adenosine is only short lasting, being replaced within 1–2 min by vasodilatation. It appears that the steady-state response to the increase of plasma adenosine levels above normal resulting from the infusion is global renal vasorelaxation that is the result of A2AR activation in most parts of the renal vasculature, including larger renal arteries, juxtamedullary afferent arterioles, efferent arterioles, and medullary vessels. A2AR-mediated vasorelaxation is probably facilitated by endothelial receptors that cause the release of nitric oxide and other endothelial relaxing factors. In contrast, isolated perfused afferent arterioles of superficial and midcortical nephrons of rabbit and mouse, especially in their most distal segment at the entrance to the glomerulus, respond to adenosine with persistent vasoconstriction, indicating predominant or exclusive expression of A1AR. A1AR in afferent arterioles are selectively activated from the interstitial aspect of the vessel. This property can dissociate A1AR activation from changes in vascular adenosine concentration, a characteristic that is ideally suited for the role of renal adenosine as a paracrine factor in the control of glomerular function.
cDNA array identification of genes regulated in rat renal medulla in response to vasopressin infusion
Authors: Brooks Heddwen L. , Ageloff Shana , Kwon Tae-Hwan , Brandt William , Terris James M. , Seth Akhil , Michea Luis , Nielsen Søren , Fenton Robert , Knepper Mark A.
Published: March 2015

With the aim of identifying possible gene targets for direct or indirect regulation by vasopressin in the renal medulla, we have carried out cDNA array experiments in inner medullas of Brattleboro rats infused with the V2receptor-selective vasopressin analog desamino-Cys1,d-Arg8 vasopressin (dDAVP) for 72 h. Of the 1,176 genes on the array, 137 transcripts were increased by 2-fold or more, and 10 transcripts were decreased to 0.5-fold or less. Quantitative, real-time RT-PCR measurements confirmed increases seen for six selected transcripts (Wilms' tumor protein, β-arrestin 2, neurofibromin, casein kinase IIβ, aquaporin-3, and aquaporin-4). To correlate changes in mRNA expression with changes in protein expression, we carried out quantitative immunoblotting for 28 of the proteins whose cDNAs were on the array. For several targets including aquaporin-2, transcript abundance and protein abundance changes did not correlate. However, for most genes examined, changes in mRNA abundances were associated with concomitant protein abundance changes. Targets with demonstrated increases in both protein and mRNA abundances included neurofibromin, casein kinase IIβ, the β-subunit of the epithelial Na channel (β-ENaC), 11β-hydroxysteroid dehydrogenase type 2, and c-Fos. Additional cDNA arrays revealed that several transcripts that were increased in abundance after 72 h of dDAVP were also increased after 4 h, including casein kinase IIβ, β-ENaC, aquaporin-3, UT-A, and syntaxin 2. These studies have identified several transcripts whose abundances are regulated in the inner medulla in response to infusion of dDAVP and that could play roles in the regulation of salt and water excretion.
Regulation of collecting duct AQP3 expression: response to mineralocorticoid
Authors: Kwon Tae-Hwan , Nielsen Jakob , Masilamani Shyama , Hager Henrik , Knepper Mark A. , Frøkiær Jørgen , Nielsen Søren
Published: March 2015

Adrenocortical steroid hormones are importantly involved in the regulation of extracellular fluid volume. The present study was aimed at examining whether aldosterone and/or glucocorticoid regulates the abundance of aquaporin-3 (AQP3), -2, and -1 in rat kidney. In protocol 1, rats were adrenalectomized, followed by aldosterone replacement, dexamethasone replacement, or combined aldosterone and dexamethasone replacement (rats had free access to water but received a fixed amount of food). Protocol 2 was identical to protocol 1, except that all groups received fixed daily food and water intake. In both protocols 1 and 2, aldosterone deficiency was associated with increased fractional Na excretion and severe hyperkalemia. Semiquantitative immunoblotting revealed that aldosterone deficiency was associated with a dramatic downregulation of AQP3 abundance. Consistent with this, immunocytochemistry and immunoelectron microscopy revealed a marked decrease in AQP3 labeling in the basolateral plasma membranes of collecting duct principal cells. In contrast, AQP1 and AQP2 abundance and distribution were unchanged. Glucocorticoid deficiency revealed no changes in AQP3, -2, or -1 abundance. In protocol 3, Na restriction (to increase endogenous aldosterone levels) or exogenous aldosterone infusion in either normal rats or vasopressin-deficient Brattleboro rats was associated with a major increase in AQP3 abundance. In protocol 4, aldosterone levels were clamped by infusion of aldosterone, while Na intake was altered from a low to a high level. Under these circumstances, there were no changes in AQP3 or AQP2 abundance, although the level of the thiazide-sensitive Na-Cl cotransporter was decreased. In conclusion, the results uniformly demonstrate that aldosterone regulates AQP3 abundance independently of Na intake. In contrast, changes in glucocorticoid levels in these models do not influence AQP3 or AQP2 abundance. Therefore, in the collecting duct aldosterone may regulate, at least in part, AQP3 expression in addition to regulating Na and K transport.
Clusterin protects renal tubular epithelial cells from gentamicin-mediated cytotoxicity
Authors: Girton Richard A. , Sundin David P. , Rosenberg Mark E.
Published: March 2015

