bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcium oxalate monohydrate crystals are endocytosed by renal epithelial cells and induce proliferation
Authors: Lieske J. C. , Walsh-Reitz M. M. , Toback F. G.
Published: December 2017

Monkey kidney epithelial cells of the nontransformed BSC-1 line were used as a model system with which to search for biological responses to urinary crystals commonly found in renal stones. Calcium oxalate monohydrate (COM), the most common urinary crystal, was avidly internalized, initiated DNA synthesis, and stimulated cell multiplication. The increase in DNA synthesis observed after exposure to COM crystals was equivalent in magnitude to that of 10% calf serum, but occurred 8 h later. Maximal stimulation of DNA synthesis by COM was associated with crystal endocytosis by 50% of the cell monolayer. COM crystals also stimulated DNA synthesis and multiplication of canine kidney epithelial cells (MDCK line). As COM stimulated growth of both monkey and canine renal cells but not fibroblasts, the mitogenic effect of this crystal appeared cell-type specific. Hydroxyapatite also enhanced multiplication of BSC-1 cells, whereas brushite, another calcium-containing urinary crystal, did not. In the presence of nephrocalcin (NC), a glycoprotein in normal human urine that inhibits nucleation, aggregation, and growth of COM crystals, the capacity of these crystals to initiate DNA synthesis was blocked. This is the first demonstration that specific calcium-containing urinary crystals can induce proliferation of renal epithelial cells and that NC can inhibit this effect.
Attenuated expression of epithelial cell adhesion molecules in murine polycystic kidney disease
Authors: Rocco M. V. , Neilson E. G. , Hoyer J. R. , Ziyadeh F. N.
Published: December 2017

Polycystic kidney disease is an inherited disorder of parenchymal structure that leads to renal failure. Cysts begin as focal dilations in proximal tubules and collecting ducts, giving rise to cyst walls lined by a phenotypically disturbed epithelium that expresses dysfunctional transport and matrix proteins. We used an mRNA search protocol to probe efficiently for tissue-specific disturbances that might underlie the formation of cysts. This search assessed the relative abundance of transcripts encoding a variety of growth factors (transforming growth factor-beta 1, interleukin-6, tumor necrosis factor, and endothelin-1), structural proteins (collagen IV, nidogen, fibronectin, and laminins A and B1), and cell adhesion molecules (CAMs; E-cadherin, N-CAM, laminin receptor, and fibronectin receptor) in the cystic kidneys of cpk/cpk mice and uncovered a previously unrecognized early reduction in mRNA encoding N-CAM (54%) and E-cadherin (56%) (n = 5; P less than 0.001). Levels of transcripts for growth factors, structural proteins, and for fibronectin and laminin receptors in normal and cystic kidneys were generally similar. The reduction in transcripts for N-CAM and E-cadherin in kidneys from cystic mice was not observed in autologous liver. The immunofluorescent staining of cystic kidneys confirmed that the decrease in N-CAM and E-cadherin was generally confined to regions abundant in developing cystic epithelium. The presence of both N-CAM and E-cadherin appears to guide the sequential differentiation and polarization of normal renal epithelium, and their attenuated expression in the kidney of cpk/cpk mice may be a material factor contributing to the pathogenesis of cyst formation.
Phenol red inhibits growth of renal epithelial cells
Authors: Walsh-Reitz M. M. , Toback F. G.
Published: December 2017

Phenol red coeluted with a novel kidney cell growth factor during its isolation from conditioned medium by high-performance liquid chromatography. The possibility that phenol red rather than the putative factor mediated the growth-promoting activity was tested, because this pH indicator is known to possess estrogenic properties including a mitogenic effect. Unexpectedly, phenol red at the concentration found in Dulbecco's modified Eagle's medium (DMEM) inhibited growth of monkey (BSC-1) and canine kidney (MDCK) epithelial cells by 20-30%, but stimulated multiplication of 3T3 fibroblasts. The growth-inhibitory effect of phenol red for BSC-1 cells was reversible, concentration dependent, and cell-density independent when multiplication was examined in the presence and absence of the dye. Phenol red partially masked the mitogenic effect of calf serum and epidermal growth factor and was additive to the growth-inhibitory effect of transforming growth factor-beta 2. These observations indicate that phenol red at the concentration in DMEM both underestimates the potency of mitogens and overestimates the strength of an inhibitor of kidney epithelial cell growth and suggest that the dye be omitted from the culture medium when a new growth-regulatory compound is under study.
Spatial and temporal expression of kallikrein and its mRNA during nephron maturation
Authors: el-Dahr S. S. , Chao J.
Published: December 2017

To determine the relationships between nephron maturation and the ontogeny of intrarenal kallikrein, the expression and distribution of kallikrein and its mRNA were examined in fetal, neonatal, and adult Sprague-Dawley rats. Kallikrein-like immunoreactivity was first expressed 1 day before birth in the upper limb of S-shaped bodies. Immediately after birth, kallikrein immunostaining was present along the apical membranes of inner cortical distal tubules, whereas subcapsular less mature comma-shaped vesicles did not express kallikrein. After completion of nephrogenesis, kallikrein was also seen in the distal and connecting tubules of the outer cortex. In situ hybridization localized the kallikrein message in the distal tubules of inner cortical nephrons in newborns and outer cortex in adults. The transition from newborn to adult life was associated with six- and fourfold increase in renal kallikrein content and mRNA accumulation, respectively. We conclude that immature distal tubules have the transcriptional and translational capacity to express the kallikrein gene and protein and that the renal kallikrein gene is developmentally regulated. The spatial and temporal changes in intrarenal kallikrein expression during ontogeny are consistent with the hypothesis that the kallikrein-kinin system plays a role in the maturation of renal functions.
Apolipoprotein gene expression in analbuminemic rats and in rats with Heymann nephritis
Authors: Sun X. , Jones H. , Joles J. A. , van Tol A. , Kaysen G. A.
Published: December 2017

