bluuebunny/crossref_metadata_2025 huggingface dataset:
Thresholds for cellular disruption and activation of the stress response in renal epithelia
Authors: van Why Scott K. , Kim Sunmi , Geibel John , Seebach Frank A. , Kashgarian Michael , Siegel Norman J.
Published: December 2017

Renal ischemia causes a rapid fall in cellular ATP, increased intracellular calcium (Cai), and dissociation of Na+-K+-ATPase from the cytoskeleton along with initiation of a stress response. We examined changes in Cai, Na+-K+-ATPase detergent solubility, and activation of heat-shock transcription factor (HSF) in relation to graded reduction of ATP in LLC-PK1cells to determine whether initiation of the stress response was related to any one of these perturbations alone. Caiincreased first at 75% of control ATP. Triton X-100 solubility of Na+-K+-ATPase increased below 70% control ATP. Reducing cellular ATP below 50% control consistently activated HSF. Stepped decrements in cellular ATP below the respective thresholds caused incremental increases in Cai, Na+-K+-ATPase solubility, and HSF activation. ATP depletion activated both HSF1 and HSF2. Proteasome inhibition caused activation of HSF1 and HSF2 in a pattern similar to ATP depletion. Lactate dehydrogenase release remained at control levels irrespective of the degree of ATP depletion. Progressive accumulation of nonnative proteins may be the critical signal for the adaptive induction of the stress response in renal epithelia.
Decreased renal Na-K-2Cl cotransporter abundance in mice with heterozygous disruption of the Gsα gene
Authors: Ecelbarger Carolyn A. , Yu Shuhua , Lee Alanna J. , Weinstein Lee S. , Knepper Mark A.
Published: December 2017

Transport processes along the nephron are regulated in part by hormone stimulation of adenylyl cyclases mediated by the heterotrimeric G protein Gs. To assess the role of this pathway in the regulation of Na-K-2Cl cotransporter abundance in the renal thick ascending limb (TAL), we studied mice with heterozygous disruption of the G nas gene, which codes for the α-subunit of Gs. Outer medullary Gsα protein abundance (as assessed by semiquantitative immunoblotting) and glucagon-stimulated cAMP production were significantly reduced in the heterozygous Gsα knockout mice (GSKO) relative to their wild-type (WT) littermates. Furthermore, Na-K-2Cl cotransporter protein abundance in the outer medulla was significantly reduced (band density, 48% of WT). In addition, GSKO mice had a significantly reduced (72% of WT) urinary osmolality in response to a single injection of 1-deamino-[8-d-arginine]vasopressin (DDAVP), a vasopressin analog. In contrast, outer medullary protein expression of the type 3 Na/H exchanger (NHE-3) or Tamm-Horsfall protein did not differ between the GSKO mice and their WT littermates. However, abundance of type VI adenylyl cyclase was markedly decreased in the outer medullas of GSKO mice, suggesting a novel feed-forward regulatory mechanism. We conclude that expression of the Na-K-2Cl cotransporter of the TAL is dependent on Gsα-mediated hormone stimulation, most likely due to long-term changes in cellular cAMP levels.
Inhibition of initial transport rate of basolateral organic anion carrier in renal PT by BK and phenylephrine
Authors: Gekle Michael , Mildenberger Sigrid , Sauvant Christoph , Bednarczyk Dallas , Wright Stephen H. , Dantzler William H.
Published: December 2017

The effect of ligands for phospholipase C-coupled receptors and of protein kinase C (PKC) stimulation with phorbol ester [phorbol 12-myristate 13-acetate (PMA)] or 1,2-dioctanoyl- sn-glycerol on the activity of the basolateral organic anion transporter (OAT) in S2 segments of single, nonperfused rabbit proximal tubules (PT) was measured with the use of fluorescein and epifluorescence microscopy. The initial uptake rate (25 s, OAT activity) was measured in real time by using conditions similar to those found in vivo. Stimulation of PKC with PMA or 1,2-dioctanoyl- sn-glycerol led to an inhibition of OAT activity, which could be prevented by 10−7mol/l of the PKC-specific inhibitor bisindolylmaleimide. The α1-receptor agonist phenylephrine as well as the peptide hormone bradykinin induced a reversible decrease of OAT activity, which was prevented by bisindolylmaleimide. The observed effect was not due to a decrease in the concentration of the counterion α-ketoglutarate or to impaired α-ketoglutarate recycling, because it was unchanged in the continuous presence of α-ketoglutarate or methyl succinate. We conclude that physiological stimuli can inhibit the activity of OAT in rabbit PT via PKC. The effect is not mediated by alterations in counterion availability but by a direct action on the OAT.
Two types of K+channels at the basolateral membrane of proximal tubule: inhibitory effect of taurine
Authors: Noulin Jean-François , Brochiero Emmanuelle , Lapointe Jean-Yves , Laprade Raynald
Published: December 2017