Clusterin is a heterodimeric secreted glycoprotein that is upregulated after acute renal injury. In aminoglycoside nephrotoxicity, clusterin is induced in the tubular epithelium and increased levels are found in the urine. In this study, we developed an in vitro model of gentamicin-induced cytotoxicity in renal proximal tubule cells and tested whether clusterin protected these cells from injury. LLC-PK1cells were incubated with varying concentrations of gentamicin in serum-free media, and cytotoxicity was quantified by lactate dehydrogenase release and confirmed by vital dye exclusion. A dose-dependent increase in cytotoxicity occurred with gentamicin concentrations up to 27 mg/ml. Clusterin decreased cytotoxicity in a dose- and time-dependent manner at 6, 12, and 24 h, whereas albumin, used as a control protein, had no effect. In contrast to the aminoglycoside model, when cells were injured by depletion of ATP, clusterin had only a minimally protective effect. LLC-PK1cells did not express megalin, a receptor that can mediate the uptake of both clusterin and aminoglycosides into proximal tubule cells. Uptake of gentamicin into LLC-PK1cells was observed despite the absence of megalin. In conclusion, clusterin specifically protects against gentamicin-induced renal tubular cell injury by a megalin-independent mechanism.
Altered dietary iron intake is a strong modulator of renal DMT1 expression
Authors: Wareing Mark , Ferguson Carole J. , Delannoy Mathieu , Cox Alan G. , McMahon Raymond F. T. , Green Roger , Riccardi Daniela , Smith Craig P.
Published: March 2015

Divalent metal transporter1 (DMT1; also known as DCT1 or NRAMP2) is an important component of the cellular machinery responsible for dietary iron absorption in the duodenum. DMT1 is also highly expressed in the kidney where it has been suggested to play a role in urinary iron handling. In this study, we determined the effect on renal DMT1 expression of feeding an iron-restricted diet (50 mg/kg) or an iron-enriched diet (5 g/kg) for 4 wk and measured urinary and fecal iron excretion rates. Feeding the low-iron diet caused a reduction in serum iron concentration and fecal iron output rate with an increase in renal DMT1 expression. Feeding an iron-enriched diet had the converse effect. Therefore, DMT1 expression in the kidney is sensitive to dietary iron intake, and the level of expression is inversely related to the dietary iron content. Changes in DMT1 expression occurred intracellularly in the proximal tubule and in the apical membrane and subapical region of the distal convoluted tubule. Increased DMT1 expression was accompanied by a decrease in urinary iron excretion rate and vice versa when DMT1 expression was reduced. Together, these findings suggest that modulation of renal DMT1 expression may influence renal iron excretion rate.
Molecular cloning, expression, and function of osteoclastic calcineurin Aα
Authors: Sun Li , Moonga Baljit S. , Lu Min , Zaidi Neeha , Iqbal Jameel , Blair Harry C. , Epstein Solomon , Abe Etsuko , Troen Bruce R. , Huang Christopher L.-H. , Zaidi Mone
Published: March 2015

This study explores the role of the calmodulin- and Ca2+-sensitive phosphatase calcineurin A in the control of bone resorption by mature osteoclasts. We first cloned full-length calcineurin Aα and Aβ cDNA from a rabbit osteoclast library. Sequence analysis revealed an ∼95 and 86% homology between the amino acid and the nucleotide sequences, respectively, of the two isoforms. The two rabbit isoforms also showed significant homology with the mouse, rat, and human homologs. In situ RT-PCR showed evidence of high levels of expression of calcineurin Aα mRNA in freshly isolated rat osteoclasts. Semiquantitative analysis of staining intensity revealed no significant difference in calcineurin Aα expression in cells treated with vehicle vs. those treated with the calcineurin (activity) inhibitors cyclosporin A (8 × 10−7M) and FK506 (5 × 10−9and 5 × 10−7M). We then constructed a fusion protein comprising calcineurin Aα and TAT, a 12-amino acid-long arginine-rich sequence of the human immunodeficiency virus protein. Others have previously shown that the fusion of proteins to this sequence results in their receptor-less transduction into cells, including osteoclasts. Similarly, unfolding of the TAT-calcineurin Aα fusion protein by shocking with 8 M urea resulted in its rapid influx, within minutes, into as many as 90% of all freshly isolated rat osteoclasts, as was evident on double immunostaining with anti-calcineurin Aα and anti-TAT antibodies. Pit assays performed with TAT-calcineurin Aα-positive osteoclasts revealed a concentration-dependent (10–200 nM) attenuation of bone resorption in the absence of cell cytotoxicity or changes in cell number. TAT-hemaglutinin did not produce significant effects on bone resorption or cell number. The study suggests the following: 1) the 61-kDa protein phosphatase calcineurin Aα can be effectively tranduced into osteoclasts by using the TAT-based approach, and 2) the transduced protein retains its capacity to inhibit osteoclastic bone resorption.
Basolateral and apical A1 adenosine receptors mediate sodium transport in cultured renal epithelial (A6) cells
Authors: Macala Lawrence J. , Hayslett John P.
Published: March 2015

