bluuebunny/crossref_metadata_2025 huggingface dataset:
In vitro effects of simvastatin on tubulointerstitial cells in a human model of cyclosporin nephrotoxicity
Authors: Johnson David W. , Saunders Heather J. , Field Michael J. , Pollock Carol A.
Published: December 2017

To investigate the possibility that 3-hydroxy-3-methylglutaryl CoA (HMGCoA) reductase inhibitors ameliorate renal disease via direct effects on the tubulointerstitium, primary cultures of human proximal tubule cells (PTC) and renal cortical fibroblasts (CF) were exposed for 24 h to simvastatin (0.1–10 μmol/l) under basal conditions and in the presence of 1,000 ng/ml of cyclosporin (CsA), which we have previously shown to promote in vitro interstitial matrix accumulation at least partially via activation of local cytokine networks. Simvastatin, in micromolar concentrations, engendered cholesterol-independent inhibition of CF and PTC thymidine incorporation and cholesterol-dependent suppression of PTC apical Na+/H+exchange (NHE) (ethylisopropylamiloride-sensitive apical22Na+uptake). Similarly, CF secretion of insulin-like growth factor-I (IGF-I) and IGF binding protein-3 were depressed, whereas CF collagen synthesis ([3H]proline incorporation) and PTC secretion of the fibrogenic cytokines, transforming growth factor-β1, and platelet-derived growth factor were unaffected. A lower concentration (0.1 μmol/l) of simvastatin did not affect any of the above parameters under basal conditions but completely prevented CsA-stimulated CF collagen synthesis (control, 6.6 ± 0.6; CsA, 8.3 ± 0.6; CsA+simvastatin, 6.2 ± 0.5%; P < 0.05) and IGF-I secretion (89.5 ± 16.6, 204.7 ± 57.0, and 94.6 ± 22.3 ng ⋅ mg protein−1⋅ day−1, respectively; P < 0.05). The results suggest that simvastatin exerts direct cholesterol-dependent and -independent effects on the human kidney tubulointerstitium. HMGCoA reductase inhibitors may ameliorate interstitial fibrosis complicating CsA therapy via direct actions on human renal cortical fibroblasts.
Molecular regulation of nephron endowment
Authors: Clark Amander T. , Bertram John F.
Published: December 2017

Recent data suggests that the number of nephrons in normal adult human kidneys ranges from ∼300,000 to more than 1 million. There is increasing evidence that reduced nephron number, either inherited or acquired, is associated with the development of essential hypertension, chronic renal failure, renal disease in transitional indigenous populations, and possibly the long-term success of renal allografts. Three processes ultimately govern the number of nephrons formed during the development of the permanent kidney (metanephros): branching of the ureteric duct in the metanephric mesenchyme; condensation of mesenchymal cells at the tips of the ureteric branches; and conversion of the mesenchymal condensates into epithelium. This epithelium then grows and differentiates to form nephrons. In recent years, we have learned a great deal about the molecular regulation of these three central processes and hence the molecular regulation of nephron endowment. Data has come from studies on cell lines, isolated ureteric duct epithelial cells, isolated metanephric mesenchyme, and whole metanephric organ culture, as well as from studies of heterozygous and homozygous null mutant mice. With accurate and precise methods now available for estimating the total number of nephrons in kidneys, more advances in our understanding of the molecular regulation of nephron endowment can be expected in the near future.
The nongastric H+-K+-ATPases: molecular and functional properties
Authors: Jaisser Frederic , Beggah Ahmed T.
Published: December 2017

The Na-K/H-K-ATPase gene family is divided in three subgroups including the Na-K-ATPases, mainly involved in whole body and cellular ion homeostasis, the gastric H-K-ATPase involved in gastric fluid acidification, and the newly described nongastric H-K-ATPases for which the identification of physiological roles is still in its infancy. The first member of this last subfamily was first identified in 1992, rapidly followed by the molecular cloning of several other members. The relationship between each member remains unclear. The functional properties of these H-K-ATPases have been studied after their ex vivo expression in various functional expression systems, including the Xenopus laevisoocyte, the insect Sf9 cell line, and the human HEK 293 cells. All these H-K-ATPase α-subunits appear to encode H-K-ATPases when exogenously expressed in such expression systems. Recent data suggest that these H-K-ATPases could also transport Na+in exchange for K+, revealing a complex cation transport selectivity. Moreover, they display a unique pharmacological profile compared with the canonical Na-K-ATPases or the gastric H-K-ATPase. In addition to their molecular and functional characterizations, a major goal is to correlate the molecular expression of these cloned H-K-ATPases with the native K-ATPases activities described in vivo. This appears to be more complex than anticipated. The discrepancies between the functional data obtained by exogenous expression of the nongastric H-K-ATPases and the physiological data obtained in native organs could have several explanations as discussed in the present review. Extensive studies will be required in the future to better understand the physiological role of these H-K-ATPases, especially in disease processes including ionic or acid-base disorders.
Characterization of acute reversible systemic hypertension in a model of heme protein-induced renal injury
Authors: Warden David H. , Croatt Anthony J. , Katusic Zvonimir S. , Nath Karl A.
Published: December 2017