The cell-attached configuration of the patch-clamp technique was used to investigate the effects of taurine on the basolateral potassium channels of rabbit proximal convoluted tubule. In the absence of taurine, the previously reported ATP-blockable channel, KATP, was observed in 51% of patches. It is characterized by an inwardly rectifying current-voltage curve with an inward slope conductance of 49 ± 5 pS ( n = 15) and an outward slope conductance of 13 ± 6 pS ( n = 15). The KATPchannel open probability ( Po) is low, 0.15 ± 0.06 ( n = 15) at a − Vp= −100 mV ( Vpis the pipette potential), and increases slightly with depolarization. The gating kinetics are characterized by one open time constant (τo= 5.0 ± 1.9 ms, n = 6) and two closed time constants (τC1= 5.2 ± 1.5 ms, τC2= 140 ± 40 ms; n = 6). In 34% of patches, a second type of potassium channel, sK, with distinct properties was recorded. Its current-voltage curve is characterized by a sigmoidal shape, with an inward slope conductance of 12 ± 2 pS ( n = 4). Its Pois voltage independent and averages 0.67 ± 0.03 ( n = 4) at − Vp= −80 mV. Both its open time and closed time distributions are described by a single time constant (τo= 96 ± 19 ms, τC= 10.5 ± 3.6 ms; n = 4). Extracellular perfusion of 40 mM taurine fails to affect sK channels, whereas KATPchannel Podecreases by 75% (from 0.17 ± 0.06 to 0.04 ± 0.02, n = 7, P < 0.05). In conclusion, the absolute basolateral potassium conductance of rabbit proximal tubules is the resulting combination of, at least, two types of potassium channels of roughly equal importance: a high-conductance low-open probability KATPchannel and a low-conductance high-open probability sK channel. The previously described decrease in the basolateral absolute potassium conductance by taurine is, however, mediated by a single type of K channel: the ATP-blockable K channel.
Renal endosomes contain angiotensin peptides, converting enzyme, and AT1Areceptors
Authors: Imig John D. , Navar Gabriel L. , Zou Li-Xian , O’Reilly Katie C. , Allen Patricia L. , Kaysen James H. , Hammond Timothy G. , Navar L. Gabriel
Published: December 2017

Kidney cortex and proximal tubular angiotensin II (ANG II) levels are greater than can be explained on the basis of circulating ANG II, suggesting intrarenal compartmentalization of these peptides. One possible site of intracellular accumulation is the endosomes. In the present study, we tested for endosomal ANG I, ANG II, angiotensin type 1A receptor (AT1A), and angiotensin converting enzyme (ACE) activity and determined whether these levels are regulated by salt intake. Male Sprague-Dawley rats were fed chow containing either high or low dietary sodium for 10–14 days. Blood and kidneys were harvested and processed for measurement of plasma, kidney, and renal intermicrovillar cleft and endosomal angiotensin levels. Kidney ANG I averaged 179 ± 20 fmol/g and ANG II averaged 258 ± 36 fmol/g in rats fed a high-sodium diet and were significantly higher, averaging 347 ± 58 fmol/g and 386 ± 55 fmol/g, respectively, in rats fed a low-salt diet. Renal intermicrovillar clefts and endosomes contained ANG I and ANG II. Intermicrovillar cleft ANG I and ANG II levels averaged 8.4 ± 2.6 and 74 ± 26 fmol/mg, respectively, in rats fed a high-salt diet and 7.6 ± 1.7 and 70 ± 25 fmol/mg in rats fed a low-salt diet. Endosomal ANG I and ANG II levels averaged 12.3 ± 4.4 and 43 ± 19 fmol/mg, respectively, in rats fed a high-salt diet, and these levels were similar to those observed in rats fed a low-salt diet. Renal endosomes from rats fed a low-salt diet demonstrated significantly more AT1Areceptor binding compared with rats fed a high-salt diet. ACE activity was detectable in renal intermicrovillar clefts and was 2.5-fold higher than the levels observed in renal endosomes. Acute enalaprilat treatment decreased ACE activity in renal intermicrovillar clefts by 90% and in renal endosomes by 84%. Likewise, intermicrovillar cleft and endosomal ANG II levels decreased by 61% and 52%, respectively, in enalaprilat-treated animals. These data demonstrate the presence of intact angiotensin peptides and ACE activity in renal intermicrovillar clefts and endosomes, indicating that intact angiotensin peptides are formed and/or trafficked through intracellular endosomal compartments and are dependent on ACE activity.
PAH extraction and estimation of plasma flow in human postischemic acute renal failure
Authors: Corrigan Geraldine , Ramaswamy Deepa , Kwon Osun , Sommer F. Graham , Alfrey Edward J. , Dafoe Donald C. , Olshen Richard A. , Scandling John D. , Myers Bryan D.
Published: December 2017

We determined the effect of postischemic injury to the human renal allograft on p-aminohippurate (PAH) extraction (EPAH) and renal blood flow. We evaluated renal function in 44 allograft recipients on two occasions: 1–3 h after reperfusion ( day 0) and again on postoperative day 7. On day 0 subsets underwent intraoperative determination of renal blood flow ( n = 35) by Doppler flow meter and EPAH( n = 25) by renal venous assay. Blood flow was also determined in another subset of 16 recipients on postoperative day 7 by phase contrast-cine-magnetic resonance imaging, and EPAHwas computed from the simultaneous PAH clearance. Glomerular filtration rate (GFR) on day 7 was used to divide subjects into recovering ( n = 23) and sustained ( n = 21) acute renal failure (ARF) groups, respectively. Despite profound depression of GFR in the sustained ARF group, renal plasma flow was only slightly depressed, averaging 296 ± 162 ml ⋅ min−1⋅ 1.73 m−2on day 0 and 202 ± 72 ml ⋅ min−1⋅ 1.73 m−2on day 7, respectively. These values did not differ from corresponding values in the recovering ARF group: 252 ± 133 and 280 ± 109 ml ⋅ min−1⋅ 1.73 m−2, respectively. EPAHwas profoundly depressed on day 0, averaging 18 ± 14 and 10 ± 7% in recovering and sustained ARF groups, respectively, vs. 86 ± 6% in normal controls ( P < 0.001). Corresponding values on day 7remained significantly depressed at 65 ± 20 and 11 ± 22%, respectively. We conclude that postischemic injury to the renal allograft results in profound impairment of EPAHthat persists for at least 7 days, even after the onset of recovery. An ensuing reduction in urinary PAH clearance results in a gross underestimate of renal plasma flow, which is close to the normal range in the initiation, maintenance, and recovery stages of this injury.
Urinary excretion of urodilatin is increased during pressure natriuresis in the isolated perfused rat kidney
Authors: Heringlake Matthias , Wagner Klaus , Schumacher Jan , Pagel Horst
Published: December 2017

