bluuebunny/crossref_metadata_2025 huggingface dataset:
Two apical multidrug transporters, P-gp and MRP2, are differently altered in chronic renal failure
Authors: Laouari Denise , Yang Ruchun , Veau Celine , Blanke Isabelle , Friedlander Gérard
Published: December 2017

Tubular function is altered in chronic renal failure (CRF). Whether drug secretion by renal tubules is modified in CRF is questioned because of frequent accumulation of various toxins in CRF. This function mainly involves ATP-dependent drug transporters, particularly P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) 2, both present in apical membrane of epithelial cells. The present study was aimed at determining the changes in P-gp and MRP2 expression induced by experimental CRF in kidney and liver. The relationship between MRP2 and glutathione metabolism changes was examined because MRP2 transports GSSG and glutathione conjugates. Rats underwent either 80% subtotal nephrectomy (Nx) or sham operation, and determinations were performed 3 and 6 wk later. CRF induced a 70–200% rise in protein and mRNA expression of MRP2 after 3 and 6 wk post-Nx in remnant kidney and after 6 wk in liver. However, P-gp expression was unchanged by CRF. Relative to whole kidney mass, total MRP2 levels decreased by only 27% in Nx rats whereas total P-gp levels were reduced by 60%. Renal GSSG and total glutathione levels were increased by 30% in Nx rats, but glutathione- S-transferase (GST) activity was normal; liver GSSG levels and GST activity were reduced in Nx rats. In conclusion, CRF resulted in specific overexpression of MRP2 in kidney and liver. This could be an adaptative response to some elevated circulating toxins. The later MRP2 induction and different glutathione changes in liver compared with kidney suggest different mechanisms for MRP2 induction and/or action in these two tissues.
Aldosterone induces rapid apical translocation of ENaC in early portion of renal collecting system: possible role of SGK
Authors: Loffing Johannes , Zecevic Marija , Féraille Eric , Kaissling Brigitte , Asher Carol , Rossier Bernard C. , Firestone Gary L. , Pearce David , Verrey François
Published: December 2017

Aldosterone controls sodium reabsorption and potassium secretion in the aldosterone-sensitive distal nephron (ASDN). Although clearance measurements have shown that aldosterone induces these transports within 30–60 min, no early effects have been demonstrated in vivo at the level of the apical epithelial sodium channel (ENaC), the main effector of this regulation. Here we show by real-time RT-PCR and immunofluorescence that an aldosterone injection in adrenalectomized rats induces α-ENaC subunit expression along the entire ASDN within 2 h, whereas β- and γ-ENaC are constitutively expressed. In the proximal ASDN portions only, ENaC is shifted toward the apical cellular pole and the apical plasma membrane within 2 and 4 h, respectively. To address the question of whether the early aldosterone-induced serum and glucocorticoid-regulated kinase (SGK) might mediate this apical shift of ENaC, we analyzed SGK induction in vivo. Two hours after aldosterone, SGK was highly induced in all segment-specific cells of the ASDN, and its level decreased thereafter. In Xenopus laevis oocytes, SGK induced ENaC activation and surface expression by a kinase activity-dependent mechanism. In conclusion, the rapid in vivo accumulation of SGK and α-ENaC after aldosterone injection takes place along the entire ASDN, whereas the translocation of α,β,γ-ENaC to the apical plasma membrane is restricted to its proximal portions. Results from oocyte experiments suggest the hypothesis that a localized activation of SGK may play a role in the mediation of ENaC translocation.
Post-cyclosporine-mediated hypertension and nephropathy: amelioration by vascular endothelial growth factor
Authors: Kang Duk-Hee , Kim Yoon-Goo , Andoh Takeshi F. , Gordon Katherine L. , Suga Shin-Ichi , Mazzali Marilda , Jefferson J. Ashley , Hughes Jeremy , Bennett William , Schreiner George F. , Johnson Richard J.
Published: December 2017

Recent studies have demonstrated a role for microvascular and tubulointerstitial injury in some models of salt-sensitive hypertension. We utilized a model of post-cyclosporin A (CsA) nephropathy and hypertension to test the hypothesis that treatment with an angiogenic factor aimed at ameliorating the microvascular and renal injury would prevent the development of hypertension. CsA was administered with a low-salt diet for 45 days, resulting in a renal lesion characterized by afferent arteriolopathy, focal peritubular capillary loss, and tubulointerstitial fibrosis. Rats were then placed on a high-salt diet and randomized to receive either vascular endothelial growth factor (VEGF121) or vehicle for 14 days. Placement of rats with established CsA nephropathy on a high-salt diet results in the rapid development of salt-sensitive hypertension. VEGF121treatment resulted in lower blood pressure, and this persisted on discontinuing the VEGF. VEGF121treatment was also associated with a decrease in osteopontin expression, macrophage infiltration, and collagen III deposition and markedly stimulated resolution of the arteriolopathy (20.9 ± 7.8 vs. 36.9 ± 6.1%, VEGF vs. vehicle, P < 0.05). In conclusion, CsA-associated renal microvascular and tubulointerstitial injury results in the development of salt-sensitive hypertension. Treatment of animals with established CsA nephropathy with VEGF reduces the hypertensive response and accelerates histological recovery. The vascular protective effect of VEGF may be due to the improvement of arteriolopathy. Angiogenic growth factors may represent a novel strategy for treating CsA-associated hypertension and renal disease.
Ion permeation and selectivity in ClC-type chloride channels
Authors: Fahlke Christoph
Published: December 2017