In the glycerol model of renal injury we describe an acute rise in systemic arterial pressure which is attended by a reduced vasodilatory response to acetylcholine in vivo; vasodilatory responses to verapamil, however, were not impaired. Neither arginine nor sodium nitroprusside diminished this rise in blood pressure; N ω-nitro-l-arginine methyl ester (l-NAME) elevated basal mean arterial pressure and markedly blunted the rise in mean arterial pressure following the administration of glycerol. Aortic rings from the glycerol-treated rat demonstrate an impaired vasodilatory response to acetylcholine, an effect not repaired by arginine; the vasodilatory responses to nitric oxide donors, sodium nitroprusside and SIN-1, were also impaired; 8-bromo-cGMP, at higher doses, evinced a vasodilatory response comparable to that observed in the control rings. This pattern of responses was not a nonspecific effect of aortic injury, since aortic rings treated with mercuric chloride, a potent oxidant, displayed an impaired vasodilatory response to acetylcholine but not to sodium nitroprusside. We conclude that in the glycerol model of heme protein-induced tissue injury, there is an acute elevation in mean arterial pressure attended by impaired endothelium-dependent vasodilatation in vitro and in vivo. We suggest that the acute scavenging of nitric oxide by heme proteins depletes the blood vessel wall of its endogenous vasodilator and permeation of heme proteins into the blood vessel wall may contribute to such sustained effects as observed in vitro.
Angiotensin-(1–7) can interact with the rat proximal tubule AT4receptor system
Authors: Handa Rajash K.
Published: December 2017

This study was undertaken to identify the non-AT1, non-AT2angiotensin receptor that mediates the ANG-(1–7) inhibitory action on rat proximal tubule transport processes. ANG-(1–7) inhibited nystatin-stimulated, ouabain-suppressible O2consumption (Qo2) rates in freshly isolated rat proximal tubules (reflecting reduced basolateral Na+-K+-ATPase activity). Selective angiotensin-receptor subtype antagonists revealed that AT1and AT4receptors mediated the response of ANG-(1–7). Receptor autoradiography of the rat kidney demonstrated a high density of AT1and AT4receptors and no specific125I-ANG(1–7) binding sites. Competition assays in rat kidney sections indicated that ANG-(1–7) competed predominantly for the AT1receptor site, whereas its NH2-terminal-deleted metabolite, ANG-(3–7), competed primarily for the AT4-receptor site. Metabolism of125I-ANG-(1–7) in rat proximal tubules generated peptide fragments that included ANG-(3–7), with the pentapeptide producing a concentration-dependent inhibition of nystatin-stimulated proximal tubule Qo2that was abolished by AT4-receptor blockade. These results suggest that the generation of ANG-(3–7) from the NH2-terminal metabolism of ANG-(1–7) caused the interaction of the parent peptide with the proximal tubule AT4receptor, which elicited a decrease in energy-dependent solute transport.
Vitamin D
Authors: Brown Alex J. , Dusso Adriana , Slatopolsky Eduardo
Published: December 2017

The vitamin D endocrine systems plays a critical role in calcium and phosphate homeostasis. The active form of vitamin D, 1,25-dihydroxyvitamin D3[1,25(OH)2D3], binds with high affinity to a specific cellular receptor that acts as a ligand-activated transcription factor. The activated vitamin D receptor (VDR) dimerizes with another nuclear receptor, the retinoid X receptor (RXR), and the heterodimer binds to specific DNA motifs (vitamin D response elements, VDREs) in the promoter region of target genes. This heterodimer recruits nuclear coactivators and components of the transcriptional preinitiation complex to alter the rate of gene transcription. 1,25(OH)2D3also binds to a cell-surface receptor that mediates the activation of second messenger pathways, some of which may modulate the activity of the VDR. Recent studies with VDR-ablated mice confirm that the most critical role of 1,25(OH)2D3is the activation of genes that control intestinal calcium transport. However, 1,25(OH)2D3can control the expression of many genes involved in a plethora of biological actions. Many of these nonclassic responses have suggested a number of therapeutic applications for 1,25(OH)2D3and its analogs.
Pretreatment with inducers of ER molecular chaperones protects epithelial cells subjected to ATP depletion
Authors: Bush Kevin T. , George Sathish K. , Zhang Ping L. , Nigam Sanjay K.
Published: December 2017

We have investigated the potential cytoprotective role of endoplasmic reticulum (ER) molecular chaperones in a cultured cell model of renal ischemia. Madin-Darby canine kidney (MDCK) cells were pretreated with tunicamycin (an inducer of ER but not cytosolic molecular chaperones) for 12–16 h, followed by 6 h of ATP depletion. A rapid and severe depletion of cellular ATP was noted in both control and tunicamycin-treated cells. Trypan blue exclusion assays indicated that pretreatment of MDCK cells with tunicamycin reduced ATP depletion-induced cell damage by ∼80% compared with nonpretreated controls. This apparent cytoprotective effect was also found following pretreatment with another inducer of ER molecular chaperones (i.e., A23187). For example, A23187 was found to reduce lactate dehydrogenase release by ∼50% compared with untreated controls, whereas E-64, a cysteine protease inhibitor which may affect degradation of some proteins in the ER, had little or no effect on cell injury. Moreover, a fluorescent assay confirmed the marked reduction in cell damage following ATP depletion (up to 80% reduction in tunicamycin-pretreated cells). Together, these findings are consistent with the notion that induction of ER molecular chaperones leads to the acquisition of cytoprotection in the face of ATP depletion. However, inhibition of protein translation by cycloheximide was found to only partially attenuate the observed cytoprotective effect, raising the possibility that other, as yet to be identified, nonprotein synthesis-dependent mechanisms may also play a role in the observed cytoprotection.
Cyclooxygenase-2 expression and function in the medullary thick ascending limb
Authors: Ferreri Nicholas R. , An Shao-Jian , McGiff John C.
Published: December 2017