The nephrotic syndrome results from altered glomerular permselectivity, causing urinary protein loss, reduced albumin concentration, oncotic pressure (pi), and hyperlipidemia. Hepatic lipid and apolipoprotein synthesis increases and lipoprotein catabolism decreases. Decreased lipoprotein catabolism follows the onset of proteinuria but is not associated with hereditary analbuminemia [Nagase analbuminemic rat (NAR)] if proteinuria is absent. We measured plasma apolipoproteins (apo) AI, B, and E levels, their mRNA concentrations in liver, and the transcription rate of each mRNA in rats with Heymann nephritis (HN) or NAR to determine which alterations occurred in NAR alone without proteinuria. Plasma apo AI, B, and E were increased in both HN and NAR. Cholesterol and apo AI were inversely proportional to pi and independent of urinary protein loss or the presence of albumin in plasma. In contrast, triglycerides (TGs) were significantly greater in HN and were increased out of proportion to apo B. The concentration of apo AI mRNA increased in liver of both HN and NAR as did apo AI transcription. Apo E mRNA increased in neither HN nor NAR, whereas apo B mRNA increased only in HN. Transcription of neither apo B nor E increased. Plasma apo AI levels are likely to be regulated transcriptionally at the level of protein synthesis, whereas plasma apo B and E levels are regulated either posttranscriptionally, at the level of protein catabolism, or at both sites. Lipoproteins rich in TG and poor in apo B appear after the development of proteinuria but not as a consequence of analbuminemia alone. The accumulation of TG-rich apo B containing lipoproteins in rats with HN may result from impaired lipolysis occurring as a consequence of proteinuria.
Anti-Fx1A antibody recognizes a beta 1-integrin on glomerular epithelial cells and inhibits adhesion and growth
Authors: Adler S. , Chen X.
Published: December 2017

Cell-matrix interactions play an important role in regulating cell growth and metabolic activity and potentially in maintaining the integrity of the glomerular filtration barrier. The effect of anti-Fx1A antibody (Ab), the pathogenetic Ab of passive Heymann nephritis, on the interaction of cultured rat glomerular visceral epithelial cells (GEC) with matrix components was examined in an effort to determine whether it affects cell adhesion. Affinity chromatography demonstrated that anti-Fx1A recognizes an alpha 3 beta 1-integrin-type matrix receptor on GEC. Anti-Fx1A inhibited the adhesion of GEC to several substrates (collagen IV approximately equal to collagen I less than laminin approximately equal to fibronectin) in a dose-dependent manner and produced reversible cell detachment and "rounding up" when added to adherent cells. Maximal inhibition of adhesion was similar with anti-Fx1A and anti-beta 1, and competition studies showed no additive effects between anti-Fx1A and anti-beta 1 in inhibiting adhesion, suggesting that the effect of anti-Fx1A on GEC adhesion is attributable to its anti-beta 1 activity. Anti-Fx1A Ab also inhibited the growth of GEC in culture without evidence of cytotoxicity, and cells resumed normal growth on removal of Ab. These studies suggest that anti-Fx1A Ab can bind to matrix receptors on GEC, resulting in inhibition of cell attachment and growth, as well as producing detachment of cells that are already adherent. Interference with GEC-glomerular basement membrane interactions in vivo might have significant effects on glomerular permeability to proteins.
Time course and kinetics of proximal tubular processing of insulin
Authors: Nielsen S.
Published: December 2017

The present study was undertaken to determine the time courses and kinetics of the subcellular processing of 125I-insulin in isolated and in vitro perfused proximal tubules. Morphometric analysis demonstrated well-preserved ultrastructure after 90 min of perfusion. After luminal perfusion for 90 min the absorption was constant with time and reached steady state within 5 min (177 +/- 7 fg.min-1.mm-1). Also the hydrolysis rate and tubular accumulation rate were constant and averaged 84 +/- 8 and 93 +/- 10 fg.min-1.mm-1, respectively. Free 125I appeared already within 5 min of perfusion and reached steady state within 10 min. From proximal tubules perfused with 125I-insulin for 30 min and chased for 60 min, a compartmental analysis revealed two compartments; half time (t1/2) for delivery of insulin to the lysosomes was determined to be 8.5 min, and t1/2 for lysosomal degradation was 72 min. The results demonstrated that internalization by endocytic invaginations, incorporation in endocytic vacuoles, fusion with lysosomes, and hydrolysis were rapid processes and reached maximum rates within few minutes. A significant transtubular transport of insulin to the peritubular compartment was determined to be a constant rate of 11.2 +/- 0.7 fg.min-1.mm-1. Perfusion of tubules with insulin at high concentrations in the perfusate revealed that the transport was dependent on the absorbed amount and not on the perfused load, compatible with transport through the cells and not via a paracellular mechanism. The intactness of the tight junctions was supported by the following: 1) [14C]inulin leak did not increase with time and 2) enzyme-free intercellular spaces were evident after perfusion for only 5 min with microperoxidase (mol wt of 1,700). The transported 125I-insulin was trichloroacetic acid precipitable and immunoprecipitable.
Mercury blocks Na-K-ATPase by a ligand-dependent and reversible mechanism
Authors: Anner B. M. , Moosmayer M. , Imesch E.
Published: December 2017