Voltage-gated anion channels are present in almost every living cell and have many physiological functions. Recently, a novel gene family encoding voltage-gated chloride channels, the ClC family, was identified. The knowledge of primary amino acid sequences has allowed for the study of these anion channels in heterologous expression systems and made possible the combination of site-directed mutagenesis and high-resolution electrophysiological measurements as a means of gaining insights into the molecular basis of channel function. This review focuses on one particular aspect of chloride channel function, the selective transport of anions through biological membranes. I will describe recent experiments using a combination of cellular electrophysiology, molecular genetics, and recombinant DNA technology to study the molecular basis of ion permeation and selection in ClC-type chloride channels. These novel tools have provided new insights into basic mechanisms underlying the function of these biologically important channels.
Flow-dependent K+secretion in the cortical collecting duct is mediated by a maxi-K channel
Authors: Woda Craig B. , Bragin Alvina , Kleyman Thomas R. , Satlin Lisa M.
Published: December 2017

K+secretion by the cortical collecting duct (CCD) is stimulated at high flow rates. Patch-clamp analysis has identified a small-conductance secretory K+(SK) and a high-conductance Ca2+-activated K+(maxi-K) channel in the apical membrane of the CCD. The SK channel, encoded by ROMK, is believed to mediate baseline K+secretion. The role of the stretch- and Ca2+-activated maxi-K channel is still uncertain. The purpose of this study was to identify the K+channel mediating flow-dependent K+secretion in the CCD. Segments isolated from New Zealand White rabbits were microperfused in the absence and presence of luminal tetraethylammonium (TEA) or charybdotoxin, both inhibitors of maxi-K but not SK channels, or apamin, an inhibitor of small-conductance maxi-K+channels. Net K+secretion and Na+absorption were measured at varying flow rates. In the absence of TEA, net K+secretion increased from 8.3 ± 1.0 to 23.4 ± 4.7 pmol · min−1· mm−1( P < 0.03) as the tubular flow rate was increased from 0.5 to 6 nl · min−1· mm−1. Flow stimulation of net K+secretion was blocked by luminal TEA (8.2 ± 1.2 vs. 9.9 ± 2.7 pmol · min−1· mm−1at 0.6 and 6 nl · min−1· mm−1flow rates, respectively) or charybdotoxin (6.8 ± 1.6 vs. 8.3 ± 1.6 pmol · min−1· mm−1at 1 and 4 nl · min−1· mm−1flow rates, respectively) but not by apamin. These results suggest that flow-dependent K+secretion is mediated by a maxi-K channel, whereas baseline K+secretion occurs through a TEA- and charybdotoxin-insensitive SK (ROMK) channel.
The calcium-sensing receptor regulates calcium absorption in MDCK cells by inhibition of PMCA
Authors: Blankenship Kristy A. , Williams J. Jason , Lawrence Martha S. , McLeish Kenneth R. , Dean William L. , Arthur John M.
Published: December 2017

Calcium transport across a monolayer of Madin-Darby canine kidney (MDCK) cells was measured in response to stimulation of the basal surface with calcium-sensing receptor (CaR) agonists. Stimulation of the CaR resulted in a time- and concentration-dependent inhibition of calcium transport but did not change transepithelial voltage or resistance. Inhibition of transport was not altered by pretreatment of cells with pertussis toxin but was blocked by the phospholipase C (PLC) inhibitor U-73122. To determine a potential mechanism by which the CaR could inhibit calcium transport, we measured activity of the plasma membrane calcium ATPase (PMCA). Stimulation of the CaR on the basal surface resulted in an inhibition of the PMCA in a concentration- and PLC-dependent manner. Thus stimulation of the CaR inhibits both calcium transport and PMCA activity through a PLC-dependent pathway. These studies provide the first direct evidence that calcium can inhibit its own transcellular absorption in a model of the distal tubule. In addition, they provide a potential mechanism for the CaR to inhibit calcium transport, inhibition of PMCA.
SFKs, Ras, and the classic MAPK pathway couple muscarinic receptor activation to increased Na-HCO3cotransport activity in renal epithelial cells
Authors: Robey R. Brooks , Ruiz Ofelia S. , Baniqued Jessica , Mahmud Dolores , Espiritu Doris Joy D. , Bernardo Angelito A. , Arruda Jose A. L. ,   With the Technical Assistance of Yi-Yong Qiu
Published: December 2017

Cholinergic agents are known to affect the epithelial transport of H2O and electrolytes in the kidney. In proximal tubule cells, cholinergic agonists increase basolateral Na-HCO3cotransport activity via M1muscarinic receptor activation. The signaling intermediates that couple these G protein-coupled receptors to cotransporter activation, however, are not well defined. We therefore sought to identify distal effectors of muscarinic receptor activation that contribute to increased NBC activity in cultured proximal tubule cells. As demonstrated previously for acute CO2-regulated cotransport activity, we found that inhibitors of Src family kinases (SFKs) or the classic mitogen-activated protein kinase (MAPK) pathway prevented the stimulation of NBC activity by carbachol. The ability of carbachol to activate Src, as well as the proximal (Raf) and distal [extracellular signal-regulated kinases 1 and 2 (ERK1/2)] elements of the classic MAPK module, was compatible with these findings. Cholinergic stimulation of ERK1/2 activity was also completely prevented by overexpression of a dominant negative mutant of Ras (N17-Ras). Taken together, these findings suggest a requirement for the sequential activation of SFKs, Ras, and the classic MAPK pathway [Raf→MAPK/ERK kinase (MEK)→ERK]. These findings provide important insights into the molecular mechanisms underlying cholinergic regulation of NBC activity in renal epithelial cells. They also suggest a specific mechanism whereby cholinergic stimulation of the kidney can contribute to pH homeostasis.
Localization of GFP-tagged concentrative nucleoside transporters in a renal polarized epithelial cell line
Authors: Mangravite Lara M. , Lipschutz Joshua H. , Mostov Keith E. , Giacomini Kathleen M.
Published: December 2017