The medullary thick ascending limb (MTAL) metabolizes arachidonic acid (AA) via cytochrome P-450 (CyP450)- and cyclooxygenase (COX)-dependent pathways. In the present study, we demonstrated that the COX-2-selective inhibitor, NS-398, prevented tumor necrosis factor-α (TNF)- and phorbol myristate acetate (PMA)-mediated increases in PGE2production by cultured MTAL cells. Accumulation of COX-2, but not COX-1, mRNA increased when cells were challenged with TNF (1 nM) or PMA (1 μM). Pretreatment of cells for 30 min with actinomycin D (AcD, 1 μM) had little effect on COX-2 mRNA accumulation in unstimulated cells or in cells challenged with either TNF or PMA. Moreover, a posttranscriptional mechanism(s) appears to contribute significantly to COX-2 mRNA accumulation as pretreatment for 15 min with cycloheximide (CHX, 1 μM) caused a superinduction of COX-2 mRNA accumulation in unstimulated cells as well as in cells challenged with either TNF or PMA. Expression of COX-2 protein in unstimulated MTAL cells was attenuated by preincubation for 2 h with dexamethasone (Dex, 2 μM); however, Dex had little or no effect on COX-2 expression in cells challenged with either PMA or TNF. The time-dependent inhibition of86Rb uptake by MTAL cells challenged with TNF was diminished by pretreating cells with NS-398. These data suggest that TNF-mediated induction of COX-2 protein expression accounted for the lag-time required for this cytokine to inhibit86Rb uptake in MTAL cells.
Newer aspects of glutamine/glutamate metabolism: the role of acute pH changes
Authors: Nissim Itzhak
Published: December 2017

This review focuses on the role of acute pH changes in the regulation of Gln/Glu metabolism in the kidney, liver, and brain. Alterations of proton concentration ([H+]) profoundly affect flux through phosphate-dependent glutaminase (PDG) or glutamate dehydrogenase (GDH), the primary enzymes responsible for mitochondrial metabolism of glutamine and glutamate, respectively. In the kidney, acute acidosis stimulates Gln uptake and its metabolism via the PDG pathway. The Glu formed from Gln can be removed via 1) oxidative deamination through the GDH reaction, 2) transamination reactions, and 3) transport of Glu from intracellular to extracellular compartment, thereby diminishing the intramitochondrial pool of glutamate sufficiently to stimulate flux through the PDG pathway. Converse changes may occur with increased pH. In the liver, acidosis diminishes the rate of Gln and Glu metabolism via the PDG and GDH pathways, but stimulates glutamine synthesis (i.e., glutamine recycling). Alkalosis has little effect. Hepatic Gln metabolism via the PDG pathway has a central role in ureagenesis via 1) supplementation of nitrogen for the synthesis of carbamyl phosphate, and 2) providing glutamate for N-acetylglutamate synthesis. In the brain, Gln/Glu metabolism links ammonia detoxification and energy metabolism via 1) detoxification of ammonia and excess glutamate by glutamine synthesis in astrocytes, 2) formation and export of glutamine to neurons where it is metabolized to glutamate and GABA, and 3) production of α-ketoglutarate and lactate from Glu and their transport to neurons. Changes in intracellular pH associated with changes in cellular [K+] may have a key role in the regulation of these processes of glial-neuronal metabolism of Gln/Glu metabolism.
Regulation of high-affinity glutamate transport by amino acid deprivation and hyperosmotic stress
Authors: McGivan J. D. , Nicholson B.
Published: December 2017

High-affinity glutamate transport activity is induced by stress in NBL-1 cells. Exposure of cells to hyperosmotic medium led to an induction of the EAAC1 glutamate transporter, preceded by a large increase in EAAC1 mRNA levels. Culture of cells in amino acid-free medium also caused a protein synthesis-dependent increase in glutamate transport activity, but this was not accompanied by an increase of either EAAC1 mRNA or protein. Indirect evidence suggests that the increase in EAAC1 activity in the latter case may be due to the synthesis of an activator protein in response to decreased intracellular glutamate concentrations.
Role of TNFR1 and TNFR2 receptors in tubulointerstitial fibrosis of obstructive nephropathy
Authors: Guo Guangjie , Morrissey Jeremiah , McCracken Ruth , Tolley Timothy , Klahr Saulo
Published: December 2017

Unilateral ureteral obstruction (UUO) results in tubulointerstitial fibrosis of the obstructed kidney. In this study, we report the contribution of tumor necrosis factor-α (TNF-α) to the fibrosis that develops after ureteral obstruction. Mice in which individual TNF-α receptors TNFR1 or TNFR2 had been genetically knocked out were used, and results were compared with mice of C57Bl/6 background after 5 days UUO. Both kidneys were removed and examined histologically for changes in interstitial volume (Vvint), collagen IV deposition, α-smooth muscle actin (α-SMA) matrix score, nuclear factor-κB (NF-κB) activity, and TNF-α mRNA levels. We found that the Vvintof contralateral unobstructed kidneys averaged ∼7% and was indistinguishable among the three genotypes of mice. Vvintof ureteral obstructed kidney of C57Bl/6 mice averaged 33 ± 3.9% after 5 days of UUO. Vvintof obstructed kidneys of TNFR1 mice was significantly reduced to 19.4 ± 3.1%, whereas that of TNFR2 mice was significantly decreased to 25.4% ± 4.8%. There was a modest but significant difference between Vvintof TNFR1 and TNFR2 ( P < 0.047). Both collagen IV and α-SMA matrix scores were decreased significantly in obstructed kidney of TNFR1 mouse compared with that of C57Bl/6 and TNFR2 mice. Nuclear extracts prepared from kidney cortex were found to have a significant increase in NF-κB binding activity in obstructed kidney compared with contralateral kidney. Individual knockout of the TNFR1 or TNFR2 genes resulted in significantly less NF-κB activation compared with the wild type, with TNFR1 being less than TNFR2 knockout. There was a significant increase in TNF-α mRNA in the kidney with ureteral obstruction in all three genotypes. TNFR1 knockout displayed a significant reduction in amount of TNF-α mRNA induced compared with wild-type or TNFR2 knockout mice. Treatment of TNFR1 knockout mice with an angiotensin converting enzyme inhibitor further decreased Vvintand TNF-α mRNA induction, suggesting an interaction of ANG II and TNF-α systems. These results suggest that TNF-α contributes, in part, to changes in interstitial volume, myofibroblast differentiation, and NF-κB activation in the kidney during ureteral obstruction. These changes appear to be mediated through both TNFR1 and TNFR2 gene products with effects through the TNFR1 receptor predominating. Furthermore, ANG II appears to stimulate TNF-α pathophysiological events leading to renal fibrosis.
Increased activity and expression of Ca2+-dependent NOS in renal cortex of ANG II-infused hypertensive rats
Authors: Chin So Yeon , Pandey Kailash N. , Shi Shang-Jin , Kobori Hiroyuki , Moreno Carol , Navar L. Gabriel
Published: December 2017