An inhibitory receptor for cardioactive steroids such as digoxin and ouabain is located at the extracellular surface of the Na-K-adenosinetriphosphatase (ATPase) molecule. Besides cardioactive steroids, mercury is a potent inhibitor of the Na-K-ATPase activity. The half-maximal inhibitory concentration (IC50), determined within 30 min at 37 degrees C at 1 microgram protein/ml, was 200 nM, despite the presence of 1 mM EDTA; the IC50 decreased with increasing protein/inhibitor ratio, and it reached 2.7 microM at 0.1 mg protein/ml and 20 microM at 1 mg protein/ml. The IC50 for Na-K-ATPase inhibition by the diuretic compound mersalyl was 4 and 5 microM for the nondiuretic p-chloromercuribenzenesulfonic acid at 0.1 mg protein/ml. The IC50 for HgCl2 inhibition was modulated by the presence of EDTA as well as by the pump ligands Mg, Na, K, and ATP. The E2 conformation of the Na-K-ATPase molecule was more sensitive to HgCl2 than the E1 conformation. The mercury antidote 2,3-dimercapto-1-propanesulfonic acid was able to reactivate approximately 70% of the blocked enzyme. In conclusion, a metal-binding domain of the Na-K-ATPase molecule with particular high affinity for Hg(II) was described functionally in the present work. Therefore Na-K-ATPase belongs to the metal-binding proteins. Metals may modulate the cellular expression and activity of the system by interacting with its metal-binding interface.
Mercury weakens membrane anchoring of Na-K-ATPase
Authors: Imesch E. , Moosmayer M. , Anner B. M.
Published: December 2017

The presence of circulating inhibitors able to decrease the renal Na-K-adenosinetriphosphatase (ATPase) activity (natriuretic hormones) was postulated some 30 years ago. In the present work, the natriuretic inhibitor HgCl2 was selected as a model compound for the structural characterization of a possible natriuretic pathway for Na-K-ATPase modification. The structural effects of Na-K-ATPase inhibition by HgCl2 were assessed by trypsinolysis of the blocked enzyme in comparison with untreated preparations. The results show that inactivation of Na-K-ATPase by HgCl2 leads to the release of the alpha-subunit from the membrane preferentially in the E2 conformation but also in the E1 conformation. Apparently, HgCl2 weakens the membrane anchoring of the alpha-subunit, presumably by loosening the alpha-beta-subunit interaction. By this mechanism, the sensitivity of the Na-K-ATPase to extracellular drugs, hormones, and antibodies, as well as to intracellular proteases and other regulatory factors, could be altered.
Regulation of glutamine metabolism in dog kidney cortex: effect of pH and chronic acidosis
Authors: Schoolwerth A. C. , Smith B. C. , Drewnowska K.
Published: December 2017

To examine the interrelationships of proton compartmentation and ammoniagenesis, experiments were performed in tubules and mitochondria isolated from dog kidney cortex. Tubules were incubated in Krebs-Henseleit buffer at different pH (pHe), and cytosolic pH (pHi) was estimated with the fluorescent probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Mitochondrial pH (pHm) was determined simultaneously in intact tubules by use of dimethyloxazolidine-2,4-dione. Over the pHe range 6.9-7.7, pHi was similar in control and acidotic dogs and linearly related to pHe. At pHe 7.4 in control tubules. pHm was 7.78 +/- 0.07, and varied little over the pHe range of 7.0-7.7. The pH gradient across the mitochondrial membrane rose at acid pHe. pHm was more alkaline when estimated in tubules from acidotic dogs compared with controls. Ammonium and glucose productions from glutamine were inversely related to pHe and pHi in tubules from both control and acidotic animals and were higher in acidosis. In contrast, ammonium production by isolated mitochondria did not vary as pHe was altered. Enzyme fluxes, calculated from metabolite changes, demonstrated that glutamate dehydrogenase (GDH) flux was altered. Enzyme fluxes, calculated from metabolite changes, demonstrated that glutamate dehydrogenase (GDH) flux was inversely and glutaminase (PDG) flux was linearly related to pHe. Ammonium production was significantly greater in mitochondria from acidotic dogs because of accelerated flux through PDG but not GDH. The present study demonstrates significant difference between proton compartmentation and regulation of ammoniagenesis in kidneys from acidotic dog compared with rat.
Intracellular band 3 immunostaining in type A intercalated cells of rabbit kidney
Authors: Madsen K. M. , Kim J. , Tisher C. C.
Published: December 2017

Intercalated cells (ICs) in the collecting duct and the connecting tubule (CNT) are involved in H+ secretion and HCO3- reabsorption. H+ secretion is mediated by an H(+)-adenosinetriphosphatase in the apical plasma membrane, whereas a band 3-like Cl(-)-HCO3- exchanger in the basolateral membrane is responsible for HCO3- reabsorption. Recent studies have reported that a band 3-like protein is also present in mitochondria in rabbit ICs. The purpose of this study was to establish the subcellular location of the band 3-like Cl(-)-HCO3- exchanger in rabbit ICs by electron microscopic immunocytochemistry using a monoclonal antibody, IVF12, against erythrocyte band 3 protein. Rabbit kidneys were preserved by in vivo perfusion with a paraformaldehyde-lysine-periodate solution and processed for immunocytochemistry using a horseradish peroxidase preembedding technique. Band 3 immunostaining was observed on the basolateral plasma membrane of ICs in the outer medullary collecting duct and type A cells in the cortical collecting duct (CCD) and CNT. In addition, distinct staining for band 3 was present in numerous small vesicles and in multivesicular bodies in type A ICs in the CCD and CNT. However, there was no evidence of band 3 immunostaining of mitochondria or of the apical plasma membrane in any cells of the collecting duct. These observations suggest that basolateral Cl(-)-HCO3- exchangers in type A ICs in the rabbit kidney are stored in intracellular vesicles and possibly degraded in the vascular-lysosomal system when these cells are in a resting state. The previously reported band 3 immunolabeling of mitochondria could not be confirmed.
Luminal influences on potassium secretion: chloride, sodium, and thiazide diuretics
Authors: Velazquez H. , Ellison D. H. , Wright F. S.
Published: December 2017