Many nucleosides undergo active reabsorption within the kidney, probably via nucleoside transporters. To date, two concentrative nucleoside transporters have been cloned, the sodium-dependent purine-selective nucleoside transporter (SPNT) and concentrative nucleoside transporter 1 (CNT1). We report the stable expression of green fluorescence protein (GFP)-tagged SPNT and CNT1 in Madin-Darby canine kidney (MDCK) cells, a polarized renal epithelial line. We demonstrate that the GFP tag does not alter the substrate selectivity and only modestly affects the kinetic activity of the transporters. By using confocal microscopy and functional studies, both SPNT and CNT1 are localized primarily to the apical membrane of MDCK and LLC-PK1cells. Apical localization of these transporters suggests a role in renal nucleoside reabsorption and regulation of tubular function via the adenosine pathway.
Regulation of proximal tubule sodium/hydrogen antiporter with chronic volume contraction
Authors: Fisher Kenneth A. , Lee Sung H. , Walker Jeffrey , Dileto-Fang Christine , Ginsberg Leonard , Stapleton Susan R.
Published: December 2017

We developed a model of volume contraction in rabbits by using a furosemide/low-salt diet to follow changes, if any, in proximal tubule Na+/H+exchanger 3 (NHE3) mRNA and brush-border protein. The rabbits' plasma renin, aldosterone, and urine sodium content confirmed the volume-contracted state. RNase protection assays demonstrated increases in treated-animal NHE3 mRNA as a percentage of control with 172 ± 23, 154 ± 15, 153 ± 14, and 141 ± 7 (SE) % ( P < 0.05) at 1, 5, 10, and 31 days, respectively. Western analysis of brush-border membrane with NHE3 antibody revealed increased immunoreactivity in treated animals as a percentage of control with 120 ± 30, 190 ± 59, 307 ± 72, and 427 ± 41% ( P < 0.05) at 1, 5, 10, and 31 days, respectively. There was no significant difference in serum potassium, bicarbonate, and cortisol in control vs. experimental animals. These data suggest that there is chronic upregulation of NHE3 in the volume-contracted state.
Expression of fibronectin splice variants in the postischemic rat kidney
Authors: Zuk Anna , Bonventre Joseph V. , Matlin Karl S.
Published: December 2017

Using an in vivo rat model of unilateral renal ischemia, we previously showed that the expression and distribution of fibronectin (FN), a major glycoprotein of plasma and the extracellular matrix, dramatically changes in response to ischemia-reperfusion. In the distal nephron in particular, FN accumulates in tubular lumens, where it may contribute to obstruction. In the present study, we examine whether the tubular FN is the plasma or cellular form, each of which is produced by alternative splicing of a single gene transcript. We demonstrate that FN in tubular lumens does not contain the extra type III A (EIIIA) and/or the extra type III B (EIIIB) region, both of which are unique to cellular FN. It does, however, contain the V95 region, which in the rat is a component of FNs in both plasma and the extracellular matrix. Expression of FN containing EIIIA increases dramatically in the renal interstitium after ischemic injury and continues to be produced at high levels 6 wk later. V95-containing FN also increases in the interstitial space, albeit more slowly and at lower levels than FN containing EIIIA; it also persists 6 wk later. FN containing the EIIIB region is not expressed in the injured kidney. The presence of V95 but not the EIIIA or EIIIB regions of FN in tubular lumens identifies the origin of FN in this location as the plasma; tubular FN is ultimately voided in the urine. The data indicate that both plasma and cellular FNs containing the V95 and/or EIIIA regions may contribute to the pathogenesis of acute renal failure and to the repair of the injured kidney.
Dynamic characteristics and underlying mechanisms of renal blood flow autoregulation in the conscious dog
Authors: Just Armin , Ehmke Heimo , Toktomambetova Lira , Kirchheim Hartmut R.
Published: December 2017

The time course of the autoregulatory response of renal blood flow (RBF) to a step increase in renal arterial pressure (RAP) was studied in conscious dogs. After RAP was reduced to 50 mmHg for 60 s, renal vascular resistance (RVR) decreased by 50%. When RAP was suddenly increased again, RVR returned to baseline with a characteristic time course (control; n = 15): within the first 10 s, it rose rapidly to 70% of baseline ( response 1), thus already comprising 40% of the total RVR response. Thereafter, it increased at a much slower rate until it started to rise rapidly again at 20–30 s after the pressure step ( response 2). After passing an overshoot of 117% at 43 s, RVR returned to baseline values. Similar responses were observed after RAP reduction for 5 min or after complete occlusions for 60 s. When tubuloglomerular feedback (TGF) was inhibited by furosemide (40 mg iv, n = 12), response 1 was enhanced, providing 60% of the total response, whereas response 2 was completely abolished. Instead, RVR slowly rose to reach the baseline at 60 s ( response 3). The same pattern was observed when furosemide was given at a much higher dose (>600 mg iv; n = 6) or in combination with clamping of the plasma levels of nitric oxide ( n = 6). In contrast to RVR, vascular resistance in the external iliac artery after a 60-s complete occlusion started to rise with a delay of 4 s and returned to baseline within 30 s. It is concluded that, in addition to the myogenic response and the TGF, a third regulatory mechanism significantly contributes to RBF autoregulation, independently of nitric oxide. The three mechanisms contribute about equally to resting RVR. The myogenic response is faster in the kidney than in the hindlimb.
Albumin overload induces apoptosis in LLC-PK1cells
Authors: Erkan Elif , De Leon Maryely , Devarajan Prasad
Published: December 2017