We have previously demonstrated that nitric oxide (NO) exerts a greater modulatory influence on renal cortical blood flow in ANG II-infused hypertensive rats compared with normotensive rats. In the present study, we determined nitric oxide synthase (NOS) activities and protein levels in the renal cortex and medulla of normotensive and ANG II-infused hypertensive rats. Enzyme activity was determined by measuring the rate of formation ofl-[14C]citrulline froml-[14C]arginine. Western blot analysis was performed to determine the regional expression of endothelial (eNOS), neuronal (nNOS), and inducible (iNOS) isoforms in the renal cortex and medulla of control and ANG II-infused rats. Male Sprague-Dawley rats were prepared by the infusion of ANG II at a rate of 65 ng/min via osmotic minipumps implanted subcutaneously for 13 days and compared with sham-operated rats. Systolic arterial pressures were 127 ± 2 and 182 ± 3 mmHg in control ( n = 13) and ANG II-infused rats ( n = 13), respectively. The Ca2+-dependent NOS activity, expressed as picomoles of citrulline formed per minute per gram wet weight, was higher in the renal cortex of ANG II-infused rats (91 ± 11) than in control rats (42 ± 12). Likewise, both eNOS and nNOS were markedly elevated in the renal cortex of the ANG II-treated rats. In both groups of rats, Ca2+-dependent NOS activity was higher in the renal medulla than in the cortex; however, no differences in medullary NOS activity were observed between the groups. Also, no differences in medullary eNOS levels were observed between the groups; however, medullary nNOS was decreased by 45% in the ANG II-infused rats. For the Ca2+-independent NOS activities, the renal cortex exhibited a greater activity in the control rats (174 ± 23) than in ANG II-infused rats (101 ± 10). Similarly, cortical iNOS was greater by 47% in the control rats than in ANG II-treated rats. No differences in the activity were found for the renal medulla between the groups. There was no detectable signal for iNOS in the renal medulla for both groups. These data indicate that there is a differential distribution of NOS activity, with the Ca2+-dependent activity and protein expression higher in the renal cortex of ANG II-infused rats compared with control rats, and support the hypothesis that increased constitutive NOS activity exerts a protective effect in ANG II-induced hypertension to maintain adequate renal cortical blood flow.
The rat Pkd2 protein assumes distinct subcellular distributions in different organs
Authors: Obermüller Nicholas , Gallagher A. Rachel , Cai Yiqiang , Gassler Nikolaus , Gretz Norbert , Somlo Stefan , Witzgall Ralph
Published: December 2017

Mutations in the PKD2 gene account for ∼15% of all cases of autosomal-dominant polycystic kidney disease. In the present study the cellular distribution of the Pkd2 protein was investigated by immunohistochemistry in different rat organs. Although the Pkd2 protein showed a widespread expression, a strikingly different distribution of the protein was observed between individual organs. Whereas in renal distal tubules and in striated ducts of salivary glands a basal-to-basolateral distribution of Pkd2 was found, a punctate cytoplasmic location was detected in the adrenal gland, ovary, cornea, and smooth muscle cells of blood vessels. Interestingly, in the adrenal gland and ovary, the rat Pkd2 protein was more heavily N-glycosylated than in the kidney and salivary gland. These results suggest that Pkd2 accomplishes its functions by interacting with proteins located in different cellular compartments. The extrarenal expression pattern of the Pkd2 protein hints at other candidate sites of disease manifestations in patients carrying PKD2 mutations.
Osteopontin expressed by renal tubular epithelium mediates interstitial monocyte infiltration in rats
Authors: Okada Hirokazu , Moriwaki Kenshi , Kalluri Raghuram , Takenaka Tsuneo , Imai Hiroe , Ban Shinichi , Takahama Motohide , Suzuki Hiromichi
Published: December 2017

In this study, we have shown that intravenously administered antisense oligodeoxynucleotide (ODN) was demonstrated to be taken up by tubular epithelium, after which it blocked mRNA expression of target genes in normal and nephritic rats. Therefore, we injected osteopontin (OPN) antisense ODN to Goodpasture syndrome (GPS) rats every second day between days 27 and 35, the time when renal OPN expression increased and interstitial monocyte infiltration was aggravated. In parallel to blockade of tubular OPN expression, this treatment significantly attenuated monocyte infiltration and preserved renal plasma flow in GPS rats at day 37, compared with sense ODN-treated and untreated GPS rats. No significant changes were observed in OPN mRNA level by RT-PCR and histopathology of the glomeruli after ODN treatment, which was compatible with an absence of differences in the urinary protein excretion rate. In conclusion, OPN expressed by tubular epithelium played a pivotal role in mediating peritubular monocyte infiltration consequent to glomerular disease.
Structure and function of aquaporin water channels
Authors: Verkman A. S. , Mitra Alok K.
Published: December 2017