The findings about mechanisms regulating production and excretion of urodilatin [ANP-(95–126)], a member of the atrial natriuretic peptide (ANP) family, are controversial. To elucidate a possible relationship between arterial blood pressure and renal urodilatin excretion, we studied the effects of different perfusion pressures on urine flow (UV), urinary sodium (UNaV), urinary potassium (UKV), and urodilatin excretion (UUROV), and the concentration of urodilatin in the perfusate (PURO) of isolated perfused rat kidneys. Kidneys were perfused for 180 min with constant perfusion pressures (80 and 120 mmHg, respectively; each, n = 4) in a closed circuit system. Samples of urine and perfusate were taken every 30 min. Mean UV, UNaV, UKV, and UUROV values were significantly higher with a perfusion pressure of 120 mmHg than with 80 mmHg, whereas PUROdid not change significantly. Serial measurements revealed no direct relation of UUROV with either UNaV or UV. This suggests that renal perfusion pressure is a determinant of UUROV and that urinary and venous effluent concentrations of urodilatin (probably production) are not coupled directly and that UUROV and UNaV may dissociate during acute variations of sodium excretion and UV.
Expression of the β2-subunit and apical localization of Na+-K+-ATPase in metanephric kidney
Authors: Burrow Christopher R. , Devuyst Olivier , Li Xiaohong , Gatti Laura , Wilson Patricia D.
Published: December 2017

During kidney organogenesis, the Na+-K+-ATPase pump is not restricted to the basolateral plasma membrane of the renal epithelial cell but is instead either localized to the apical and lateral membrane sites of the early nephron or expressed in a nonpolarized distribution in the newly formed collecting ducts. The importance of Na+-K+-ATPase β-subunit expression in the translocation of the Na+-K+-ATPase to the plasma membrane raises the question as to which β-subunit isoform is expressed during kidney organogenesis. Immunocytochemical, Western analysis and RNase protection studies showed that both β2-subunit protein and β2 mRNA are expressed in the early gestation to midgestation human metanephric kidney. In contrast, although β1 mRNA abundance is equivalent to that of the β2-subunit in the metanephric kidney, the β1-subunit protein was not detected in early to midgestation metanephric kidney samples. Immunocytochemical analysis revealed that both α1- and β2-subunits were present in the apical epithelial plasma membranes of distal nephron segments of early stage nephrons, maturing loops of Henle, and collecting ducts during kidney development. We also detected a significant increase in α1 and β1 mRNA after birth with a marked reduction in β2 mRNA abundance associated with an increase in α1- and β1-subunit proteins and loss of β2 protein expression. These studies support the conclusion that the expression of the β2-subunit in the fetal kidney may be an important mechanism controlling polarization of the Na+-K+-ATPase pump in the epithelia of the developing nephron during kidney organogenesis.
Expression of NCAM recapitulates tubulogenic development in kidneys recovering from acute ischemia
Authors: Abbate Mauro , Brown Dennis , Bonventre Joseph V.
Published: December 2017

Recovery of the kidney from acute renal failure relies on a sequence of events including epithelial cell dedifferentiation and proliferation followed by differentiation and restoration of the functional integrity of the nephron. The factors responsible for, and the significance of, reversion to a less differentiated cell phenotype and its relationship to the proliferative response after ischemia are poorly understood. In an attempt to identify adhesion molecules that may be influential in the recovery process, the expression of neural cell adhesion molecule (NCAM) and markers of epithelial differentiation and proliferation were analyzed at various times after an ischemic insult. In maturing nephrons, NCAM is detectable by immunohistochemistry in renal vesicles, S-shaped bodies, and early tubules. There is minimal cellular NCAM expression in normal tubules of the adult kidney. In contrast, in postischemic kidneys, NCAM expression is abundant in S3 proximal tubule cells 5 days after reperfusion. As in developing tubules, NCAM is concentrated in basal and lateral aspects of cells that have no apical gp330 or dipeptidyl peptidase IV detectable on their brush border. The expression of NCAM is preceded by disassembly of the brush border and proliferation of surviving S3 cells, which is most prominent at 2 days postischemia. NCAM expression persists in some flattened and dedifferentiated cells for up to 7 wk after ischemia. Thus proximal tubule epithelial cells of the postischemic kidney express NCAM in a pattern that recapitulates the expression of NCAM in the developing kidney. Such reversion of phenotype extends at least back to the early stages of renal vesicle formation, and this reversion may represent a critical step in the reestablishment of a normal tubule. NCAM-matrix interactions may mediate the motogenic and mitogenic responses of the dedifferentiated epithelium that are critical to reestablishment of a functional proximal tubule.
Functional diversity of excitatory amino acid transporters: ion channel and transport modes
Authors: Fairman W. A. , Amara S. G.
Published: December 2017

Recent studies of glutamate transporters in the central nervous system indicate that in addition to their fundamental role in mediating neurotransmitter uptake, these proteins may contribute to the modulation of a variety of cellular processes. Activation of the excitatory amino acid (EAA) carriers generates an electrogenic current attibutable to ion-coupled cotransport. In addition to this transport-associated current, a substrate-gated thermodynamically uncoupled anion flux has been identified that has been proposed to dampen neuronal excitability. Arachidonic acid has been reported to modulate a variety of membrane proteins involved in cellular signaling. Here we discuss recent findings that indicate arachidonic acid stimulates a previously uncharacterized proton-selective conductance in the Purkinje cell-specific subtype, EAAT4. The unique channel-like porperties of the EAATs, their unexpected localization, and physiological evidence propose a modulatory role for the EAATs in neuronal signaling and suggest a broader role for glutamate transporters than simply the clearance of synaptically released glutamate. Thus, the identification of this arachidonate-stimulated proton conductance extends the complexity of mechanisms through which glutamate transporters modulate neuronal excitability.
Glutamate transport and renal function
Authors: Welbourne Tomas C. , Matthews James C.
Published: December 2017