In the presence of Cl-, K+ secretion by the distal tubule saturates with increasing luminal Na+ concentration. Apparent maximal K+ secretion is attained with luminal Na+ concentrations of 40 mM. The results of the present study show that lowering the Cl- concentration of luminal fluid can increase the level of Na(+)-stimulated K+ secretion beyond the maximal level attained in the presence of Cl-. The effect of lowering luminal Cl- concentration to less than 10 mM on K+ secretion is greater with higher Na+ concentration. Under these conditions, chlorothiazide decreases K+ secretion. When chlorothiazide is present, changing the Na+ concentration does not affect K+ secretion. Because in rats a thiazide effect is attributed primarily to the distal convoluted tubule (DCT), we postulate that it is primarily DCT cells that increase K+ secretion when Na+ concentration is raised in the presence of low luminal Cl- concentration. We propose that the rat DCT cells have both an absorptive Na(+)-Cl- cotransport mechanism and a secretory K(+)-Cl- cotransport mechanism in the luminal membrane that can mediate the apparent exchange of Na+ for K+.
cDNA cloning of a renal Na(+)-Ca2+ exchanger
Authors: Reilly R. F. , Shugrue C. A.
Published: December 2017

In the present study, the polymerase chain reaction (PCR) and library screening were used to clone a cDNA for a rabbit kidney Na(+)-Ca2+ exchanger on the basis of homology with the canine cardiac sarcolemmal sequence (D. A. Nicoll, S. Longoni, and K. D. Philipson. Science Wash. DC 250:562-565, 1990). There is a high degree of similarity between the two sequences, with nucleotide identities of 95, 89, and 90% in the hydrophobic membrane-associated domain, cytoplasmic domain, and 3'-untranslated region, respectively. The rabbit kidney cDNA encodes a predicted protein of 941 amino acids, 29 amino acids shorter than the canine sequence, with a relative molecular weight of 105,121. The deduced amino acid sequence is 96% identical in the membrane-associated domain and 94% identical in the cytoplasmic domain. Northern blot analysis reveals that the cDNA is expressed in the renal cortex. No expression is detected in the medulla. This result is in agreement with micropuncture studies that show Na(+)-Ca2+ exchanger activity in cortical but not medullary nephron segments.
Corticosterone 6 beta-hydroxylation correlates with blood pressure in spontaneously hypertensive rats
Authors: Watlington C. O. , Kramer L. B. , Schuetz E. G. , Zilai J. , Grogan W. M. , Guzelian P. , Gizek F. , Schoolwerth A. C.
Published: December 2017

Evidence for increased glucocorticoid 6 beta-hydroxylation (enhanced family 3A cytochrome P-450 activity) is found in certain reversible forms of human hypertension. This association was investigated in the spontaneously hypertensive rat (SHR). The proportion of injected [3H]corticosterone excreted in urine as 6 beta-[3H]OH-corticosterone was four- to fivefold higher in SHR than in control Wistar-Kyoto rats, before and after development of overt hypertension. Both hypertension and 6 beta-hydroxylation were inhibited by troleandomycin (a selective inhibitor of family 3A cytochromes P-450), consistent with a role for increased steroid 6 beta-hydroxylation in the genesis of hypertension in the SHR.
Inhibition of renal nitric oxide synthesis with NG-monomethyl-L-arginine and NG-nitro-L-arginine
Authors: Naess P. A. , Kirkeboen K. A. , Christensen G. , Kiil F.
Published: December 2017

In barbiturate-anesthetized dogs, the effects of intrarenal infusion of the two selective inhibitors of nitric oxide synthesis, NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine (NO-Arg), were compared. Basal renal blood flow (RBF) was reduced by 15 +/- 2% after L-NMMA at 0.28 mumol/ml, by 20 +/- 3% after NO-Arg at 0.07 mumol/ml, and by 31 +/- 5% after NO-Arg at 0.56 mumol/ml. Endothelium-dependent vasodilation induced by intrarenal infusion of acetylcholine was unaltered after L-NMMA, reduced by 24 +/- 3% after NO-Arg at 0.07 mumol/ml, and reduced by 59 +/- 13% after NO-Arg at 0.56 mumol/ml. Endothelium-independent vasodilation induced by intrarenal infusion of atrial natriuretic factor was not reduced after L-NMMA and NO-Arg. This study shows that NO-Arg is more potent than L-NMMA in inhibiting basal renal nitric oxide synthesis. In contrast to L-NMMA, NO-Arg exerted an inhibitory effect on acetylcholine-induced renal vasodilation. Our findings indicate that one-third of the basal RBF and more than one-half of the increase in RBF during acetylcholine infusion are dependent on nitric oxide synthesis.
Sequential changes in renal oxygen consumption and sodium transport during hyperdynamic sepsis in sheep
Authors: Weber A. , Schwieger I. M. , Poinsot O. , Klohn M. , Gaumann D. M. , Morel D. R.
Published: December 2017