There are conflicting reports in the literature regarding the adenosine receptor that mediates the increase in sodium transport in the A6 cell. In this study we used specific A1 and A2 adenosine receptor agonists and antagonists, as well as two different subclones of the A6 cell, to determine which adenosine receptor mediates the increase in sodium transport. In the A6S2 subclone, basolateral and apical N 6-cyclohexyladenosine (CHA), a selective A1 receptor agonist, stimulated sodium transport at a threshold concentration <10−7 M, whereas CGS-21680, a selective A2 receptor agonist, had a threshold concentration that was at least 10−5 M. The A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) was found to have a nonspecific effect on CHA-stimulated sodium transport, whereas the A2 receptor antagonist 8-(3-chlorostyryl)caffeine (CSC) had no effect. As with the A6S2 subclone, basolateral and apical CHA stimulated sodium transport at a nanomolar concentration in the A6C1 subclone and the threshold concentration for CGS-21680 was in the high micromolar range. Concurrent with the increase in sodium transport, CHA also stimulated anion secretion in the A6C1 subclone. These data demonstrate that adenosine increases sodium transport via the A1 receptor in different subclones of the A6 cell, including a subclone capable of anion secretion.
Intracellular distribution of labile Zn(II) and zinc transporter expression in kidney and MDCK cells
Authors: Ranaldi Giulia , Perozzi Giuditta , Truong-Tran Ai , Zalewski Peter , Murgia Chiara
Published: March 2015

Kidneys play a key role in zinc balance. The portion of Zn(II) that enters the glomerular filtrate is efficiently reabsorbed along the nephron through a mechanism yet to be identified. We used the Zn(II)-specific fluorophore Zinquin to visualize intracellular Zn(II) accumulated in the kidney epithelium and compared it with the intracellular localization of the vesicular zinc transporter ZnT4 both in vivo and in vitro. The Madin-Darby canine kidney (MDCK) cell line, stably overexpressing rat ZnT4, was used as a tissue culture model of the kidney epithelium. Zinquin labeling of MDCK cells revealed rapid internalization of Zn(II) and compartmentalization in intracellular bodies interspersed throughout the cytoplasm. In polarized kidney cells, ZnT4 protein was localized on the membrane of intracellular vesicles concentrated around the nucleus, mostly on the basal side. Results of double stainings demonstrated that ZnT4-containing vesicles do not overlap with Zn(II) bodies. Zinquin fluorescence, confirmed by autometallography in rat kidney, indicated that consistent with its physiological role, the central glomerulus was weakly stained, whereas the epithelium that lines convoluted tubules was strongly labeled. Double staining of rat kidney with Zinquin and anti-ZnT4 antibodies confirmed the in vitro observations, as Zinquin fluorescence appeared to be distinct from ZnT4 immunofluorescence. To gain further insight into which of the known zinc transporters might be involved in Zn(II) metabolism in the kidney, we have also characterized by RT-PCR the expression of other proteins involved in Zn(II) transport. All of the mRNAs examined [ZnT1, -T2, -T4, and human Zrt, Irt-like protein 1 (hZIP1)], with the exception of hZIP2, were present in adult rat kidney.
Characterization of an amiloride binding region in the α-subunit of ENaC
Authors: Kelly Ollie , Lin Chaomei , Ramkumar Mohan , Saxena Nina C. , Kleyman Thomas R. , Eaton Douglas C.
Published: March 2015