The degree of albuminuria is a well-known adverse prognostic indicator in human glomerular diseases. However, the mechanisms by which albuminuria by itself contributes to tubulointerstitial injury and progression of renal disease remain unclear. We tested the hypothesis that apoptosis may represent one of the mechanisms by which tubule epithelial cells are damaged after albumin overload in vitro. Cultured LLC-PK1proximal tubule cells were incubated with varying concentrations of BSA. This resulted in a dose- and duration-dependent induction of apoptosis, as evidenced by internucleosomal DNA cleavage (DNA laddering and nick-end labeling), externalization of plasma membrane phosphatidylserine (annexin labeling), and characteristic morphological changes (cell shrinkage and nuclear condensation). Albumin overload also resulted in a dose-dependent upregulation of Fas and Fas-associated protein with death domain (FADD), and activation of caspase 8. Incubation with the caspase 8 inhibitor IETD ameliorated the albumin-induced apoptosis. Collectively, our results indicate that albumin overload induces apoptosis of cultured LLC-PK1cells, mediated at least in part by the Fas-FADD-caspase 8 pathway.
Adrenomedullin gene delivery attenuates renal damage and cardiac hypertrophy in Goldblatt hypertensive rats
Authors: Wang Cindy , Dobrzynski Eric , Chao Julie , Chao Lee
Published: December 2017

Adrenomedullin (AM) is a potent vasodilator and natriuretic peptide that plays an important role in cardiovascular function. In this study, we employed a somatic gene delivery approach to explore its potential protective role in renovascular hypertension. A single tail vein injection of adenovirus harboring the human AM gene significantly blunted a blood pressure increase that lasted for more than 3 wk in two-kidney one-clip (2K1C) hypertensive rats. The expression of human AM mRNA was detected in the kidney, adrenal gland, heart, lung, and liver, and immunoreactive human AM was detected in the plasma and urine of 2K1C rats after human AM gene delivery. A maximal blood pressure difference of 28 mmHg was observed 10 days after AM gene delivery, compared with that in rats injected with the control virus carrying the LacZ gene. Human AM gene delivery significantly attenuated increases in the ratio of left ventricular weight to heart weight, cardiomyocyte diameter, and fibrosis in the heart, as well as glomerular sclerosis, tubular injuries, and protein casts in the kidney. The beneficial effects of AM gene delivery were accompanied by increased urinary cAMP levels, indicating activation of AM receptors. These findings provide new insights into the role of AM in renovascular hypertension and may have significance in therapeutic applications in cardiovascular diseases.
Uremic levels of urea inhibitl-arginine transport in cultured endothelial cells
Authors: Xiao Shen , Wagner Laszlo , Mahaney James , Baylis Chris
Published: December 2017

Hypertension in end-stage renal disease (ESRD) may involve lack of endothelial nitric oxide (NO), as suggested by reduced total NO synthase (NOS) in dialysis patients. One reason might be due to substrate deficiency. To test the hypothesis that uremia is a state of intracellular l-arginine deficiency, uremic plasma was obtained from dialysis patients, and its effect was tested on arginine transport in cultured vascular endothelial cells. l-arginine transport ( P< 0.01) was reduced in human dermal microvascular endothelial cells (HDMEC) incubated for 6 h with 20% uremic plasma from peritoneal dialysis and hemodialysis patients obtained immediately predialysis. Similar transport inhibition was seen with ESRD plasma in human glomerular capillary and bovine aortic endothelial cells. Hemodialysis partially reversed inhibition of l-arginine transport. HDMECs incubated for 6 h with synthetic media containing high (uremic) urea concentrations showed inhibition ofl-arginine transport, but this was not competitive because acute exposure to urea had no impact on l-arginine transport. Over a 6-h period, urea-induced inhibition ofl-arginine transport was not sufficient to inhibit NOS activity, but after 7 days NOS activity was reduced. These cellular findings suggest that substrate delivery may be lowered, thus reducing endothelial NOS activity and contributing to hypertension in ESRD patients.
97- and 117-kDa forms of collecting duct urea transporter UT-A1 are due to different states of glycosylation
Authors: Bradford Ako D. , Terris James M. , Ecelbarger Carolyn A. , Klein Janet D. , Sands Jeff M. , Chou Chung-Lin , Knepper Mark A.
Published: December 2017

UT-A1 is an extremely hydrophobic 929-amino acid integral membrane protein, expressed in the renal inner medullary collecting duct, with a central role in the urinary concentrating mechanism. Previous immunoblotting studies in rats have revealed that UT-A1 is present in kidney in 97- and 117-kDa monomeric forms and that the relative abundance of the two forms is altered by vasopressin treatment and other treatments that altered urinary inner medullary urea concentration. The present studies were carried out using protein chemistry techniques to determine the origin of the two forms. Peptide-directed polyclonal antibodies targeted to five sites along the polypeptide sequence from the NH2to the COOH terminus labeled both forms, thus failing to demonstrate a significant deletion in the primary amino acid chain. The 97- and 117-kDa monomeric forms were both reduced to 88 kDa by deglycosylation with N-glycosidase F, indicating that a single polypeptide chain is glycosylated to two different extents. Studies using nonionic detergents for membrane solubilization or using homobifunctional cross-linkers demonstrated that UT-A1 exists as a 206-kDa protein complex in native kidney membranes. The mobility of this complex was also increased by deglycosylation. Both the 97- and 117-kDa proteins, as well as the 206-kDa complex, were immunoprecipitated with UT-A1 antibodies. We conclude that UT-A1 is a glycoprotein and that the two monomeric forms (97 and 117 kDa) in inner medullary collecting duct are the consequence of different states of glycosylation.
Evidence for endothelin involvement in the response to high salt
Authors: Pollock David M. , Pollock Jennifer S.
Published: December 2017