The sequence of changes in systemic and renal oxygen delivery (QO2) and consumption (VO2) and renal function in an ovine model of progressive hyperdynamic sepsis was investigated. Nine chronically instrumented awake sheep were given a continuous intravenous Escherichia coli endotoxin infusion (20 ng.kg-1.min-1) for 3 days. After 8 h of the infusion, systemic arterial blood pressure and vascular resistance stayed decreased by 30% (P less than 0.001). Systemic QO2 progressively increased to a maximum of 157% of baseline values at 24 h and was associated with a decreased O2 extraction ratio from 33 +/- 2 (SE) to 23 +/- 2% (P less than 0.05), resulting in an unchanged systemic VO2. Renal blood flow and renal QO2 decreased by 40% during the first 12 h, returning to and staying at baseline values after 24 h. Renal VO2 decreased significantly by 35% at 12 h and then partially recovered to baseline values. Plasma creatinine clearance was maximally reduced to 25% of baseline values at 12 h and thereafter remained significantly (P less than 0.01) below 50% of baseline values. Both total and fractional sodium excretion fell at 12 h by 95 and 74%, respectively, and remained reduced over time, indicating conserved tubular function. The ratio of moles of sodium reabsorbed to moles of O2 consumed by the kidney was transiently reduced, from 33.4 +/- 4.1 to 12.4 +/- 3.6 at 12 h (P less than 0.05), indicating a relative increase in energy expenditure for tubular transport or renal synthetic activities, but recovered to baseline values after 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)
Effects of endothelin on renal hemodynamics and tubuloglomerular feedback
Authors: Takabatake T. , Ise T. , Ohta K. , Kobayashi K.
Published: December 2017

The effect of endothelin (ET)-1 infusion on renal hemodynamics and the tubuloglomerular feedback (TGF) mechanism was examined in rats. ET-1 reduced early proximal flow rate (EPFR) measured in the absence of distal flow from 28 +/- 1 to 23 +/- 1 nl/min at a subpressor dose (100 pmol.100 g body wt-1.h-1 iv) and from 27 +/- 2 to 19 +/- 2 nl/min at a pressor dose (200 pmol.100 g body wt-1.h-1). Reductions of EPFR induced by loop perfusion at 40 nl/min were 11 +/- 1 and 12 +/- 1 nl/min at the subpressor and pressor doses and were not different from controls. The stop-flow pressure response to loop perfusion was not altered by the pressor dose of ET-1. The subpressor dose of ET-1 increased renal vascular resistance (RVR) by 40% but left glomerular filtration rate (GFR) and urinary excretion of sodium (UNaV) unaltered. The pressor dose of ET-1 not only increased RVR by 140% and decreased GFR and renal plasma flow by 37 and 52%, but it increased UNaV proportional to the rise in blood pressure (r = 0.724, P less than 0.01). ET-1 is a potent renal vasoconstrictor and induces pressure natriuresis. ET-1 increases both pre- and postglomerular resistances but does not affect TGF response.
Eicosanoids modulate apical Ca(2+)-dependent K+ channels in cultured rabbit principal cells
Authors: Ling B. N. , Webster C. L. , Eaton D. C.
Published: December 2017

Patch clamp technology was utilized to study the effects of apical phospholipase A2 (PLA2) metabolites on “maxi K” channels in the principal cell apical membrane of rabbit cortical collecting tubule (CCT) primary cultures (B. N. Ling, C. F. Hinton, and D. C. Eaton. Kidney Int. 40: 441–452, 1991). At resting membrane potential, this channel is quiescent in the cell-attached configuration. Apical application of the PLA2 agonist melittin (1 microgram/ml) for 10 min increased single-channel open probability (Po) from 0.0004 +/- 0.0010 to 0.11 +/- 0.05. Similarly, apical exposure to 50 microM arachidonic acid (AA) or 0.5 microM prostaglandin (PG) E2, but not 0.5 microM PGF2 alpha, also increased channel activity. Conversely, 10 microM of the PLA2 antagonist quinacrine applied apically decreased Po. Removal of apical bath Ca2+ did not prevent melittin-, AA-, or PGE2-induced channel activation. We then examined the role of maxi K channels and eicosanoids in principal cell volume regulation. Within seconds of reducing basolateral bath tonicity (285 to 214 mosmol/kgH2O), NPo (i.e., no. of channels x Po) initially increased approximately 200%, followed by a delayed but prolonged activation phase that was attenuated by removal of apical bath Ca2+. Pretreatment with 10 microM quinacrine, 100 microM indomethacin (cyclooxygenase inhibitor), or 0.25 microM thapsigargin (to deplete intracellular Ca2+ stores) abolished the initial phase of swelling-induced channel activation.(ABSTRACT TRUNCATED AT 250 WORDS)
Activation of proximal tubular Na(+)-H+ exchange by angiotensin II
Authors: Bloch R. D. , Zikos D. , Fisher K. A. , Schleicher L. , Oyama M. , Cheng J. C. , Skopicki H. A. , Sukowski E. J. , Cragoe E. J. , Peterson D. R.
Published: December 2017

Stimulation of Na(+)-H+ exchange by angiotensin II (ANG II) was characterized in renal proximal tubular cells. Rabbit proximal nephron segments were incubated in the presence or absence of ANG II (5 x 10(-10) M), after which brush-border membrane vesicles (BBMV) were isolated and assayed for Na(+)-H+ antiporter activity using the acridine orange technique. Both the affinity (for sodium) and capacity of the carrier were elevated significantly (P less than 0.05) within 15 min of incubation with ANG II. To determine whether the stimulation of transport capacity involved a change in Na(+)-H+ antiporter density in the luminal membrane, binding of tritiated 5-(N-methyl-N-isobutyl)amiloride ([3H]MIA) was measured in BBMV derived from control and ANG II-treated nephron segments, following maximal stimulation. This demonstrated a significant (P less than 0.05) increase in the maximal specific binding (Bmax) of [3H]MIA binding in the ANG II-treated group compared with control, of a magnitude sufficient to account for the observed change in maximal velocity (Vmax). The data indicate that the Vmax effect is caused by an apparent increase in the number (density) of active Na(+)-H+ carriers present in the luminal membrane. Finally, to test the possibility that the observed kinetic change involves an exocytic mechanism, the effect of colchicine on ANG II-stimulated antiporter activity was examined. The increase in Vmax due to ANG II was blocked by the addition of 0.5 mM colchicine to the incubation medium, whereas colchicine alone had no significant effect on the Vmax of Na(+)-H+ kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)
cDNA sequence and tissue expression of bovine vacuolar H(+)-ATPase M(r) 70,000 subunit
Authors: Marushack M. M. , Lee B. S. , Masood K. , Gluck S.
Published: December 2017