One of the defining characteristics of the epithelial sodium channel (ENaC) is its block by the diuretic amiloride. This study investigates the role of the extracellular loop of the α-subunit of ENaC in amiloride binding and stabilization. Mutations were generated in a region of the extracellular loop, residues 278–283. Deletion of this region, WYRFHY, resulted in a loss of amiloride binding to the channel. Channels formed from wild-type α-subunits or α-subunits containing point mutations in this region were examined and compared at the single-channel level. The open probabilities ( Po) of wild-type channels were distributed into two populations: one with a high Po and one with a low Po. The mean open times of all the mutant channels were shorter than the mean open time of the wild-type (high- Po) channel. Besides mutations Y279A and H282D, which had amiloride binding affinities similar to that of wild-type α-ENaC, all other mutations in this region caused changes in the amiloride binding affinity of the channels compared with the wild-type channel. These data provide new insight into the relative position of the extracellular loop with respect to the pore of ENaC and its role in amiloride binding and channel gating.
Natriuretic peptide receptor A mediates renal sodium excretory responses to blood volume expansion
Authors: Shi Shang-Jin , Vellaichamy Elangovan , Chin So Yeon , Smithies Oliver , Navar L. Gabriel , Pandey Kailash N.
Published: March 2015

The deficiency of Npr1 [genetic determinant of natriuretic peptide receptor A (NPRA)] increases arterial pressures and causes hypertensive heart disease in mice similar to those seen in untreated human hypertensive patients. However, the quantitative role of NPRA in mediating the renal responses to blood volume expansion remains uncertain. To determine the specific contribution of NPRA in mediating the signaling mechanisms responsible for natriuretic and diuretic responses to nondilutional intravascular expansion, we administered whole blood to anesthetized Npr1 homozygous null mutant (0-copy), wild-type (2-copy), and gene-duplicated (4-copy) mice. In wild-type (2-copy) animals, urinary flow (μl · min–1· g kidney wt–1) increased from 4.9 ± 1.0 to 14.4 ± 1.8 and sodium excretion (μeq · min–1· g kidney wt–1) from 1.15 ± 0.22 to 3.11 ± 0.60, associated with a rise in glomerular filtration rate (GFR; ml · min–1· g kidney wt–1) from 0.63 ± 0.03 to 0.82 ± 0.09 and renal plasma flow (RPF; ml · min–1· g kidney wt–1) from 2.96 ± 0.17 to 4.36 ± 0.41, whereas arterial pressure did not significantly increase. After volume expansion, 0-copy mice showed significantly lesser increases in urinary flow ( P < 0.001) and sodium excretory ( P < 0.001) responses even though the increases in arterial pressures were greater ( P < 0.001) compared with 2-copy mice. The 4-copy mice showed augmented responses in urinary flow ( P < 0.01) and sodium excretion ( P < 0.001) along with rises in both GFR ( P < 0.01) and RPF ( P < 0.01) compared with 2-copy wild-type mice. These results establish that NPRA activation is the predominant mechanism mediating the natriuretic, diuretic, and renal hemodynamic responses to acute blood volume expansion.
Dissociation of spectrin-ankyrin complex as a basis for loss of Na-K-ATPase polarity after ischemia
Authors: Woroniecki Robert , Ferdinand Jean R. , Morrow Jon S. , Devarajan Prasad
Published: March 2015

The polarized distribution of Na-K-ATPase at the basolateral membranes of renal tubule epithelial cells is maintained via a tethering interaction with the underlying spectrin-ankyrin cytoskeleton. In this study, we have explored the mechanism underlying the loss of Na-K-ATPase polarity after ischemic injury in Madin-Darby canine kidney (MDCK) cells, utilizing a novel antibody raised against a recently described kidney-specific isoform of ankyrin. In control MDCK cells, ankyrin was colocalized with Na-K-ATPase at the basolateral membrane. ATP depletion resulted in a duration-dependent mislocation of Na-K-ATPase and ankyrin throughout the cytoplasm. Colocalization studies showed a partial overlap between the distribution of ankyrin and Na-K-ATPase at all periods after ATP depletion. By immunoprecipitation with anti-ankyrin antibody, the mislocated Na-K-ATPase remained bound to ankyrin at all time points after ATP depletion. However, the interaction between ankyrin and spectrin was markedly diminished within 3 h of ATP depletion and was completely lost after 6 h. In solution binding assays using a fusion peptide of glutathione S-transferase with the ankyrin binding domain of Na-K-ATPase, a complex with ankyrin was detected at all time points after ATP depletion, but spectrin was lost from the complex in a duration-dependent manner. The loss of spectrin binding was not attributable to spectrin degradation but was associated with hyperphosphorylation of ankyrin. The results suggest that a dissociation of the membrane-cytoskeleton complex at the spectrin-ankyrin interface may contribute to the loss of Na-K-ATPase polarity after ischemic injury and reaffirm a critical adapter role for ankyrin in the normal maintenance of Na-K-ATPase polarity.
TNFR2-mediated apoptosis and necrosis in cisplatin-induced acute renal failure
Authors: Ramesh Ganesan , Reeves W. Brian
Published: March 2015