Recent evidence suggests that endothelin-1 (ET-1), perhaps through the ETB receptor, may participate in blood pressure regulation through the control of sodium excretion. Mean arterial pressure (MAP) was continuously measured via telemetry implants in male Sprague-Dawley rats. After 1 wk of baseline measurements, rats were given either high (10%) or low (0.08%) NaCl in chow for the remainder of the experiment ( n = 5 in each group). MAP was significantly increased in rats on a high-salt diet (115 ± 2 mmHg) compared with rats on the low-salt diet (103 ± 2 mmHg; P < 0.05). All rats were then treated with the ETB receptor antagonist A-192621 mixed with the food and adjusted daily to ensure a dose of 30 mg · kg−1 · day−1. ETB blockade produced an increase in MAP within a few hours of treatment and was significantly higher in rats on the high-salt diet over a 1-wk period (170 ± 3 vs. 115 ± 3 mmHg, P < 0.01). To determine whether the increase in MAP during A-192621 treatment was due to increased ETA receptor activation, all rats were then given the ETA-selective antagonist ABT-627 in the drinking water while a low-salt/high-salt diet and ETB blockade were continued. ABT-627 decreased MAP within a few hours in rats on either the high-salt (113 ± 3 mmHg) or low-salt (101 ± 3 mmHg) diet. These results support the hypothesis that endothelin, through the ETB receptor, participates in blood pressure regulation in the response to salt loading.
Angiotensin II activates the GFAT promoter in mesangial cells
Authors: James Leighton R. , Ingram Alistair , Ly Hao , Thai Kerri , Cai Lu , Scholey James W.
Published: December 2017

Expression of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme for glucose entry into the hexosamine pathway, is transcriptionally regulated. Immunohistochemical studies of human kidney biopsies demonstrate increased GFAT expression in diabetic glomeruli, but the mechanism responsible for this overexpression is unknown. Given the role of ANG II in diabetic kidney disease, we chose to study the effect of ANG II on GFAT promoter activity in mesangial cells (MC). Exposure of MC to ANG II (10−7M) increased GFAT promoter activity (2.5-fold), mRNA (3-fold), and protein (1.6-fold). ANG II-mediated GFAT promoter activation was inhibited by the ANG II type I receptor antagonist candesartan (10−8M) but was unaffected by the ANG II type II receptor antagonist PD-123319 (10−8M). The intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid (10−6M), protein kinase C (PKC) inhibitors bisindoylmaleimide-4 (10−6M) and calphostin C (10−7M), protein tyrosine kinase (PTK) inhibitor genistein (10−4M), Src family kinase inhibitor PP2 (2.5 × 10−7M), p42/44 mitogen-activated protein kinase (MAPK) inhibitor PD-98059 (10−5M), and the epidermal growth factor (EGF) inhibitor AG-1478 all attenuated GFAT promoter activation by ANG II. We conclude that the GFAT promoter is activated by ANG II via the AT1receptor. Promoter activation is calcium dependent and PKC dependent but also involves PTK signaling pathways including Src, the EGF receptor, and p42/44 MAPK.
Tetrahydrobiopterin reverses the inhibition of nitric oxide by high glucose in cultured murine mesangial cells
Authors: Prabhakar Sharma S.
Published: December 2017

Alterations of intrarenal nitric oxide (NO) synthesis play an important role in the pathogenesis and progression of diabetic nephropathy. We tested the hypothesis that hyperglycemia modulates intrarenal NO synthesis, which might mediate the mesangial cell proliferation and matrix production. Murine mesangial cells were grown in media containing varying glucose concentrations, and cytokine-induced NO synthesis was assayed by chemiluminescence using an NO analyzer. High media glucose (25 mM) inhibited NO synthesis in a time-dependent fashion. This inhibition was posttranslational as revealed by analysis of inducible nitric oxide synthase (iNOS) gene and protein expression. l-Arginine supplementation partially reversed the inhibition whereas addition of tetrahydrobiopterin (BH4), a cofactor for NOS, restored the inducibility of NO synthesis. The in vitro [3H]citrulline assay for iNOS activity indicated that high glucose decreased BH4availability whereas examination of the BH4synthetic pathway suggested decreased BH4stability rather than synthesis, a defect that was corrected by ascorbic acid. We conclude that hyperglycemia inhibits NO synthesis in mesangial cells by a posttranslational defect that might involve the stability and hence availability of BH4.
Mycophenolate mofetil prevents salt-sensitive hypertension resulting from nitric oxide synthesis inhibition
Authors: Quiroz Yasmir , Pons Héctor , Gordon Katherine L. , Rincón Jaimar , Chávez Maribel , Parra Gustavo , Herrera-Acosta Jaime , Gómez-Garre Dulcenombre , Largo Raquel , Egido Jesus , Johnson Richard J. , Rodríguez-Iturbe Bernardo
Published: December 2017

Recent studies have suggested that subtle microvascular and tubulointerstitial injury in the kidney can cause salt-sensitive hypertension. To test this hypothesis, we determined whether the mild renal disease induced by transient blockade of nitric oxide (NO) synthesis would result in salt-sensitive hypertension and whether prevention of the renal injury by coadministration of the immunosuppressive agent mycophenolate mofetil (MMF) would block the development of salt sensitivity. Nω-nitro-l-arginine-methyl ester (l-NAME; 70 mg/100 ml in the drinking water) was administered for 3 wk to rats with or without MMF (30 mg · kg−1· day−1by gastric gavage), followed by a 1-wk “washout” period in which the MMF was continued, which was followed in turn by placement on a high-salt (4% NaCl) diet for an additional 4 wk. Renal histology was examined at 3 and 8 wk, and blood pressure was measured serially.l-NAME treatment resulted in acute hypertension and the development of mild renal injury. During the washout period, blood pressure returned to normal, only to return to the hypertensive range on exposure of the animals to a high-salt diet. MMF treatment prevented the development of hypertension in response to a high-salt diet. This correlated with the ability of MMF to inhibit specific aspects of the renal injury, including the development of segmental glomerulosclerosis, the infiltration of T cells and ANG II-positive cells, and the thickening of afferent arterioles.
cADP-ribose/ryanodine channel/Ca2+-release signal transduction pathway in mesangial cells
Authors: Yusufi Ahad N. K. , Cheng Jingfei , Thompson Michael A. , Dousa Thomas P. , Warner Gina M. , Walker Henry J. , Grande Joseph P.
Published: December 2017