cDNA clones encoding the 70,000 relative molecular weight (M(r)) subunit of the bovine vacuolar proton-adenosine triphosphatase (H(+)-ATPase) were isolated, and a total length of 3.1 kb in overlapping clones from bovine kidney and brain libraries was sequenced. The cDNA contains a 1.9-kb coding region and yields a deduced protein sequence of 618 amino acids. The subunit sequence has 50% amino acid identity with the corresponding subunit from yeast, carrot, and Neurospora vacuolar H(+)-ATPases. The internal regions of the protein are most highly conserved, whereas the NH2- and COOH-terminals exhibit variability. mRNA levels of the M(r) 70,000 subunit were examined in multiple bovine tissues and were found to be expressed at highest levels in kidney medulla and cortex, at moderate levels in brain and adrenal gland, and at low levels in liver, muscle, and heart.
Vanadate causes hypokalemic distal renal tubular acidosis
Authors: Dafnis E. , Spohn M. , Lonis B. , Kurtzman N. A. , Sabatini S.
Published: December 2017

Pages F449–F453: E. Dafnis, M. Spohn, B. Lonis, N. A. Kurtzman, and S. Sabatini. “Vanadate causes hypokalemic distal renal tubular acidosis.” The value for urine anion gap in NH4Cl-treated animals on page F450 (last sentence in first paragraph of results), as well as on page F51 (Table 2, last value in last line), should be -82 ± 7 meq/l instead of -232 ± 27 meq/l.
Effects of glucagon on glomerular filtration rate and urea and water excretion
Authors: Ahloulay M. , Bouby N. , Machet F. , Kubrusly M. , Coutaud C. , Bankir L.
Published: December 2017

Clearance experiments were performed in anesthetized male Wistar rats to reevaluate the renal effects of glucagon (Gluc) on glomerular filtration rate (GFR) and solute and water excretion. After an 80-min control period, these effects were evaluated in the last 80 min of a 2-h intravenous Gluc infusion. Gluc induced significant increases in GFR (+20%), urine flow rate (+150%), free water reabsorption (+50%), urea synthesis and urea excretion (+66%), and nonurea solute excretion (+67%). In addition, fractional urea excretion (FEurea) increased by 43% (P less than 0.01). Additional experiments showed that increases in either urea excretion or urine flow rate (induced by appropriate infusion of urea or half-dilute saline), similar to those seen after Gluc, could not account for the increased FEurea. All significant effects of Gluc were also observed during infusion of antidiuretic hormone or during water diuresis. The tubular effects of Gluc could be explained by a reduction in proximal reabsorption. The dose of Gluc required to induce all the effects described above was 12 ng.min-1.100 g body wt-1, a dose producing an approximately 10-fold supraphysiological peripheral plasma concentration but a “physiological” level for the liver. Infusion of 1.2 ng induced almost no change in renal function, and infusion of 120 ng induced no greater effects than 12 ng. These results suggest 1) that Gluc, a hormone liberated after protein ingestion, exerts coordinated effects on liver and kidney to increase simultaneously urea synthesis and excretion and to promote water conservation and 2) that these effects could, at least in part, be indirect and depend on the Gluc-induced stimulation of hepatocyte metabolism.
Effect of selenium-deficient diet in experimental glomerular disease
Authors: Baliga R. , Baliga M. , Shah S. V.
Published: December 2017

We examined the effect of a selenium-deficient diet on two experimental models of glomerular disease, the puromycin aminonucleoside (PAN)-induced nephrotic syndrome, a model of minimal change disease, and passive Heymann nephritis, a complement-dependent and neutrophil-independent model that resembles membranous nephropathy. The specific activity of selenium-dependent glutathione peroxidase was markedly reduced in the liver, the kidney cortex, and in glomeruli in weanling male Sprague-Dawley rats placed on a selenium-deficient diet for 6 wk compared with rats fed a selenium-replete diet, with no significant differences in the specific activities of superoxide dismutase or catalase. PAN-injected selenium-deficient rats had a marked and significantly greater proteinuria throughout the course of the experiment compared with PAN-injected selenium-replete rats with no significant histological differences. In the passive Heymann nephritis model induced by injecting anti-Fx1A immunoglobulin G, rats fed a selenium-deficient diet had significantly higher urinary protein (day 5: 91 +/- 16 mg/24 h, n = 10) compared with rats fed a selenium-replete diet (52 +/- 5 mg/24 h, n = 11) with no differences in the amount of antibody deposited in the kidney. The most likely explanation for the effect of a selenium-deficient diet is that selenium deficiency resulted in a marked reduction of glutathione peroxidase, thus indicating an important role of glutathione peroxidase in these models of glomerular injury.
Regulation of mesangial cell cyclooxygenase synthesis by cytokines and glucocorticoids
Authors: Coyne D. W. , Nickols M. , Bertrand W. , Morrison A. R.
Published: December 2017