Brush border γ-glutamyltransferase-glutaminase activity and the high-affinity glutamate transporter EAAC1 function as a unit in generating and transporting extracellular glutamate into proximal tubules as a signal that modulates intracellular glutamine/glutamate metabolism, paracellular permeability, and urinary acidification. The reported presence of a second glutamate transporter, GLT1, on the antiluminal tubule surface points to specific functional roles for each subtype in physiological and pathophysiological processes.
Nitric oxide and renal nerve-mediated proximal tubular reabsorption in normotensive and hypertensive rats
Authors: Wu Xiao Chun , Harris Peter J. , Johns Edward J.
Published: December 2017

In Inactin-anesthetized Wistar rats with an intact renal innervation, intratubular nitro-l-arginine methyl ester (l-NAME, 10−4M) increased proximal fluid uptake ( Jva, at 2.47 ± 0.61 × 10−4mm3⋅ mm−2⋅ s−1) by 17% ( P < 0.05), whereas coadministration with sodium nitroprusside (SNP, 10−4M) decreased Jvaby 18% ( P < 0.01). Similar manipulation of NO generation was without effect in groups of Wistar rats subjected to acute renal denervation. Intratubular aminoguanidine (10−4M), a selective inducible nitric oxide synthase (NOS) blocker, had no effect on Jvain intact kidneys of Wistar rats, but the neuronal NOS (nNOS) blocker, 7-nitroindazole (10−4M and 10−6M) increased Jvaby 19–23% (both P < 0.001). In stroke-prone spontaneously hypertensive rats (SHRSP), Jvavalues in the innervated kidneys were lower ( P< 0.05) than in the corresponding Wistar groups and were unchanged by intratubular l-NAME or l-NAME plus SNP. The tonic attenuation of proximal epithelial transport by NO was dependent on the renal sympathetic nerves and appeared to be generated by the nNOS isoform of the enzyme. This role of NO was not evident in the SHRSP.
Cation and voltage dependence of rat kidney electrogenic Na+- HCO 3 − cotransporter, rkNBC, expressed in oocytes
Authors: Sciortino Christopher M. , Romero Michael F.
Published: December 2017

Recently, we reported the cloning and expression of the rat renal electrogenic Na+-[Formula: see text]cotransporter (rkNBC) in Xenopusoocytes [M. F. Romero, P. Fong, U. V. Berger, M. A. Hediger, and W. F. Boron. Am. J. Physiol. 274 ( Renal Physiol. 43): F425–F432, 1998]. Thus far, all NBC cDNAs are at least 95% homologous. Additionally, when expressed in oocytes the NBCs are 1) electrogenic, 2) Na+dependent, 3)[Formula: see text] dependent, and 4) inhibited by stilbenes such as DIDS. The apparent[Formula: see text]:Na+coupling ratio ranges from 3:1 in kidney to 2:1 in pancreas and brain to 1:1 in the heart. This study investigates the cation and voltage dependence of rkNBC expressed in Xenopus oocytes to better understand NBC’s apparent tissue-specific physiology. Using two-electrode voltage clamp, we studied the cation specificity, Na+dependence, and the current-voltage ( I- V) profile of rkNBC. These experiments indicate that K+and choline do not stimulate[Formula: see text]-sensitive currents via rkNBC, and Li+elicits only 3 ± 2% of the total Na+current. The Na+dose response studies show that the apparent affinity of rkNBC for extracellular Na+(∼30 mM [Na+]o) is voltage and [Formula: see text] independent, whereas the rkNBC I- Vrelationship is Na+dependent. At [Na+]ovmax(96 mM), the I- Vresponse is approximately linear; both inward and outward Na+-[Formula: see text]cotransport are observed. In contrast, only outward cotransport occurs at low [Na+]o(<1 mM [Na+]o). All rkNBC currents are inhibited by extracellular application of DIDS, independent of voltage and [Na+]o. Using ion-selective microelectrodes, we monitored intracellular pH and Na+activity. We then calculated intracellular [[Formula: see text]] and, with the observed reversal potentials, calculated the stoichiometry of rkNBC over a range of [Na+]ovalues from 10 to 96 mM at 10 and 33 mM [[Formula: see text]]o. rkNBC stoichiometry is 2 [Formula: see text]:1 Na+over this entire Na+range at both[Formula: see text] concentrations. Our results indicate that rkNBC is highly selective for Na+, with transport direction and magnitude sensitive to [Na+]oas well as membrane potential. Since the rkNBC protein alone in oocytes exhibits a stoichiometry of less than the 3[Formula: see text]:1 Na+thought necessary for[Formula: see text] reabsorption by the renal proximal tubule, a control mechanism or signal that alters its in vivo function is hypothesized.
Defective processing and expression of thiazide-sensitive Na-Cl cotransporter as a cause of Gitelman’s syndrome
Authors: Kunchaparty Shanti , Palcso Matthew , Berkman Jennifer , Velázquez Heino , Desir Gary V. , Bernstein Paul , Reilly Robert F. , Ellison David H.
Published: December 2017

Gitelman’s syndrome is an autosomal recessive disorder of salt wasting and hypokalemia caused by mutations in the thiazide-sensitive Na-Cl cotransporter. To investigate the pathogenesis of Gitelman’s syndrome, eight disease mutations were introduced into the mouse thiazide-sensitive Na-Cl cotransporter and studied by functional expression in Xenopus oocytes. Sodium uptake into oocytes that expressed the wild-type clone was more than sevenfold greater than uptake into control oocytes. Uptake into oocytes that expressed the mutated transporters was not different from control. Hydrochlorothiazide reduced Na uptake by oocytes expressing the wild-type gene to control values but had no effect on oocytes expressing the mutant clones. Western blots of oocytes injected with the wild-type clone showed bands representing glycosylated (125 kDa) and unglycosylated (110 kDa) forms of the transport protein. Immunoblot of oocytes expressing the mutated clones showed only the unglycosylated protein, indicating that protein processing was disrupted. Immunocytochemistry with an antibody against the transport protein showed intense membrane staining of oocytes expressing the wild-type protein. Membrane staining was completely absent from oocytes expressing mNCCR948X; instead, diffuse cytoplasmic staining was evident. In summary, the results show that several mutations that cause Gitelman’s syndrome are nonfunctional because the mutant thiazide-sensitive Na-Cl cotransporter is not processed normally, probably activating the “quality control” mechanism of the endoplasmic reticulum.
Evolution of gene expression patterns in a model of branching morphogenesis
Authors: Pavlova Anna , Stuart Robert O. , Pohl Martin , Nigam Sanjay K.
Published: December 2017