Signaling via release of Ca2+from intracellular stores is mediated by several systems, including the inositol 1,4,5-trisphosphate (IP3) and cADP-ribose (cADPR) pathway. We recently discovered a high capacity for cADPR synthesis in rat glomeruli and cultured mesangial cells (MC). We sought to determine whether 1) cADPR synthesis in MC is regulated by cytokines and hormones, 2) ryanodine receptors (RyRs) are expressed in MC, and 3) Ca2+is released through RyRs in response to cADPR. We found that ADP-ribosyl cyclase, a CD38-like enzyme that catalyzes cADPR synthesis, is upregulated in MC by tumor necrosis factor-α, interleukin-1β, and all- trans retinoic acid (atRA). [3H]ryanodine binds to microsomal fractions from MC with high affinity in a Ca2+-dependent manner; binding is enhanced by specific RyR agonists and blocked by ruthenium red and cADPR. Western blot analysis confirmed the presence of RyR in MC. Release of45Ca2+from MC microsomes was stimulated by cADPR; release was blocked by ruthenium red and 8-bromo-cADPR. ADPR (non-cyclic) was without effect. In MC, TNF-α and atRA amplified the increment of cytoplasmic Ca2+elicited by vasopressin. We conclude that MC possess elements of a novel ADP-ribosyl cyclase→cADPR→RyR→Ca2+-release signaling pathway subject to regulation by proinflammatory cytokines and steroid superfamily hormones.
Regulation of ANP clearance receptors by EGF in mesangial cells from NOD mice
Authors: Placier Sandrine , Bretot Xavier , Ardaillou Nicole , Dussaule Jean-Claude , Ardaillou Raymond
Published: December 2017

Mesangial cells from nonobese diabetic (NOD) mice (D-NOD) that develop diabetes at 2–4 mo express an increased density of atrial natriuretic peptide (ANP) clearance receptors [natriuretic peptide C receptor (NPR-C)] and produce less GMP in response to ANP than their nondiabetic counterparts (ND-NOD). Our purpose was to investigate how both phenotypic characteristics were regulated. Epidermal growth factor (EGF) and heparin-binding (HB)-EGF, but not platelet-derived growth factor or insulin-like growth factor I, inhibited125I-ANP binding to ND-NOD and D-NOD mesangial cells, particularly in the latter. NPR-C density decreased with no change in the apparent dissociation constant, and there was also a decrease in NPR-C mRNA expression. The EGF effect depended on activation of its receptor tyrosine kinase but not on that of protein kinase C, mitogen-activated protein kinases, or phosphoinositide-3 kinase. Activation of activator protein-1 (AP-1) was necessary, as shown by the inhibitory effect of curcumin and the results of the gel-shift assay. The cGMP response to physiological concentrations of ANP was greater in EGF-treated D-NOD cells. These studies suggest that EGF potentiates the ANP glomerular effects in diabetes by inhibition of its degradation by mesangial NPR-C via a mechanism involving AP-1.
Norepinephrine-induced calcium signaling pathways in afferent arterioles of genetically hypertensive rats
Authors: Salomonsson Max , Arendshorst William J.
Published: December 2017

This study provides new information about the relative importance of calcium mobilization and entry in the renal vascular response to adrenoceptor activation in afferent arterioles isolated from 7- to 8-wk-old Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Intracellular free calcium concentration ([Ca2+]i) was measured in microdissected arterioles utilizing ratiometric photometry of fura 2 fluorescence. There was no significant strain difference in baseline [Ca2+]i. Norepinephrine (NE; 10−6and 10−7M) elicited immediate, sustained increases in [Ca2+]i. The general temporal pattern of response to 10−6M NE consisted of an initial peak and a maintained plateau phase. The response to NE was partially blocked by nifedipine (10−6M) or 8-( N,N-diethylamino) octyl-3,4,5-trimetoxybenzoate (TMB-8; 10−5M). A calcium-free external solution abolished the sustained [Ca2+]iplateau response to NE, with less influence on the peak response. In the absence of calcium entry, TMB-8 (10−5M) completely blocked the calcium response to NE in WKY but not SHR, suggesting strain differences in mobilization. A higher concentration of TMB-8 (10−4M), however, blocked all discernible mobilization in both strains. We conclude that there are differences in Ca2+handling in renal resistance vessels between young WKY and SHR with respect to mobilization stimulated by α-adrenoceptors. Afferent arterioles of young SHR appear to have a larger inositol-1,4,5-trisphosphate-sensitive pool or release from a site less accessible to TMB-8.
Independent organic cation transport activity of Na+-l-carnitine cotransport system in LLC-PK1 cells
Authors: Ohnishi Shuichi , Saito Hideyuki , Fukada Atsuko , Inui Ken-Ichi
Published: December 2017

We investigated expression of the Na+-l-carnitine cotransport system and its role in transport of tetraethylammonium in a kidney epithelial cell line, LLC-PK1. l-Carnitine uptake in the LLC-PK1 cells was markedly stimulated in the presence of Na+. The uptake was saturable, with Michaelis constant and maximal uptake velocity values of 7.8 μM and 153.7 pmol · mg protein−1 · 15 min−1, respectively. Cationic drugs such as tetraethylammonium, cimetidine, and quinidine inhibited l-carnitine uptake. The basolateral-to-apical transport of [14C]tetraethylammonium was enhanced markedly in the presence of an H+ gradient on the apical side at a pH of 5.9. Under the conditions in which Na+/l-carnitine cotransport activity was saturable by the addition of 100 μM l-carnitine to the apical-side medium, the basolateral-to-apical transcellular transport of [14C]tetraethylammonium was unaffected. These results suggested that the Na+-l-carnitine cotransporter is expressed in the apical membranes of LLC-PK1 cells, and is not responsible for efflux of tetraethylammonium from the cells. Transport of tetraethylammonium appeared to be mediated predominantly by an H+/organic cation antiporter in the apical membranes.
K+-induced HSP-72 expression is mediated via rapid Ca2+influx in renal epithelial cells
Authors: Eickelberg Oliver , Geibel John , Seebach Frank , Giebisch Gerhard , Kashgarian Michael
Published: December 2017