The cytokines, interleukin-1 (IL-1) and tumor necrosis factor (TNF), potently induce prostaglandin formation in glomerular mesangial cells. Mechanisms by which these cytokines stimulate prostaglandin formation vary among cell types. We investigated whether alterations in phospholipase A2 (PLA2) or cyclooxygenase (COX) mass and activity contribute to the changes in mesangial cell prostaglandin production. These cytokines induced COX activity and mass in a time-dependent manner, which paralleled prostaglandin production. IL-1 increased COX mass approximately threefold by 24 h. TNF had a much smaller effect, although it appeared to be additive with IL-1. IL-1-induced COX mass was maintained at an increased level for at least 48 h. The glucocorticoid dexamethasone (DEX) virtually abolished prostaglandin production and blocked cytokine induction of COX activity and mass. DEX did not reduce COX activity or mass below the basal, serum-fed levels, however. By utilizing stable isotope methods, we could demonstrate that IL-1 increased free arachidonate levels, implying new PLA2 synthesis over a time course that was maximal at 6 h and was cycloheximide and actinomycin D sensitive. These data demonstrate that the cytokines IL-1 and TNF enhance synthesis of COX and PLA2, contributing to increased prostaglandin production. Cytokine-stimulated prostaglandin production ceases when cells are also treated with DEX, although control levels of COX activity and mass remain. This occurs because DEX inhibits the IL-1-induced enhanced arachidonate release.
Prostanoid biosynthesis and mechanisms of action
Authors: Smith W. L.
Published: December 2017

Prostanoids are local hormones formed from arachidonic acid that coordinate responses to circulating hormones which elicit prostanoid synthesis. For example, in the kidney, prostaglandin (PG) E2 synthesized by collecting tubule epithelia in response to arginine vasopressin (AVP) acts on the parent collecting tubule as well as the neighboring thick limb to modulate NaCl and water reabsorption occurring in response to AVP. Studies performed over the last 15 years have defined the major cellular and subcellular sites of PG synthesis in the kidney. In addition, it is now recognized that the multiple cellular actions of prostanoids in the kidney are mediated through receptors coupled to guanine nucleotide regulatory proteins. The goal of this review is to summarize recent biochemical and molecular biological studies on prostanoid biosynthetic enzymes and on prostanoid receptors. The major topics to be addressed are 1) phospholipid precursors of arachidonate, 2) membrane-associated and cytosolic phospholipase A2s, 3) PG endoperoxide (PGH) synthase isozymes, 4) thromboxane A (TxA) synthase, and 5) TxA/PGH and PGE receptors.
Lipid peroxidation in isolated rat nephron segments
Authors: Ha H. , Endou H.
Published: December 2017

Elevated levels of lipid peroxides (LPO) in tissues have been considered an index of increased reactive oxygen metabolites, which are important pathological mediators also found in the kidney. By adopting the quantification of malondialdehyde-thiobarbituric acid adduct as a standard, using a fluorometer, a microassay was developed that enabled us to measure LPO in tissue having less than 1 microgram protein. By this method, basal levels of LPO along the rat nephron showed that proximal tubules bear more LPO per millimeter of tubule than distally located segments (approximately 0.2 pmol/mm tubule for proximal tubules and 0.02 for thick ascending limbs) and that S3 was the highest LPO per tissue protein (2.2 +/- 0.1 pmol/microgram protein, n = 8). In addition, the levels of LPO were stimulated by 10 microM phorbol 12-myristate 13-acetate (PMA) in both glomeruli and S3 (P less than 0.001) and in S2 (P less than 0.05). Furthermore, sphingosine (100 microM), a protein kinase C (PKC) inhibitor, totally blocked the LPO increment by PMA without any effect on the basal LPO in glomeruli, suggesting the involvement of PKC in LPO formation. Taken together, the results indicate the applicability of LPO assay to the nephron for evaluation of site-specific nephrotoxic insult and its mechanisms in renal pathophysiology.
Paradoxical exacerbation of leukocyte-mediated glomerulonephritis with cyclooxygenase inhibition
Authors: Nagamatsu T. , Pippin J. , Schreiner G. F. , Lefkowith J. B.
Published: December 2017

Nephrotoxic nephritis (NTN) is characterized by glomerular inflammation, an increase in glomerular eicosanoid synthesis, and renal dysfunction. Data further suggest that eicosanoids may play a critical role in the inflammatory response. In the current study, we examined the effects of in vivo manipulation of arachidonate metabolism on the cellular component of the inflammatory response in NTN. We found that inhibition of cyclooxygenase with indomethacin in mild NTN caused a two- to fourfold increase in the leukocyte influx into glomeruli with a change histologically from a focal to a more diffuse lesion. Both the accompanying proteinuria and the increase in ex vivo glomerular eicosanoid production were also augmented by the administration of indomethacin. The effect of indomethacin was reversible and not limited to the acute phase of NTN. The administration of aspirin, like indomethacin, augmented the glomerular inflammation of NTN. Neither OKY-046 (a thromboxane synthase inhibitor) nor MK-886 (a 5-lipoxygenase inhibitor) altered the glomerular inflammation of NTN. Administration of exogenous prostaglandin E (in the form of misoprostol) did diminish the proteinuria accompanying NTN; however, glomerular inflammation was not significantly affected. Incubation of glomeruli with [14C]arachidonate demonstrated the presence of noncyclooxygenase pathways of arachidonate metabolism (11-, 12-, and 15-lipoxygenases) with increased activity in NTN. These data demonstrate that cyclooxygenase inhibition may paradoxically worsen glomerular inflammation and suggest a potential role for noncyclooxygenase/non-5-lipoxygenase pathways of arachidonate metabolism.
Coordinate development of oxidative enzymes and Na-K-ATPase in thick ascending limb: role of corticosteroids
Authors: Djouadi F. , Wijkhuisen A. , Bastin J.
Published: December 2017