Cisplatin produces acute renal failure in humans and mice. Previous studies have shown that cisplatin upregulates the expression of TNF-α in mouse kidney and that inhibition of either the release or action of TNF-α protects the kidney from cisplatin-induced nephrotoxicity. In this study, we examined the effect of cisplatin on the expression of TNF receptors TNFR1 and TNFR2 in the kidney and the role of each receptor in mediating cisplatin nephrotoxicity. Injection of cisplatin into C57BL/6 mice led to an upregulation of TNFR1 and TNFR2 mRNA levels in the kidney. The upregulation of TNFR2 but not TNFR1 was blunted in TNF-α-deficient mice, indicating ligand-dependent upregulation of TNFR2. To study the roles of each receptor, we administered cisplatin to TNFR1- or TNFR2-deficient mice. TNFR2-deficient mice developed less severe renal dysfunction and showed reduced necrosis and apoptosis and leukocyte infiltration into the kidney compared with either TNFR1-deficient or wild-type mice. Moreover, renal TNF-α expression, ICAM-1 expression, and serum TNF-α levels were lower in TNFR2-deficient mice compared with wild-type or TNFR1-deficient mice treated with cisplatin. These results indicate that TNFR2 participates in cisplatin-induced renal injury in mice and may play an important role in TNF-α-mediated inflammation in the kidney in response to cisplatin.
Three GADD45 isoforms contribute to hypertonic stress phenotype of murine renal inner medullary cells
Authors: Chakravarty Devulapalli , Cai Qi , Ferraris Joan D. , Michea Luis , Burg Maurice B. , Kültz Dietmar
Published: March 2015

Mammalian renal inner medullary (IM) cells routinely face and resist hypertonic stress. Such stress causes DNA damage to which IM cells respond with cell cycle arrest. We report that three growth arrest and DNA damage-inducible 45 (GADD45) isoforms (GADD45α, GADDD45β, and GADD45γ) are induced by acute hypertonicity in murine IM cells. Maximum induction occurs 16–18 h after the onset of hypertonicity. GADD45γ is induced more strongly (7-fold) than GADD45β (3-fold) and GADD45α (2-fold). GADD45α and GADD45β protein induction is more pronounced and stable compared with the corresponding transcripts. Hypertonicity of various forms (NaCl, KCl, sorbitol, or mannitol) always induces GADD45 transcripts, whereas nonhypertonic hyperosmolality (urea) has no effect. Actinomycin D does not prevent hypertonic GADD45 induction, indicating that mRNA stabilization is the mechanism that mediates this induction. GADD45 induction patterns in IM cells exposed to 10 different stresses suggest isoform specificity, but similar functions, of individual isoforms during hypertonicity, heat shock, and heavy metal stress, when GADD45γ induction is strongest (17-fold). These data associate all known GADD45 isoforms with the hypertonicity phenotype of renal IM cells.
Impaired ability of the Na+/Ca2+exchanger from the Dahl/Rapp salt-sensitive rat to regulate cytosolic calcium
Authors: Hwang E. F. , Williams I. , Kovacs G. , Peti-Peterdi J. , Siroky B. , Rice W. C. , Bates E. , Schwiebert E. M. , Unlap M. T. , Bell P. D.
Published: March 2015

We previously cloned Na+/Ca2+exchanger (NCX1) from mesangial cells of salt-sensitive (SNCX = NCX1.7) and salt-resistant (RNCX = NCX1.3) Dahl/Rapp rats. The abilities of these isoforms to regulate cytosolic Ca2+concentration ([Ca2+]i) were assessed in fura 2-loaded OK cells expressing the vector (VOK), RNCX (ROK), and SNCX (SOK). Baseline [Ca2+]iwas 98 ± 20 nM ( n = 12) in VOK and was significantly lower in ROK (44 ± 5 nM; n = 12) and SOK (47 ± 13 nM; n = 12) cells. ATP at 100 μM increased [Ca2+]iby 189 ± 55 nM ( n = 12), 21 ± 9 nM ( n = 12), and 69 ± 18 nM ( n = 12) in VOK, ROK, and SOK cells, respectively. ATP (1 mM) or bradykinin (0.1 mM) caused large increases in [Ca2+]iand ROK but not SOK cells were much more efficient in reducing [Ca2+]iback to baseline levels. Parental Sprague-Dawley rat mesangial cells express both RNCX (SDRNCX) and SNCX (SDSNCX). SDRNCX and RNCX are identical at every amino acid residue, but SDSNCX and SNCX differ at amino acid 218 where it is isoleucine in SDSNCX and not phenylalanine. OK cells expressing SDSNCX (SDSOK) reduced ATP (1 mM)-induced [Ca2+]iincrease back to baseline at a rate equivalent to that for ROK cells. PKC downregulation significantly attenuated the rate at which ROK and SDSOK cells reduced ATP-induced [Ca2+]iincrease but had no effect in SOK cells. The reduced efficiency of SNCX to regulate [Ca2+]iis attributed, in part, to the isoleucine-to-phenylalanine mutation at amino acid 218.
Role of connective tissue growth factor in fibronectin expression and tubulointerstitial fibrosis
Authors: Yokoi Hideki , Mukoyama Masashi , Sugawara Akira , Mori Kiyoshi , Nagae Tetsuya , Makino Hisashi , Suganami Takayoshi , Yahata Kensei , Fujinaga Yuriko , Tanaka Issei , Nakao Kazuwa
Published: March 2015