Pathophysiological stimuli, including hypoxia, lead to K+efflux from the intracellular lumen to the extracellular space, thereby increasing local tissue K+concentrations and depolarizing resident cells. In this study, we investigated the effects of increased extracellular K+concentrations ([K+]e) on heat shock protein (HSP) expression in the porcine proximal tubule epithelial cell line LLC-PK1. We analyzed HSP-25, HSP-72, HSC-73, and HSP-90 protein expression by Western blot analyses and HSP-72 promoter activity by luciferase reporter gene assays using the proximal 1,440 bp of the HSP-72 promoter. Elevating [K+]efrom 20 to 50 mM increased HSP-72 protein expression and promoter activity but did not affect HSP-25, HSC-73, or HSP-90 levels. Addition of identical concentrations of sodium chloride did not increase HSP-72 expression to a similar amount. The Ca2+channel blocker diltiazem and the Ca2+-specific chelator EGTA-AM abolished high [K+]e-induced HSP-72 expression by 69.7 and 75.2%, respectively, indicating that the transcriptional induction of HSP-72 involves Ca2+influx. As measured by confocal microscopy using the Ca2+dye fluo 3-AM, we also observed a rapid increase of intracellular Ca2+concentration as early as 30 s after placing LLC-PK1cells in high [K+]e. We further analyzed whether Ca2+influx was necessary for induction of HSP-72 expression by high [K+]eusing Ca2+-free medium. Here, induction of HSP-72 in response to high [K+]ewas completely abolished. Our data thus demonstrate activation of a protective cellular response to ionic stress, e.g., elevated K+concentrations, by specifically increasing protein levels of HSP-72.
Upregulated expression of human membrane type-5 matrix metalloproteinase in kidneys from diabetic patients
Authors: Romanic Anne M. , Burns-Kurtis Cynthia L. , Ao Zhaohui , Arleth Anthony J. , Ohlstein Eliot H.
Published: December 2017

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade the extracellular matrix (ECM). The membrane-type matrix metalloproteinases (MT-MMPs) are a new family of MMPs that differ from other MMPs in that they have a transmembrane domain that anchors them to the cell surface. MT-MMPs have been shown to function as receptors and activators for other MMPs and to localize extracellular matrix proteolysis at the pericellular region. Here we report on mRNA and protein expression of the fifth human MT-MMP (MT5-MMP), a 64-kDa protein that is capable of converting pro-MMP-2 to its active form, in human kidney as well as its upregulation in diabetes. We also demonstrate upregulation of the active form of MMP-2 in kidney samples from patients with diabetes. Through immunohistochemistry, MT5-MMP expression was localized to the epithelial cells of the proximal and distal tubules, the collecting duct, and the loop of Henle. Furthermore, the tubular epithelial cells that expressed MT5-MMP were associated with tubular atrophy. Because renal tubular atrophy is a significant factor in the pathogenesis of diabetic nephropathy and renal failure and the molecular mechanisms regulating this process remain unknown, it is hypothesized that the elevated expression of MT5-MMP contributes to the activation of pro-MMP-2, which participates in the remodeling of the proximal and distal tubules as well as in the collecting duct. These results provide the first evidence of the expression of a MT-MMP in diabetes and suggest a novel role for MT5-MMP in the pathogenesis of renal tubular atrophy and end-stage renal disease.
Immunohistochemical localization of colonic H-K-ATPase to the apical membrane of connecting tubule cells
Authors: Fejes-Tóth Géza , Náray-Fejes-Tóth Anikó
Published: December 2017

Previous studies indicate that the colonic H-K-ATPase mRNA is expressed as the distal nephron. However, the exact intrarenal localization of the colonic H-K-ATPase protein is still unclear. The goal of the present study was to determine the cellular and subcellular localization of the colonic H-K-ATPase protein in the rabbit kidney. We used three monoclonal antibodies (MAbs) directed against different epitopes of the rabbit colonic H-K-ATPase α-subunit (HKα2) to localize HKα2protein by immunofluorescence labeling of kidney sections and laser-scanning confocal microscopy. The specificity of the MAbs was confirmed by reaction with a single ∼100-kDa band on Western blots of distal colon. Specific immunohistochemical reaction with the apical membrane of surface epithelial cells was observed with all three MAbs on distal colon sections. In rabbit kidney, immunofluorescence was detected only on the apical membrane of connecting tubule cells. Immunofluorescence was not detected in the cortical-, outer-, and inner-medullary collecting ducts. Furthermore, costaining with principal- and intercalated cell-specific MAbs and a MAb against the thick ascending limb suggests that these cell types express HKα2protein at levels that are below the detection limit with this method. We conclude that in the rabbit kidney, under normal dietary conditions, the HKα2protein is expressed in the apical membrane of connecting tubule cells.
Specific association of nitric oxide synthase-2 with Rac isoforms in activated murine macrophages
Authors: Kuncewicz Teresa , Balakrishnan Priya , Snuggs Mark B. , Kone Bruce C.
Published: December 2017