The postnatal development of mitochondrial ATP-producing pathways and Na-K-adenosinetriphosphatase (ATPase) in the rat medullary thick ascending limb of Henle (MTAL) was studied by measuring the activities of 3-ketoacid-CoA transferase, fumarase, citrate synthase, and Na-K-ATPase in microdissected MTAL of 16, 21, and 30-day-old pups and in adults. The role of adrenal steroids in the development of these four markers was also investigated by studying 21-day-old rats adrenalectomized on day 16 and given dexamethasone or aldosterone or NaCl injections from day 16 to day 21. There were large and correlated increases in the activities of the oxidative enzymes in the MTAL of control rat kidneys between 16 and 30 days after birth; Na-K-ATPase activity in the MTAL also greatly increased during the same period. Adrenalectomy completely prevented the developmental increases in MTAL oxidative enzymes and Na-K-ATPase; dexamethasone restored the development of all four enzymes, whereas aldosterone had no effect. We conclude that the postnatal maturation of Na+ reabsorption functions in MTAL cells involves coordinated increases in the capacity to produce ATP by oxidative metabolism and in Na-K-ATPase activity. This maturation process is probably triggered by the rise in circulating glucocorticoids that occurs during the weaning period.
Basolateral membrane potassium channels in rabbit cortical thick ascending limb
Authors: Hurst A. M. , Duplain M. , Lapointe J. Y.
Published: December 2017

The nature of K exit across the basolateral membrane of rabbit cortical thick ascending limb (CTAL) was investigated using the patch clamp technique. The basolateral membrane was exposed by mild collagenase treatment (0.1 U/ml), and a K-selective inwardly rectifying channel was identified. In cell-attached patches (140 mM K pipette) the inward conductance was 35.0 +/- 1.3 pS (n = 9) compared with an outward conductance of 7.0 +/- 0.9 pS (n = 5), and the current reversed at a pipette potential of -63.5 +/- 3.1 mV (n = 9). The channel is strongly voltage dependent, showing an e-fold increase in open probability per 18-mV depolarization. Barium blocked the channel, reducing both mean open probability and single-channel current amplitude; however, the channel was not Ca sensitive. On excision the channel exhibited rundown, which could not be prevented by 0.1 mM ATP or ATP plus 20 U/ml catalytic subunit of protein kinase A. A few excised patch recordings were possible, which confirmed the presence of a highly K-selective channel with a K-to-Na permeability ratio of 100. In conclusion, 1) it is possible to obtain patch clamp recordings from the rabbit CTAL basolateral membrane using a very mild collagenase treatment, and 2) the exit of K across the basolateral membrane is mediated at least in part by the presence of voltage-sensitive K channels.
Both peripheral chylomicron catabolism and hepatic uptake of remnants are defective in nephrosis
Authors: Kaysen G. A. , Mehendru L. , Pan X. M. , Staprans I.
Published: December 2017

We showed previously that proteinuria caused delayed chylomicron (CM) clearance in the rat and postulated the existence of a primary defect in CM hydrolysis. It was possible that reduced CM clearance resulted from increased lipogenesis causing saturation of catabolic sites and not from a primary defect in CM catabolism. To clarify this point we measured kinetically the absolute rate of triglyceride (TG) uptake from CM in rats with Heymann nephritis (HN) and normal Sprague-Dawley rats (SD) and determined TG uptake in individual tissues using [3H]TG- and [14C]cholesterol-labeled CM. Hepatic [14C]cholesterol uptake was reduced in HN (69.3 +/- 6 vs. 7.2 +/- 2% of dose, P less than 0.001). TG uptake was reduced in HN measured kinetically (1.01 +/- 0.09 vs. 0.213 +/- 0.028 mg TG.min-1.100 g body wt-1, P less than 0.001) and reduced in all tissues (heart, skeletal muscle, fat, and liver). CM are catabolized on the vascular endothelium to atherogenic, cholesterol-rich remnant (CM remnant) particles, which are then rapidly taken up by the liver. We measured hepatic CM remnant uptake in SD and in HN using [14C]cholesterol-labeled CM remnant. CM remnant uptake was significantly reduced in HN (58 +/- 1.2 vs. 20 +/- 0.86% uptake, P less than 0.01). CM remnants were increased significantly in plasma of HN. Thus the nephrotic syndrome causes a primary defect in the uptake of TG from CM that is expressed in all tissues and a separate defect in hepatic CM remnant uptake. Although CM remnant generation is impaired because of defective CM hydrolysis, the defect in hepatic CM remnant uptake is so severe that these particles accumulate in blood, posing a potential risk for atherogenesis.
PAH/alpha-KG countertransport stimulates PAH uptake and net secretion in isolated rabbit renal tubules
Authors: Chatsudthipong V. , Dantzler W. H.
Published: December 2017

Possible stimulation of both basolateral uptake and net transepithelial secretion of p-aminohippurate (PAH) by PAH/alpha-ketoglutarate (alpha-KG) countertransport was examined in intact perfused and nonperfused rabbit proximal S2 renal tubules. Preloading tubules with alpha-KG (100 microM) for 30 min increased [14C]-PAH rate of uptake by nonperfused tubules and rate of net secretion by perfused tubules approximately three- to sixfold. During stimulation of net secretion, intracellular [14C]PAH concentration increased to about the same extent as net secretion. Presence of Li+ (2 mM) or absence of Na+ (inhibitors of Na(+)-dicarboxylate cotransport) in bathing medium during alpha-KG preloading eliminated stimulation of PAH transport. Addition of unlabeled alpha-KG (1 mM) to bathing medium stimulated efflux of [14C]PAH across the basolateral membrane of tubules with oil-filled lumina, further supporting the concept of PAH/alpha-KG countertransport. Preloading with succinate (100 microM) also stimulated rates of [14C]PAH uptake by nonperfused tubules and net secretion by perfused tubules, but stimulation was only approximately 1.5-fold. Moreover, preloading with methyl succinate, a slowly metabolized derivative of succinate, did not stimulate [14C]PAH uptake by nonperfused tubules or net secretion by perfused tubules. Thus it seems most likely that succinate preloading stimulates PAH transport via metabolism, possibly by conversion to alpha-KG, not by direct countertransport for PAH. This study indicates for the first time in intact mammalian proximal S2 renal tubules that PAH/alpha-KG countertransport can stimulate net PAH secretion by generating an increased intracellular PAH concentration.