Branching morphogenesis of the ureteric bud in response to unknown signals from the metanephric mesenchyme gives rise to the urinary collecting system and, via inductive signals from the ureteric bud, to recruitment of nephrons from undifferentiated mesenchyme. An established cell culture model for this process employs cells of ureteric bud origin (UB) cultured in extracellular matrix and stimulated with conditioned media (BSN-CM) from a metanephric mesenchymal cell line (H. Sakurai, E. J. Barros, T. Tsukamoto, J. Barasch, and S. K. Nigam. Proc. Natl. Acad. Sci. USA 94: 6279–6284, 1997.). In the presence of BSN-CM, the UB cells form branching tubular structures reminiscent of the branching ureteric bud. The pattern of gene regulation in this model of branching morphogenesis of the kidney collecting system was investigated using high-density cDNA arrays. Software and analytical methods were developed for the quantification and clustering of genes. With the use of a computational method termed “vector analysis,” genes were clustered according to the direction and magnitude of differential expression in n-dimensional log-space. Changes in gene expression in response to the BSN-CM consisted primarily of differential expression of transcription factors with previously described roles in morphogenesis, downregulation of pro-apoptotic genes accompanied by upregulation of anti-apoptotic genes, and upregulation of a small group of secreted products including growth factors, cytokines, and extracellular proteinases. Changes in expression are discussed in the context of a general model for epithelial branching morphogenesis. In addition, the cDNA arrays were used to survey expression of epithelial markers and secreted factors in UB and BSN cells, confirming the largely epithelial character of the former and largely mesenchymal character of the later. Specific morphologies (cellular processes, branching multicellular cords, etc.) were shown to correlate with the expression of different, but overlapping, genomic subsets, suggesting differences in morphogenetic mechanisms at these various steps in the evolution of branching tubules.
Regulation of mesangial cell hexokinase activity by PKC and the classic MAPK pathway
Authors: Robey R. Brooks , Ma Jianfei , Santos Anna V. P.
Published: December 2017

Phorbol esters increase glucose (Glc) uptake and utilization in a variety of cell types, and, in some cells, these changes have been attributed to increased Glc phosphorylation and better functional coupling of hexokinases (HKs) to facilitative Glc transporters. Phorbol esters are potent mesangial cell mitogens, but their effects on HK-catalyzed Glc phosphorylation and metabolism are unknown. When examined in murine mesangial cells, active, but not inactive, phorbol esters increased HK activity in a time- and dose-dependent manner. Maximal induction of HK activity at 12–24 h was accompanied by parallel increases in both Glc utilization and lactate production and was blocked by the specific MEK1/2 inhibitor PD-98059 (IC50∼3 μM). This effect involved early activation of protein kinase C (PKC), MEK1/2, and ERK1/2, and the prolonged time course of subsequent HK induction was attributable, in part, to requirements for ongoing gene transcription and de novo protein synthesis. Mesangial cell HK activity thus exhibits novel regulatory behavior involving both PKC and classic MAPK pathway activation, suggesting specific mechanisms whereby PKC activation may influence Glc metabolism.
Thiazide induces water absorption in the inner medullary collecting duct of normal and Brattleboro rats
Authors: César Kátia R. , Magaldi Antonio J.
Published: December 2017

The reduction of urinary volume after the use of thiazide in the treatment of diabetes insipidus (DI) is known as the “paradoxical effect.” Since enhanced proximal solute and water reabsorption only partially account for the reduction in urinary volume, an additional diuretic effect on nephron terminal segments was postulated. Thus the aim of our work was to investigate the effect of hydrochlorothiazide (HCTZ) on water transport in the inner medullary collecting duct (IMCD) of normal and Brattleboro rats. Osmotic water permeability ( P f) and diffusional water permeability ( P dw) were studied at 37°C and pH 7.4 by the in vitro microperfusion technique. In the absence of antidiuretic hormone (ADH), HCTZ (10−6 M) added to the perfused fluid enhanced P f from 6.36 ± 0.56 to 19.08 ± 1.70 μm/s ( P < 0.01) and P dw from 38.01 ± 4.52 to 52.26 ± 4.38 ×10−5 cm/s ( P < 0.01) in normal rats and also stimulated P f in Brattleboro rats from 3.53 ± 1.41 to 11.16 ± 1.13 μm/s ( P < 0.01). Prostaglandin E2(PGE2) (10−5 M) added to the bath fluid inhibited HCTZ-stimulated P f (in μm/s) as follows: control, 16.93 ± 2.64; HCTZ, 29.65 ± 5.67; HCTZ+PGE2, 10.46 ± 1.84 ( P < 0.01); recovery, 16.77 ± 4.07. These data indicate that thiazides enhance water absorption in IMCD from normal rats (in the absence of ADH) and from Brattleboro rats and that the HCTZ-stimulated P f was partially blocked by PGE2. Thus we may conclude that the effect of thiazide in the treatment of DI occurs not only in the Na+-Cl−cotransport in the distal tubule but also in the IMCD.
Regulation of apical K channels in rat cortical collecting tubule during changes in dietary K intake
Authors: Palmer Lawrence G. , Frindt Gustavo
Published: December 2017