Connective tissue growth factor (CTGF) is one of the candidate factors mediating downstream events of transforming growth factor-β (TGF-β), but its role in fibrogenic properties of TGF-β and in tubulointerstitial fibrosis has not yet been clarified. Using unilateral ureteral obstruction (UUO) in rats, we analyzed gene expression of TGF-β1, CTGF, and fibronectin. We further investigated the effect of blockade of endogenous CTGF on TGF-β-induced fibronectin expression in cultured rat renal fibroblasts by antisense oligodeoxynucleotide (ODN) treatment. After UUO, CTGF mRNA expression in the obstructed kidney was significantly upregulated subsequent to TGF-β1, followed by marked induction of fibronectin mRNA. By in situ hybridization, CTGF mRNA was detected mainly in the interstitial fibrotic areas and tubular epithelial cells as well as in parietal glomerular epithelial cells in the obstructed kidney. The interstitial cells expressing CTGF mRNA were also positive for α-smooth muscle actin. CTGF antisense ODN transfected into cultured renal fibroblasts significantly attenuated TGF-β-stimulated upregulation of fibronectin mRNA and protein compared with control ODN transfection, together with inhibited synthesis of type I collagen. With the use of a reporter assay, rat fibronectin promoter activity was increased by 2.5-fold with stimulation by TGF-β1, and this increase was abolished with antisense CTGF treatment. Thus CTGF plays a crucial role in fibronectin synthesis induced by TGF-β, suggesting that CTGF blockade could be a possible therapeutic target against tubulointerstitial fibrosis.
High glucose promotes mesangial cell apoptosis by oxidant-dependent mechanism
Authors: Kang Barinder P. S. , Frencher Stanley , Reddy Venkatesh , Kessler Alex , Malhotra Ashwani , Meggs Leonard G.
Published: March 2015

Reactive oxygen species are recognized as important mediators of biological responses. Hyperglycemia promotes the intracellular generation of superoxide anion and hydrogen peroxide. In several cell lines, oxidant stress has been linked to the activation of death programs. Here, we report for the first time that high ambient glucose concentration induces apoptosis in murine and human mesangial cells by an oxidant-dependent mechanism. The signaling cascade activated by glucose-induced oxidant stress included the heterodimeric redox-sensitive transcription factor NF-κB, which exhibited an upregulation in p65/c-Rel binding activity and suppressed binding activity of the p50 dimer. Recruitment of NF-κB and mesangial cell apoptosis were both inhibited by antioxidants, implicating oxidant-induced activation of NF-κB in the transmission of the death signal. The genetic program for glucose-induced mesangial cell apoptosis was characterized by an upregulation of the Bax/Bcl-2 ratio. In addition, phosphorylation of the proapoptotic protein Bad was attenuated in mesangial cells maintained at high-glucose concentration, favoring progression of the apoptotic process. These perturbations in the expression and phosphorylation of the Bcl-2 family were coupled with the release of cytochrome c from mitochondria and caspase activation. Our findings indicate that in mesangial cells exposed to high ambient glucose concentration, oxidant stress is a proximate event in the activation of the death program, which culminates in mitochondrial dysfunction and caspase-3 activation, as the terminal event.
An internal enhancer regulates heme- and cadmium-mediated induction of human heme oxygenase-1
Authors: Hill-Kapturczak Nathalie , Sikorski Eric , Voakes Christy , Garcia Jairo , Nick Harry S. , Agarwal Anupam
Published: March 2015