The aquaporins (AQPs) are a family of small membrane-spanning proteins (monomer size ∼30 kDa) that are expressed at plasma membranes in many cells types involved in fluid transport. This review is focused on the molecular structure and function of mammalian aquaporins. Basic features of aquaporin structure have been defined using mutagenesis, epitope tagging, and spectroscopic and freeze-fracture electron microscopy methods. Aquaporins appear to assemble in membranes as homotetramers in which each monomer, consisting of six membrane-spanning α-helical domains with cytoplasmically oriented amino and carboxy termini, contains a distinct water pore. Medium-resolution structural analysis by electron cryocrystallography indicated that the six tilted helical segments form a barrel surrounding a central pore-like region that contains additional protein density. Several of the mammalian aquaporins (e.g., AQP1, AQP2, AQP4, and AQP5) appear to be highly selective for the passage of water, whereas others (recently termed aquaglyceroporins) also transport glycerol (e.g., AQP3 and AQP8) and even larger solutes (AQP9). Evidence for possible movement of ions and carbon dioxide through the aquaporins is reviewed here, as well as evidence for direct regulation of aquaporin function by posttranslational modification such as phosphorylation. Important unresolved issues include definition of the molecular pathway through which water and solutes move, the nature of monomer-monomer interactions, and the physiological significance of aquaporin-mediated solute movement. Recent results from knockout mice implicating multiple physiological roles of aquaporins suggest that the aquaporins may be suitable targets for drug discovery by structure-based and/or high-throughput screening strategies.
Immunoelectron microscopic localization of NBC3 sodium-bicarbonate cotransporter in rat kidney
Authors: Kwon Tae-Hwan , Pushkin Alexander , Abuladze Natalia , Nielsen Søren , Kurtz Ira
Published: December 2017

In the present study, we produced a rabbit peptide-derived polyclonal COOH-terminal antibody that selectively recognizes NBC3, to determine the cellular and subcellular localization of NBC3 in rat kidney, using immunocytochemistry and immunoelectron microscopy. Immunocytochemistry with cryostat sections and semithin cryosections revealed specific staining of intercalated cells (ICs) in the connecting tubule and in cortical, outer medullary, and initial inner medullary collecting ducts. In the connecting tubule and in the cortical and medullary collecting duct, the labeling was associated with both type A and type B ICs. In type A ICs, labeling was confined to the apical and subapical domains, whereas in type B ICs, basal domains were exclusively labeled. In contrast, collecting duct principal cells were consistently unlabeled, and this was confirmed using anti-aquaporin-2 antibodies, which labeled principal cells in parallel semithin cryosections. Glomeruli, proximal tubules, descending thin limbs, ascending thin limbs, thick ascending limbs, distal convoluted tubules, and vascular structures were unlabeled. For immunoelectron microscopy, tissue samples were freeze-substituted, and immunolabeling was performed on ultrathin Lowicryl HM20 sections. Immunoelectron microscopy demonstrated that NBC3 labeling was very abundant in the apical plasma membrane, in intracellular vesicles, and in tubulocisternal profiles in the subapical domains of type A ICs. In type B ICs, NBC3 was mainly present in the basolateral plasma membrane. Immunolabeling controls using peptide-absorbed antibody were consistently negative. In conclusion, NBC3 is highly abundant in the apical plasma membrane of type A ICs and in the basolateral plasma membrane of type B ICs. This suggests that NBC3 plays an important role in modulating bicarbonate transport in the connecting tubule and collecting duct.
Toward a comprehensive molecular model of active calcium reabsorption
Authors: Hoenderop Joost G. J. , Willems Peter H. G. M. , Bindels René J. M.
Published: December 2017

The fine tuning of Ca2+excretion in the kidney takes place in the distal nephron, which consists of the distal convoluted tubule, connecting tubule, and initial portion of the cortical collecting duct. In these segments, Ca2+is reabsorbed through an active transcellular pathway. The apical influx of Ca2+into the distal renal cell is presumably the rate-limiting step in this process, and its molecular identity has remained obscure so far. The recently discovered epithelial Ca2+channel (ECaC) exhibits the expected properties for being the gatekeeper in transcellular Ca2+reabsorption. The characteristics and potential physiological role of ECaC will be discussed in this review. Our knowledge of the mechanisms involved in the regulation of transcellular Ca2+transport has advanced rapidly since the development of cell models originating from distal tubular cells. Studies using these models indicate that hormones including arginine vasopressin, PGE2, adenosine, ATP, and atrial natriuretic peptide should be considered as calciotropic hormones controlling renal Ca2+handling. Evidence is now beginning to emerge that the stimulating calciotropic hormones utilize new cAMP-independent pathways to stimulate Ca2+reabsorption. These new findings allow the development of a comprehensive and detailed model of the process of transcellular calcium transport in the kidney whereby the individual contribution of the participating transporters can now be fully appreciated.
Vasopressin regulates endothelin-B receptor in rat inner medullary collecting duct
Authors: Wong Norman L. M. , Wong Bonita P. H. , Tsui Joseph K. C.
Published: December 2017