Long-term adaptation to a high-K diet is known to increase the density of conducting secretory K (SK) channels in the luminal membrane of the rat cortical collecting tubule (CCT). To examine whether these channels are involved in the short-term, day-to-day regulation of K secretion, we examined the density of K channels in animals fed a high-K diet for 6 or 48 h. CCTs were isolated and split open to provide access to the luminal membrane. Cell-attached patches were formed on principal cells with 140 mM KCl in the patch-clamp pipette. SK channels were recognized from their characteristic single-channel conductance (40–50 pS) and gating patterns. Animals fed a control diet had SK channel densities of 0.40 channels/μm2. When the diet was changed for one containing 10% KCl for 6 h, the channel density increased to 1.51 channels/μm2. Maintaining the animals on a high-K diet for 48 h resulted in a further increase in SK channels to 2.29 channels/μm2. Animals fed a low-K diet for 5 days or longer had SK densities of 0.53 channels/μm2, not significantly different from control values. The presence of conducting Na channels in the luminal membrane will also affect K secretion by the CCT by altering the electrical driving force through the K channels. The density of Na channels, measured with LiCl in the pipette, was 0.08 for controls and 1.00 and 1.08 channels/μm2 after 6 h and 48 h on a high-K diet. Plasma aldosterone increased from 15 ± 4 ng/dl (controls ) to 36 ± 8 and 98 ± 23 ng/dl after 6 and 48 h of K loading, respectively. The increase in K channel density could not be reproduced by infusion of the animals with aldosterone. We conclude that regulation of the density of conducting Na and K channels may contribute to day-to-day variation in the rate of renal K secretion and to the short-term maintenance of K balance.
Effects of in vivo metabolic acidosis on midcortical bone ion composition
Authors: Bushinsky David A. , Chabala Jan M. , Gavrilov Konstantin L. , Levi-Setti Riccardo
Published: December 2017

Chronic metabolic acidosis increases urine calcium excretion without altering intestinal calcium absorption, suggesting that bone mineral is the source of the additional urinary calcium. During metabolic acidosis there appears to be an influx of protons into bone mineral, lessening the magnitude of the decrement in pH. Although in vitro studies strongly support a marked effect of metabolic acidosis on the ion composition of bone, there are few in vivo observations. We utilized a high-resolution scanning ion microprobe with secondary ion mass spectroscopy to determine whether in vivo metabolic acidosis would alter bone mineral in a manner consistent with its purported role in buffering the increased proton concentration. Postweanling mice were provided distilled drinking water with or without 1.5% NH4Cl for 7 days; arterial blood gas was then determined. The addition of NH4Cl led to a fall in blood pH and [Formula: see text] concentration. The animals were killed on day 7, and the femurs were dissected and split longitudinally. The bulk cortical ratios Na/Ca, K/Ca, total phosphate/carbon-nitrogen bonds [(PO2 + PO3)/CN], and[Formula: see text]/CN each fell after 1 wk of metabolic acidosis. Because metabolic acidosis induces bone Ca loss, the fall in Na/Ca and K/Ca indicates a greater efflux of bone Na and K than Ca, suggesting H substitution for Na and K on the mineral. The fall in (PO2 + PO3)/CN indicates release of mineral phosphates, and the fall in[Formula: see text]/CN indicates release of mineral[Formula: see text]. Each of these mechanisms would result in buffering of the excess protons and returning the systemic pH toward normal.
In rat tIMCD, NH 4 + uptake by Na+-K+-ATPase is critical to net acid secretion during chronic hypokalemia
Authors: Wall Susan M. , Davis Bradley S. , Hassell Kathryn A. , Mehta Pramod , Park Stanley J.
Published: December 2017

The purpose of this study was to determine the magnitude of Na+pump-mediated[Formula: see text] uptake in the terminal inner medullary collecting duct (tIMCD) at K+and[Formula: see text] concentrations observed in vivo in the inner medullary interstitium of normal and in K+-restricted rats. Interstitial K+and [Formula: see text]concentrations in the terminal half of the inner medulla were taken to be 10 and 6 mM in K+-restricted rats, but 30 and 6 mM in K+-replete rats. In tubules from K+-restricted rats, when perfused at a K+concentration of 10 mM, addition of ouabain to the bath reduced total bicarbonate flux ( JtCO2) by 40% and increased intracellular pH (pHi), indicating significant[Formula: see text] uptake by the Na+-K+-ATPase. In tubules from K+-restricted rats, JtCO2was reduced with increased extracellular K+. At a K+concentration of 30 mM, ouabain addition neither reduced JtCO2nor increased pHiin tubules from rats of either treatment group. In conclusion, in the tIMCD from hypokalemic rats, 1) acute changes in extracellular K+concentration modulate net acid secretion, and 2) Na+pump-mediated [Formula: see text]uptake should be an important pathway mediating transepithelial net acid secretion in vivo.
Carbon monoxide induces vasodilation and nitric oxide release but suppresses endothelial NOS
Authors: Thorup Christian , Jones Caroline L. , Gross Steven S. , Moore Leon C. , Goligorsky Michael S.
Published: December 2017

The vascular effects of carbon monoxide (CO) resemble those of nitric oxide (NO), but it is unknown whether the two messengers converge or exhibit reciprocal feedback regulation. These questions were examined in microdissected perfused renal resistance arteries (RRA) studied using NO-sensitive microelectrodes. Perfusion of RRA with buffers containing increasing concentrations of CO resulted in a biphasic release of NO. The NO response peaked at 100 nM CO and then declined to virtually zero at 10 μM. When a series of 50-s pulses of 100 nM CO were applied repeatedly (150-s interval), the amplitude of consecutive NO responses was diminished. NO release from RRA showed dependence on l-arginine but notd-arginine, and the responses to CO were inhibited by pretreatment with NG-nitro-l-arginine methyl ester (l-NAME), an inhibitor of NO synthases (NOS). CO (100 nM) also suppressed NO release induced by 100 μM carbachol, a potent agonist for endothelial NOS (eNOS). RRA from rats in which endogenous CO production from inducible HO was elevated (cobalt chloride 12 h prior to study) also showed suppressed responses to carbachol. Furthermore, responses consistent with these findings were obtained in juxtamedullary afferent arterioles perfused in vitro, where the vasodilatory response to CO was biphasic and the response to acetylcholine was blunted. Collectively, these data suggest that the CO-induced NO release could be attributed to either stimulation of eNOS or to NO displacement from a cellular storage pool. To address this, direct in vitro measurements with an NO-selective electrode of NO production by recombinant eNOS revealed that CO dose-dependently inhibits NO synthesis. Together, the above data demonstrate that, whereas high levels of CO inhibit NOS activity and NO generation, lower concentrations of CO induce release of NO from a large intracellular pool and, therefore, may mimic the vascular effects of NO.
rOCT2 is a basolateral potential-driven carrier, not an organic cation/proton exchanger
Authors: Sweet Douglas H. , Pritchard John B.
Published: December 2017