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation, releasing iron, carbon monoxide, and biliverdin. Induction of HO-1 is an adaptive and beneficial response in renal and nonrenal settings of tissue injury. The purpose of this study was to characterize the regulation of the human HO-1 gene in renal proximal tubule and aortic endothelial cells in response to heme and cadmium. Evaluation of multiple human HO-1 promoter-reporter constructs up to -9.1 kb demonstrated only a partial response to heme and cadmium. In an effort to mimic endogenous stimulus-dependent levels of HO-1 induction, we evaluated the entire 12.5 kb of the human HO-1 gene, including introns and exons, in conjunction with a -4.5-kb human HO-1 promoter and observed significant heme- and cadmium-mediated induction of the reporter gene, suggesting the presence of an internal enhancer. Enhancer function was orientation independent and required a region between -3.5 and -4.5 kb of the human HO-1 promoter. Our studies identified a novel enhancer internal to the human HO-1 gene that, in conjunction with the HO-1 promoter, recapitulates heme- and cadmium-mediated induction of the endogenous HO-1 gene. Elucidation of the molecular regulation of the human HO-1 gene will allow for the development of therapeutic strategies to manipulate HO-1 gene expression in pathological states.
Sodium loading changes urinary protein excretion: a proteomic analysis
Authors: Thongboonkerd Visith , Klein Jon B. , Pierce William M. , Jevans Anthony W. , Arthur John M.
Published: March 2015

Plasma sodium concentration is maintained even when sodium intake is altered. Sodium homeostasis may involve changes in renal tubular protein expression that are reflected in the urine. We used proteomic analysis to investigate changes in urinary protein excretion in response to acute sodium loading. Rats were given deionized water followed by hypertonic (2.7%) saline for 28 h each. Urinary protein expression was determined during the final 4 h of each treatment. Acute sodium loading increased urinary sodium excretion (4.53 ± 1.74 vs. 1.70 ± 0.27 mmol/day, P = 0.029). Urinary proteins were separated by two-dimensional PAGE and visualized by Sypro ruby staining. Differentially expressed proteins were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry followed by peptide mass fingerprinting. The abundance of a total of 45 protein components was changed after acute sodium loading. Neutral endopeptidase, solute carrier family 3, meprin 1α, diphor-1, chaperone heat shock protein 72, vacuolar H+-ATPase, ezrin, ezrin/radixin/moesin-binding protein, glutamine synthetase, guanine nucleotide-binding protein, Rho GDI-1, and chloride intracellular channel protein 1 were decreased, whereas albumin and α-2u globulin were increased. Some of these proteins have previously been shown to be associated with tubular transport. These data indicate that alterations in the excretion of several urinary proteins occur during acute sodium loading.
Stoichiometry of organic anion/dicarboxylate exchange in membrane vesicles from rat renal cortex and hOAT1-expressing cells
Authors: Aslamkhan Amy , Han Yong-Hae , Walden Ramsey , Sweet Douglas H. , Pritchard John B.
Published: March 2015

Although membrane vesicle studies have established the driving forces that mediate renal organic anion secretion and the organic anion transporter Oat1 has now been cloned in several species, its stoichiometry has remained uncertain. In this study, we used electrophysiology, kinetic measurements, and static head experiments to determine the coupling ratio for Oat1-mediated organic anion/dicarboxylate exchange. Initial experiments demonstrated that uptake of PAH by voltage-clamped Xenopus laevis oocytes expressing rOat1 led to net entry of positive charge, suggesting that coupling was one-to-one. This conclusion was confirmed by kinetic analysis of PAH and glutarate fluxes in native basolateral membrane vesicles from the rat renal cortex, which showed a Hill coefficient of 1. Similarly, static head experiments on the rat vesicles also showed a 1:1 coupling ratio. To confirm these conclusions in a system expressing a single cloned transporter, Madin-Darby canine kidney cells were stably transfected with the human exchanger hOAT1. The hOAT1-expressing cell line showed extensive PAH transport, which was very similar in all respects to transport expressed by hOAT1 in Xenopus oocytes. Its Kmfor PAH was 8 μM and glutarate effectively trans-stimulated PAH transport. When stoichiometry was assessed using plasma membranes isolated from the hOAT1-expressing cells, both kinetic and static head data indicated that hOAT1 also demonstrated a 1:1 coupling between organic anion and dicarboxylate.
Involvement of transforming growth factor-β in regulation of calcium transients in diabetic vascular smooth muscle cells
Authors: Sharma Kumar , Deelman Leo , Madesh Muniswamy , Kurz Bernd , Ciccone Emilio , Siva Senthuran , Hu Taishan , Zhu Yanqing , Wang Lewei , Henning Robert , Ma Xinliang , Hajnoczky Gyorgy
Published: March 2015