Previous studies have shown that endothelin (ET) antagonizes the actions of arginine vasopressin (AVP) in the renal collecting ducts. On the other hand, the effects of AVP on ET function within the collecting ducts of the kidney have not been investigated extensively. Using isolated inner medullary collecting ducts (IMCD), we examined the possibility that a decrease in ETB receptor mRNA accompanied AVP-induced downregulation of ETB receptors. Binding studies revealed that overnight incubation of rat IMCD cells with AVP significantly reduced the maximal binding capacity (Bmax) of ET. Activation of adenylate cyclase by forskolin decreased the total ETB receptor density by ∼42% but did not affect the density of ETA receptors. The Rp diastereoisomer of adenosine 3′,5′-cyclic monophosphothionate, Rp-cAMPS (a specific inhibitor of protein kinase A), blocked the AVP-induced reduction in ET receptor density. Using competitive PCR method, we also observed downregulation of ETB receptor mRNA in IMCD treated with AVP. Additional studies were done with IMCD to determine whether AVP inhibited the ET-induced accumulation of cGMP. We saw a reduction in ET-induced cGMP accumulation when IMCD was incubated overnight with AVP. This inhibition of ET-induced accumulation of cGMP was blocked by Rp-cAMPS. These results suggest that AVP regulates ETB receptor expression in IMCD.
Mechanisms of inactivation of the action of aldosterone on collecting duct by TGF-β
Authors: Husted Russell F. , Sigmund Rita D. , Stokes John B.
Published: December 2017

The purpose of these experiments was to investigate the mechanisms whereby transforming growth factor-β (TGF-β) antagonizes the action of adrenocorticoid hormones on Na+transport by the rat inner medullary collecting duct in primary culture. Steroid hormones 1) increased Na+transport by three- to fourfold, 2) increased the maximum capacity of the Na+-K+pump by 30–50%, 3) increased the steady-state levels of the α1-subunit of the Na+-K+-ATPase by ∼30%, and 4) increased the steady-state levels of the α-subunit of the rat epithelial Na+channel (α-rENaC) by nearly fourfold. TGF-β blocked the effects of steroids on the increase in Na+transport and the stimulation of the Na+-K+-ATPase and pump capacity. However, there was no effect of TGF-β on the steroid-induced increase in mRNA levels of α-rENaC. The effects of TGF-β were not secondary to the decrease in Na+transport per se, inasmuch as benzamil inhibited the increase in Na+transport but did not block the increase in pump capacity or Na+-K+-ATPase mRNA. The results indicate that TGF-β does not inactivate the steroid receptor or its translocation to the nucleus. Rather, they indicate complex pathways involving interruption of the enhancement of pump activity and activation/inactivation of pathways distal to the steroid-induced increase in the transcription of α-rENaC.
Urothelial pathophysiological changes in feline interstitial cystitis: a human model
Authors: Lavelle John P. , Meyers Susan A. , Ruiz W. Giovani , Buffington C. A. Tony , Zeidel Mark L. , Apodaca Gerard
Published: December 2017

Unique barrier properties of the urothelial surface membrane permit urine storage. Interstitial cystitis causes disabling dysuria, and frequency. Similarly, feline interstitial cystitis (FIC) occurs in cats. These studies define the permeability and structural properties of normal and FIC urothelium. To determine the effects of bladder filling, groups were studied before and after hydrodistention. Normal urothelium with or without hydrodistention exhibited high transepithelial resistances (TER) and low water and urea permeabilities, resembling other species. Fluorescence confocal microscopy revealed localization of the marker AE-31 to the apical surface of all umbrella cells in normal urothelium, with the tight junction protein ZO-1 localized to tight junctions. Scanning and transmission electron microscopy revealed uniform distribution of luminal cells with characteristic apical membrane and tight junction morphology. Urothelium in FIC animals displayed reduced TER and increased water and urea permeability following hydrodistention. Structural studies in FIC revealed denuded urothelium, with appearance of AE-31 in underlying epithelial cells. The results demonstrate severe epithelial damage and dysfunction in FIC and suggest novel approaches toward examining the etiology and therapy of IC.
Coexpression of neuropilin-1, Flk1, and VEGF164in developing and mature mouse kidney glomeruli
Authors: Robert Barry , Zhao Xuemei , Abrahamson Dale R.
Published: December 2017

Neuropilin-1, a neuronal cell surface semaphorin III receptor protein important for axonal guidance in developing peripheral nervous system efferents, has also been identified as a vascular endothelial growth factor (VEGF) receptor on endothelial cells. To evaluate its expression in kidney, we carried out RT-PCR on newborn and adult total renal RNAs. A 403-bp product, which was predicted to be that from neuropilin-1 mRNA, was found in both samples. Nucleotide sequencing confirmed that these products encoded neuropilin-1. Northern analysis of newborn and adult kidney RNA showed specific hybridization to appropriately sized bands of ∼6 kb. In situ hybridization with a mouse-specific antisense neuropilin-135S-cRNA probe showed distinct glomerular localization on sections from both newborns and adults. Similar patterns of hybridization were seen in sections treated with antisense cRNA probes against another VEGF receptor, Flk1, and with VEGF probes. However, the VEGF hybridization signal was markedly less in adult glomeruli than those for neuropilin-1 and Flk1. Because neuropilin-1 specifically binds VEGF165in humans, we carried out RT-PCR on mouse kidney RNA with primers that amplified the three alternatively spliced isoforms of VEGF mRNA. Our analysis showed that for both newborn and adult kidneys, the relative abundance of VEGF mRNA was VEGF164≫ VEGF120> VEGF188. We conclude that the expression of neuropilin-1, in conjunction with Flk1 and VEGF164, jointly contributes to the development and maintenance of glomerular capillaries.
Physiology of apoptosis
Authors: Gulbins E. , Jekle A. , Ferlinz K. , Grassmé H. , Lang F.
Published: December 2017