The driving forces mediating tetraethylammonium (TEA) transport were systematically assessed in Xenopus oocytes and MDCK cells expressing organic cation transporter (OCT) 2 cloned from rat kidney (rOCT2). In rOCT2 cRNA-injected oocytes, uptake of [14C]TEA was saturable, with an estimated Michaelis constant ( K m) of 393 μM, and was specifically inhibited by organic cations. Furthermore, TEA uptake demonstrated two distinct components, one that was potential sensitive and one that was pH sensitive. When membrane potential was intact, TEA uptake was largely unaffected by changes in medium pH; when the oocyte membrane was depolarized (K+ in = out = 102.5 mM, plus valinomycin), decreasing external medium pH significantly reduced TEA uptake. Consistent with the potential sensitivity of uptake, electrophysiological analysis of rOCT2-injected oocytes demonstrated movement of positive charge into the oocyte upon TEA addition. To further evaluate the nature of the pH effect and assess the properties of rOCT2 in a renal epithelium, rOCT2 was introduced into MDCK cells. A stably transfected single cell clone (MDCK-rOCT2) showed mediated, potential-sensitive, pH-sensitive TEA uptake ( K m = 48 μM). TEA efflux from preloaded MDCK-rOCT2 cells was stimulated by externally applied ( trans) tetramethylammonium but was trans-inhibited by H+ (external pH 5.4). The effect of external H+ was to modulate rOCT2-mediated transport. Thus rOCT2 is a potential-driven transporter, not an organic cation/H+exchanger, consistent with a physiological role in the basolateral entry step in renal organic cation secretion.
Insulin stimulates Mg2+uptake in mouse distal convoluted tubule cells
Authors: Dai Long-Jun , Ritchie Gordon , Bapty Brian W. , Kerstan Dirk , Quamme Gary A.
Published: December 2017

Insulin has been shown to be a magnesium-conserving hormone acting, in part, through stimulation of magnesium absorption within the thick ascending limb. Although the distal convoluted tubule possesses the most insulin receptors, it is unclear what, if any, actions insulin has in the distal tubule. The effects of insulin were studied on immortalized mouse distal convoluted tubule (MDCT) cells by measuring cellular cAMP formation with radioimmunoassays and Mg2+uptake with fluorescence techniques using mag-fura 2. To assess Mg2+uptake, MDCT cells were first Mg2+depleted to 0.22 ± 0.01 mM by culturing in Mg2+-free media for 16 h and then placed in 1.5 mM MgCl2, and the changes in intracellular Mg2+concentration ([Mg2+]i) were measured with microfluorescence. [Mg2+]ireturned to basal levels, 0.53 ± 0.02 mM, with a mean refill rate, d([Mg2+]i)/d t, of 164 ± 5 nM/s. Insulin stimulated Mg2+entry in a concentration-dependent manner with maximal response of 214 ± 12 nM/s, which represented a 30 ± 5% increase in the mean uptake rate above control values. This was associated with a 2.5-fold increase in insulin-mediated cAMP generation (52 ± 3 pmol ⋅ mg protein−1⋅ 5 min−1). Genistein, a tyrosine kinase inhibitor, diminished insulin-stimulated Mg2+uptake (169 ± 11 nM/s), but did not change insulin-mediated cAMP formation (47 ± 5 pmol ⋅ mg protein−1⋅ 5 min−1). PTH stimulates Mg2+entry, in part, through increases in cAMP formation. Insulin and PTH increase Mg2+uptake in an additive fashion. In conclusion, insulin mediates Mg2+entry, in part, by a genistein-sensitive mechanism and by modifying hormone-responsive transport. These studies demonstrate that insulin stimulates Mg2+uptake in MDCT cells and suggest that insulin acts in concert with other peptide and steroid hormones to control magnesium conservation in the distal convoluted tubule.
PKC regulation of organic anion secretion in perfused S2 segments of rabbit proximal tubules
Authors: Shuprisha Apichai , Lynch Ronald M. , Wright Stephen H. , Dantzler William H.
Published: December 2017

To examine the role of protein kinase C (PKC) in organic anion (OA) secretion, we used epifluorescence microscopy to study steady-state transepithelial secretion of 1 μM fluorescein (FL) by isolated perfused S2 segments of rabbit renal proximal tubules. Addition of 100 nM phorbol 12-myristate 13-acetate (PMA), a known PKC activator, to the bathing medium decreased steady-state secretion of FL by ∼30% after 25 min. This inhibition was irreversible and, indeed, increased to ∼40% at 25 min following removal of PMA [10 μM 1,2-dioctanoyl- sn-glycerol (DOG) produced a comparable inhibition]. The inhibition produced by PMA was blocked when 100 nM of either staurosporine (ST) or bisindolylmaleimide I (BIM), both known PKC inhibitors, was added to the bath for a 20-min preexposure followed by the addition of PMA. ST or BIM alone had no significant effect on FL secretion, suggesting that the basal FL secretion rate was not under influence of PKC. Addition of 1 μM of either the peptide hormone bradykinin (BK) or the α1-receptor agonist phenylephrine (PE), both of which stimulate PKC via a ligand-receptor-PKC coupling reaction, to the bath also inhibited FL secretion by ∼22 and ∼27%, respectively. However, the inhibition was completely reversible after removal of BK or PE. Pretreatment of tubules with 100 nM BIM eliminated the inhibition of FL secretion produced by exposure to PE. We conclude that PKC negatively regulates the net secretion of OAs in rabbit renal proximal tubules. The data indicate that BK or catecholamines can play a physiological role in regulating OA secretion via PKC activation.
PI3K signaling in the murine kidney inner medullary cell response to urea
Authors: Zhang Zheng , Yang Xiao-Yan , Soltoff Stephen P. , Cohen David M.
Published: December 2017