Nitric oxide synthase-2 (NOS2) is responsible for high-output nitric oxide production important in renal inflammation and injury. Using a yeast two-hybrid assay, we identified Rac2, a Rho GTPase member, as a NOS2-interacting protein. NOS2 and Rac2 proteins coimmunoprecipitated from activated RAW 264.7 macrophages. The two proteins colocalized in an intracellular compartment of these cells. Glutathione- S-transferase (GST) pull-down assays revealed that both Rac1 and Rac2 associated with GST-NOS2 and that the NOS2 oxygenase domain was necessary and sufficient for the interaction. [35S]methionine-labeled NOS2 interacted directly with GST-Rac2 in the absence of GTP, calmodulin, or NOS2 substrates or cofactors. Stable overexpression of Rac2 in RAW 264.7 cells augmented LPS-induced nitrite generation (∼60%) and NOS2 activity (∼45%) without measurably affecting NOS2 protein abundance and led to a redistribution of NOS2 to a high-speed Triton X-100-insoluble fraction. We conclude that Rac1 and Rac2 physically interact with NOS2 in activated macrophages and that the interaction with Rac2 correlates with a posttranslational stimulation of NOS2 activity and likely its spatial redistribution within the cell.
Role of megalin in renal handling of aminoglycosides
Authors: Nagai Junya , Tanaka Hiroaki , Nakanishi Naoki , Murakami Teruo , Takano Mikihisa
Published: December 2017

The role of megalin in tissue distribution of aminoglycosides was examined in normal rats and maleate-treated rats that shed megalin from the renal brush-border membrane. In normal rats, amikacin administered intravenously accumulated most abundantly in the renal cortex, followed by the renal medulla. No amikacin was detected in other tissues. Tissue distributions of amikacin were well correlated with megalin levels in each tissue. Bolus administration of gentamicin increased urinary excretion of megalin ligands (vitamin D binding protein and calcium), suggesting the competition between gentamicin and these megalin ligands in renal tubules. Ligand blotting showed that binding of45Ca2+to megalin was inhibited by aminoglycosides. Both megalin levels and amikacin accumulation in renal cortex were decreased by maleate injection. Then, amikacin accumulation recovered proportionate to megalin levels. These findings suggest that megalin is involved in the renal cortical accumulation of aminoglycosides in vivo. In addition, the interaction between aminoglycosides and calcium in the kidney may be due to the competition among these compounds to bind to megalin.
Increased CO2 stimulates K/Rb reabsorption mediated by H-K-ATPase in CCD of potassium-restricted rabbit
Authors: Zhou Xiaoming , Nakamura Suguru , Xia Shen-Ling , Wingo Charles S.
Published: December 2017

Apical H-K-ATPase in the cortical collecting duct (CCD) plays an important role in urinary acidification and K reabsorption. Our previous studies demonstrated that an H-K-ATPase mediates, in part, Rb reabsorption in rabbit CCD (Zhou X and Wingo CS. Am J Physiol Renal Fluid Electrolyte Physiol 263: F1134–F1141, 1992). The purpose of these experiments was to examine using in vitro microperfused CCD from K-restricted rabbits 1) whether an acute increase in Pco 2 and, presumably, intracellular acidosis stimulate K absorptive flux; and 2) whether this stimulation was dependent on the presence of a functional H-K-ATPase. Rb reabsorption was significantly increased after exposure to 10% CO2 in CCD, and this effect was persistent for the entire 10% CO2 period, whereas 10 μM SCH-28080 in the perfusate totally abolished the stimulation of Rb reabsorption by 10% CO2. After stimulation of Rb reabsorption by 10% CO2, subsequent addition of 0.1 mM methazolamide, an inhibitor of carbonic anhydrase, failed to affect Rb reabsorption. However, simultaneous exposure to 10% CO2 and methazolamide prevented the stimulation of Rb reabsorption. Treatment with the intracellular calcium chelator MAPTAM (0.5 μM) inhibited the stimulation of Rb reabsorption by 10% CO2. Similar inhibition was also observed in the presence of either a calmodulin inhibitor, W-7 (0.5 μM), or colchicine (0.5 mM), an inhibitor of tubulin polymerization. In time control studies, the perfusion time did not significantly affect Rb reabsorption. We conclude the following: 1) stimulation of Rb reabsorption on exposure to 10% CO2 is dependent on the presence of a functional H-K-ATPase and appears to be regulated in part by the insertion of this enzyme into the apical plasma membrane by exocytosis; 2) insertion of H-K-ATPase requires changes in intracellular pH and needs a basal level of intracellular calcium concentration; and 3) H-K-ATPase insertion occurs by a microtubule-dependent process.
Cloning and characterization of the human urea transporter UT-A1 and mapping of the human Slc14a2 gene
Authors: Bagnasco Serena M. , Peng Tao , Janech Michael G. , Karakashian Alexander , Sands Jeff M.
Published: December 2017

We have isolated and characterized the human homolog of the rat largest urea transporter of the UT-A family (hUT-A1). The 4.2-kb hUT-A1 cDNA encodes a 920-amino acid peptide, which is 89% identical to the rat UT-A1 protein. By Northern hybridization, hUT-A1 expression is detected in the human inner medulla as a ∼4.4-kb mRNA transcript. By Western analysis, hUT-A1 is identified as a ∼100-kDa protein in the human inner medulla. By immunohistochemistry, hUT-A1 expression is localized to the inner medullary collecting duct (IMCD). When transfected into HEK-293 cells hUT-A1 cDNA is translated into a ∼98-kDa protein. Expression of hUT-A1 in Xenopus oocytes results in phloretin-inhibitable uptake of14C-urea, which shows only modest stimulation by cAMP, suggesting that in the human IMCD vasopressin may have a limited role in the short-term regulation of hUT-A1-mediated urea transport. We determined the organization of the human Slc14a2 gene and identified 20 exons distributed over ∼67.5 kb on chromosome 18, from which hUT-A1 and the other human urea transporter, hUT-A2, are transcribed.