Ion fluxes and volume changes of the whole cell as well as of organelles belong to the hallmarks of apoptosis; however, the molecular mechanism regulating these changes is only poorly characterized. Several ion channels in the plasma membrane, in particular the N-type K+channel, the chloride channel cystic fibrosis conductance regulator, and an outward rectifying chloride channel, as well as the mitochondrial permeability transition pore, have been implicated to be involved in signal transduction cascades regulating apoptosis. Furthermore, Bcl-2-like proteins have been suggested to function, at least in part, as ion channels, because they display some homology to bacterial pore-forming toxins. In contrast to the demonstration of the involvement of these different ion channels in apoptosis, the molecular consequences regulated by these ion channels, and finally triggering apoptosis, are almost completely unknown.
Functional expression of novel peptide transporter in renal basolateral membranes
Authors: Terada Tomohiro , Sawada Kyoko , Ito Tatsuya , Saito Hideyuki , Hashimoto Yukiya , Inui Ken-Ichi
Published: December 2017

We examined the peptide transport activity in renal basolateral membranes. [14C]glycylsarcosine (Gly-Sar) uptake in rat renal cortical slices was saturable and inhibited by excess dipeptide and aminocephalosporin cefadroxil. When several renal cell lines were screened for the basolateral peptide transport activity, Madin-Darby canine kidney (MDCK) cells were demonstrated to have the greatest transport activity. [14C]Gly-Sar uptake across the basolateral membranes of MDCK cells was inhibited by di- and tripeptide and decreased with decreases in extracellular pH from 7.4 to 5.0. The Michaelis-Menten constant value of [14C]Gly-Sar uptake across the basolateral membranes of MDCK cells was 71 μM. The basolateral peptide transporter in MDCK cells showed several different [14C]Gly-Sar transport characteristics in growth dependence, pH profile, substrate affinity, and sensitivities to chemical modifiers from those of the apical H+-peptide cotransporter of MDCK cells. The findings of the present investigation indicated that the peptide transporter was expressed in the renal basolateral membranes. In addition, from the functional characteristics, the renal basolateral peptide transporter was suggested to be distinguishable from known peptide transporters, i.e., H+-peptide cotransporters (PEPT1 and PEPT2) and the intestinal basolateral peptide transporter.
Identification and characterization of a glomerular-specific promoter from the human nephrin gene
Authors: Wong M. Andrew , Cui Shiying , Quaggin Susan E.
Published: December 2017

Podocytes are highly specialized cells that make up a major portion of the glomerular filtration barrier in the kidney. They are also believed to play a pivotal role in the progression of chronic renal disease due to diverse causes that include diabetes (3, 20, 24) and aging (1, 7). Despite the importance of podocytes for kidney function and disease, studies of this cell type have been hindered due to a lack of model systems. Recently, the gene responsible for congenital Finnish nephropathy was identified and named nephrin (13). Nephrin expression is restricted to slit diaphragms of podocytes (11, 30). Infants with congenital Finnish nephropathy develop massive proteinuria and subsequent kidney failure due to podocyte injury. We have identified a 1.25-kb DNA fragment from the human nephrin promoter and 5′-flanking region that is capable of directing podocyte-specific expression in transgenic mice; this represents the first glomerular-specific promoter to be identified. Use of this transgene will facilitate studies of the podocyte in vivo and allow the identification of transacting factors that are required for podocyte-specific expression.
Effects of experimental diabetes on renal IGF/IGFBP system during neonatal period in the rat
Authors: De la Puente A. , Goya L. , Ramos S. , Martín M. A. , Alvarez C. , Escrivá F. , Pascual-Leone A. M.
Published: December 2017

Changes in the renal synthesis and concentration of insulin-like growth factors (IGFs) and their serum-binding proteins (IGFBPs) reported in insulin-deficient diabetes mellitus may be implicated in the alterations of the kidney function and morphology accompanying this disease. Most research on this subject has been carried out in adult animals, as well as in peripubertal rats, when the regulation of the IGF system is fully dependent on serum growth hormone (GH). However, relevant differences in the regulatory pathways of IGFs between adult and neonatal periods have been described. To examine the response of the IGF/IGFBP system of neonatal kidney to diabetes, renal IGF-I and -II and IGFBP-1, -2, and -3 concentration and mRNA expression were determined in streptozotocin-induced diabetic rat neonates. Diabetic neonates exhibited a kidney weight-to-body weight ratio higher than that of control rats, together with decreased IGF-I and increased IGF-II renal concentration. Because kidney mRNA expression of both IGFs decreased, the elevated renal IGF-II might result from increased uptake from circulation. Insulin treatment recovered the altered IGFs to control values, indicating the insulin-dependent regulation of IGFs in the neonatal kidney. Elevated levels of the IGFBP-1 and -2 in the kidney of diabetic neonates did not result from changes in their kidney mRNA transcript expression, suggesting also a possible uptake from circulation.
The α1G-subunit of a voltage-dependent Ca2+ channel is localized in rat distal nephron and collecting duct
Authors: Andreasen Ditte , Jensen Boye L. , Hansen Pernille B. , Kwon Tae-Hwan , Nielsen Søren , Skøtt Ole
Published: December 2017

The molecular type and localization of calcium channels along the nephron are not well understood. In the present study, we assessed the distribution of the recently identified α1G-subunit encoding a voltage-dependent calcium channel with T-type characteristics. Using a RNase protection assay, α1G-mRNA levels in kidney regions were determined as inner medulla ≫ outer medulla ≅ cortex. RT-PCR analysis of microdissected rat nephron segments revealed α1G expression in the distal convoluted tubule (DCT), in the connecting tubule and cortical collecting duct (CT+CCD), and inner medullary collecting duct (IMCD). α1GmRNA was expressed in the IMCD cell line mIMCD-3. Single- and double-labeling immunohistochemistry and confocal laser microscopy on semithin paraffin sections of rat kidneys by using an anti-α1G antibody demonstrated a distinct labeling at the apical plasma membrane domains of DCT cells, CT principal cells, and IMCD principal cells.
Urea signaling to ERK phosphorylation in renal medullary cells requires extracellular calcium but not calcium entry
Authors: Yang Xiao-Yan , Zhao Hongyu , Zhang Zheng , Rodland Karin D. , Roullet Jean-Baptiste , Cohen David M.
Published: December 2017