Growth factors and other stimuli increase the activity of phosphatidylinositol-3 kinase (PI3K), an SH2 domain-containing lipid kinase. In the murine kidney inner medullary mIMCD3 cell line, urea (200 mM) increased PI3K activity in a time-dependent fashion as measured by immune complex kinase assay. The PI3K effector, Akt, was also activated by urea as measured by anti-phospho-Akt immunoblotting. In addition, the Akt (and PI3K) effector, p70 S6 kinase, was activated by urea treatment in a PI3K-dependent fashion. PI3K inhibition potentiated the proapoptotic effect of hypertonic and urea stress. Urea treatment also induced the tyrosine phosphorylation of Shc and the recruitment to Shc of Grb2. Coexistence of activated Shc and PI3K in a macromolecular complex was suggested by the increase in PI3K activity evident in anti-Shc immunoprecipitates prepared from urea-treated cells. Taken together, these data suggest that PI3K may regulate physiological events in the renal medullary cell response to urea stress and that an upstream tyrosine kinase conferring activation of both PI3K and Shc may govern urea signaling in these cells.
Protein tyrosine kinase regulates the number of renal secretory K channels
Authors: Wang Wenhui , Lerea Kenneth M. , Chan Mary , Giebisch Gerhard
Published: December 2017

The apical small conductance (SK) channel plays a key role in K secretion in the cortical collecting duct (CCD). A high-K intake stimulates renal K secretion and involves a significant increase in the number of SK channels in the apical membrane of the CCD. We used the patch-clamp technique to examine the role of protein tyrosine kinase (PTK) in regulating the activity of SK channels in the CCD. The application of 100 μM genistein stimulated SK channels in 11 of 12 patches in CCDs from rats on a K-deficient diet, and the mean increase in NPo, a product of channel number ( N) and open probability ( Po), was 2.5. In contrast, inhibition of PTK had no effect in tubules from animals on a high-K diet in all 10 experiments. Western blot analysis further shows that the level of cSrc, a nonreceptor type of PTK, is 261% higher in the kidneys from rats on a K-deficient diet than those on a high-K diet. However, the effect of cSrc was not the result of direct inhibition of channel itself, because addition of exogenous cSrc had no effect on SK channels in inside-out patches. In cell-attached patches, application of herbimycin A increased channel activity in 14 of 16 patches, and the mean increase in NPowas 2.4 in tubules from rats on a K-deficient diet. In contrast, herbimycin A had no effect on channel activity in any of 15 tubules from rats on a high-K diet. Furthermore, herbimycin A pretreatment increased NPoper patch from the control value (0.4) to 2.25 in CCDs from rats on a K-deficient diet, whereas herbimycin failed to increase channel activity ( NPo: control, 3.10; herbimycin A, 3.25) in the CCDs from animals on a high-K diet. We conclude that PTK is involved in regulating the number of apical SK channels in the kidney.
Oxidant stress in hyperlipidemia-induced renal damage
Authors: Scheuer Hartmut , Gwinner Wilfried , Hohbach Jan , Gröne Elisabeth F. , Brandes Ralf P. , Malle Ernst , Olbricht Christoph J. , Walli Autar K. , Gröne Hermann-Josef
Published: December 2017

Hyperlipoproteinemia can aggravate glomerulosclerosis and chronic tubulointerstitial (ti) damage in kidneys without primary immunologic disease. We evaluated whether the effect of hyperlipidemia on progression of renal damage differed between kidneys without preexisting glomerular disease and kidneys with mesangioproliferative glomerulonephritis and whether the renal actions of hyperlipidemia were dependent on oxidant-antioxidant balance. Hyperlipidemia was induced by high-fat and high-cholesterol diet in uninephrectomized rats. In rats without glomerulonephritis, hyperlipidemia led to a rise in glomerular and ti generation of reactive oxygen species (ROS). Oxygen radicals were mainly generated by enhanced xanthine oxidoreductase (XO), which rose with protein concentration and activity during hyperlipidemia; concurrently, glomerulosclerosis and chronic ti injury were noticed during hyperlipidemia [ti damage (% of total tubulointerstitium (TI) after 150 days): normolipidemia 0.1 ± 0% vs. hyperlipidemia 3.4 ± 0.9%; P < 0.05]. In mesangioproliferative Thy-1 nephritis, ti injury was significantly accelerated by hyperlipidemia (ti damage after 150 days: normolipidemic Thy-1 nephritis 2.5 ± 0.6% vs. hyperlipidemic Thy-1 nephritis 12.5 ± 3.1%; P < 0.05). Antioxidant enzyme activities decreased and XO activity rose markedly in the TI (XO activity in TI after 150 days: normolipidemic Thy-1 nephritis 2.2 ± 0.5 vs. hyperlipidemic Thy-1 nephritis 4.5 ± 0.7 cpm/μg protein; P < 0.05). In hyperlipidemic Thy-1 nephritis rats, which had a higher urinary protein excretion than normolipidemic rats, hypochlorite-modified proteins, an indirect measure for enhanced myeloperoxidase activity, were detected in renal tissue and in urine, respectively. During hyperlipidemia, chronic damage increased in renal TI. Enhanced generation of ROS, rise in oxidant enzyme activity, and generation of hypochlorite-modified proteins in renal tissue and urine were noticed. These data suggest that oxidant stress contributed to the deleterious effects of hyperlipidemia on the renal TI.