Altered calcium [Ca2+] transients of vascular smooth muscle cells to vasoconstrictors may contribute to altered regulation of blood flow in diabetes. We postulated that diabetes-induced transforming growth factor (TGF)-β production contributes to impaired ANG II response of vascular smooth muscle cells in macrovessels and microvessels. Aortic vascular smooth muscle cells isolated from diabetic rats exhibited markedly impaired ANG II-induced cytosolic calcium [Ca2+] signal that was completely restored by pretreatment with anti-TGF-β antibodies. Similar findings were noted in microvascular smooth muscle cells isolated from preglomerular vessels and cultured in high glucose. The impact of diabetes on [Ca2+] transients was replicated by addition of TGF-β1and -β2isoforms to aortic smooth muscle cells in culture and diabetic cells had enhanced production of TGF-β2. In the in vivo condition, TGF-β1was increased in diabetic glomeruli, whereas TGF-β2was increased in diabetic aorta. The characteristic increase in glomerular filtration surface area found in diabetic rats was prevented by treatment with anti-TGF-β antibodies, and impaired ANG II-induced aortic ring contraction in diabetic rats was completely restored by anti-TGF-β antibodies. Impaired vascular dysfunction may be partly due to decreased inositol 1,4,5-trisphosphate receptor (IP3R), as reduced type I IP3R expression was found in diabetic aorta and restored by anti-TGF-β antibodies. We conclude that TGF-β plays an important role in the vascular dysfunction of early diabetes by inhibiting calcium transients in vascular smooth muscle cells.
Identification of lactate as a driving force for prostanoid transport by prostaglandin transporter PGT
Authors: Chan Brenda S. , Endo Shinichi , Kanai Naoaki , Schuster Victor L.
Published: March 2015

We previously characterized the prostaglandin (PG) transporter PGT as an exchanger in which [3H]PGE2influx is coupled to the efflux of a countersubstrate. Here, we cultured HeLa cells that stably expressed human PGT under conditions known to favor glycolysis (glucose as a carbon source) or oxidative phosphorylation (glutamine as a carbon source) and studied the effect on PGT-mediated [3H]PGE2influx. PGT-expressing cells grown in glutamine exhibited a 2- to 4-fold increase in [3H]PGE2influx compared with the antisense control, whereas cells grown in glucose exhibited a 14-fold increase. In the presence of 10 vs. 25 mM glucose during the uptake, there was a dose-dependent increment in [3H]PGE2influx. Cis inhibition of [3H]PGE2influx occurred with lactate at physiological concentrations (apparent Km= 48 ± 12 mM). Preloading with lactate caused a dose-dependent trans stimulation of PGT-mediated [3H]PGE2uptake, and external lactate caused trans stimulation of PGT-mediated [3H]PGE2release. Together, these data are consistent with PGT-mediated PG-lactate exchange. Cells engaged in glycolysis would then be poised energetically for prostanoid uptake by means of PGT.
Acute acidosis-induced alteration in bone bicarbonate and phosphate
Authors: Bushinsky David A. , Smith Susan B. , Gavrilov Konstantin L. , Gavrilov Leonid F. , Li Jianwei , Levi-Setti Riccardo
Published: March 2015

During an acute fall in systemic pH due to a decrease in the concentration of serum bicarbonate ([HCO[Formula: see text]]), metabolic acidosis, there is an influx of hydrogen ions into the mineral phase of bone, buffering the decrement in pH. When bone is cultured in medium modeling acute metabolic acidosis, the influx of hydrogen ions is coupled to an efflux of sodium and potassium and a depletion of mineral carbonate. These ionic fluxes would be expected to neutralize some of the excess hydrogen ions and restore the pH toward normal. Approximately one-third of bone carbonate is located on the hydration shell of apatite, where it is readily accessible to the systemic circulation, whereas the remainder is located in less accessible areas. We hypothesize that the surface of bone would respond to acidosis in a different manner than the interior of bone, with depletion of carbonate preferentially occurring on the bone surface. We utilized a high-resolution scanning ion microprobe with secondary ion mass spectroscopy to localize the changes in bone carbonate, as measured by HCO[Formula: see text], and phosphate and determine their relative contribution to the buffering of hydrogen ions during acute metabolic acidosis. Neonatal mouse calvariae were incubated in control medium (pH ∼7.44, [HCO[Formula: see text]] ∼27 mM) or in medium acidified by a reduction in [HCO[Formula: see text]] (pH ∼7.14, [HCO[Formula: see text]] ∼13). Compared with control, after a 3-h incubation in acidic medium there is a fivefold decrease in surface HCO[Formula: see text] with respect to the carbon-carbon bond (C2) and a threefold decrease in surface HCO[Formula: see text] with respect to the carbon-nitrogen bond (CN) with no change in cross-sectional HCO[Formula: see text]. Compared with control, after a 3-h incubation in acidic medium there is a 10-fold decrease in cross-sectional phosphate with respect to C2 and a 10-fold decrease in cross-sectional phosphate with respect to CN, with no change in surface phosphate. On the bone surface, there is a fourfold depletion of HCO[Formula: see text] in relation to phosphate, and, in cross section, a sevenfold depletion of phosphate in relation to HCO[Formula: see text]. Thus acute hydrogen ion buffering by bone involves preferential dissolution of surface HCO[Formula: see text] and of cross-sectional phosphate.