The renal cell line mIMCD3 exhibits markedly upregulated phosphorylation of the extracellular signal-regulated kinase (ERK) 1 and 2 in response to urea treatment (200 mM for 5 min). Previous data have suggested the involvement of a classical protein kinase C (cPKC)-dependent pathway in downstream events related to urea signaling. We now show that urea-inducible ERK activation requires extracellular calcium; unexpectedly, it occurs independently of activation of cPKC isoforms. Pharmacological inhibitors of known intracellular calcium release pathways and extracellular calcium entry pathways fail to inhibit ERK activation by urea. Fura 2 ratiometry was used to assess the effect of urea treatment on intracellular calcium mobilization. In single-cell analyses using subconfluent monolayers and in population-wide analyses using both confluent monolayers and cells in suspension, urea failed to increase intracellular calcium concentration. Taken together, these data indicate that urea-inducible ERK activation requires calcium action but not calcium entry. Although direct evidence is lacking, one possible explanation could include involvement of a calcium-dependent extracellular moiety of a cell surface-associated protein.
Renal interstitial adenosine metabolism during ischemia in dogs
Authors: Nishiyama Akira , Kimura Shoji , He Hong , Miura Katsuyuki , Rahman Matlubur , Fujisawa Yoshihide , Fukui Toshiki , Abe Youichi
Published: December 2017

The present study was conducted to determine the metabolism of renal interstitial adenosine under resting conditions and during ischemia. By using a microdialysis method with HPLC-fluorometric analysis, renal interstitial concentrations of adenosine, inosine, and hypoxanthine were assessed in pentobarbital-anesthetized dogs. Average basal renal interstitial concentrations of adenosine, inosine, and hypoxanthine were 0.18 ± 0.04, 0.31 ± 0.05, and 0.35 ± 0.05 μmol/l, respectively. Local inhibition of adenosine kinase with iodotubercidin (10 μmol/l in perfusate) or inhibition of adenosine deaminase with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA; 100 μmol/l in perfusate) did not change adenosine concentrations in the nonischemic kidneys (0.18 ± 0.04 and 0.24 ± 0.05 μmol/l, respectively). On the other hand, treatment with iodotubercidin+EHNA significantly increased adenosine concentration (0.52 ± 0.07 μmol/l) with significant decreases in inosine and hypoxanthine levels (0.13 ± 0.03 and 0.19 ± 0.04 μmol/l, respectively). During 30 min of ischemia, adenosine, inosine, and hypoxanthine were significantly increased to 0.76 ± 0.29, 2.14 ± 0.45, and 21.8 ± 4.7 μmol/l, respectively. The treatment with iodotubercidin did not alter ischemia-induced increase in adenosine (0.84 ± 0.18 μmol/l); however, EHNA alone markedly enhanced adenosine accumulation (13.54 ± 2.16 μmol/l), the value of which was not augmented by an addition of iodotubercidin (15.80 ± 1.24 μmol/l). In contrast, ischemia-induced increases in inosine and hypoxanthine were inversely diminished by the treatment with iodotubercidin+EHNA (0.90 ± 0.20 and 9.86 ± 1.96 μmol/l, respectively). These results suggest that both adenosine kinase and adenosine deaminase contribute to the metabolism of renal interstitial adenosine under resting conditions, whereas adenosine produced during ischemia is mainly metabolized by adenosine deaminase and the rephosphorylation of adenosine by adenosine kinase is small.
Genetic deletion of COX-2 prevents increased renin expression in response to ACE inhibition
Authors: Cheng Hui-Fang , Wang Jun-Ling , Zhang Ming-Zhi , Wang Su-Wan , McKanna James. A. , Harris Raymond C.
Published: December 2017

Cyclooxygenase-2 (COX-2) is expressed in macula densa (MD) and surrounding cortical thick ascending limb of the loop of Henle (cTALH) and is involved in regulation of renin production. We and others have previously found that selective COX-2 inhibitors can inhibit renal renin production (Cheng HF, Wang JL, Zhang MZ, Miyazaki Y, Ichikawa I, McKanna JA, and Harris RC. J Clin Invest 103: 953–961, 1999; Harding P, Sigmon DH, Alfie ME, Huang PL, Fishman MC, Beierwaltes WH, and Carretero OA. Hypertension 29: 297–302, 1997; Traynor TR, Smart A, Briggs JP, and Schnermann J. Am J Physiol Renal Physiol 277: F706–F710, 1999; Wang JL, Cheng HF, and Harris RC. Hypertension 34: 96–101, 1999). In the present studies, we utilized mice with genetic deletions of the COX-2 gene in order to investigate further the potential role of COX-2 in mediation of the renin-angiotensin system (RAS). Age-matched wild-type (+/+), heterozygotes (+/−), and homozygous null mice (−/−) were administered the angiotensin-converting enzyme inhibitor (ACEI), captopril, for 7 days. ACEI failed to significantly increase plasma renin activity, renal renin mRNA expression, and renal renin activity in (−/−) mice. ACEI increased the number of cells expressing immunoreactive renin in the (+/+) mice both by inducing more juxtaglomerular cells to express immunoreactive renin and by recruiting additional renin-expressing cells in the more proximal afferent arteriole. In contrast, there was minimal recruitment of renin-expressing cells in the more proximal afferent arteriole of the −/− mice. In summary, these results indicate that ACEI-mediated increases in renal renin production were defective in COX-2 knockout (K/O) mice and provide further indication that MD COX-2 is an important mediator of the renin-angiotensin system.