bluuebunny/crossref_metadata_2025 huggingface dataset:
Feedback regulation of Na channels in rat CCT. I. Effects of inhibition of Na pump
Authors: Silver R. B. , Frindt G. , Windhager E. E. , Palmer L. G.
Published: December 2017

Na channels in the apical membrane of the rat renal cortical collecting tubule were studied using the patch-clamp technique. Channel activity was monitored in cell-attached patches on tubules that were split open to expose the luminal surface. Channel number (N), open probability (Po), and currents (i) were measured at 37 degrees C during continuous superfusion of the tubule. Addition of ouabain (1 mM) to the superfusate to increase cell Na resulted in a decrease in the mean number of open channels (NPo) to less than 20% of control values within 2 min. This effect was not reversible within 5 min after removal of ouabain. There was, in addition, a parallel decrease in i. The mechanism of inhibiton appeared to involve increased intracellular Ca (Cai). Cai was measured using the fluorescence of the Ca indicator fura-2 in principal cells of split tubules under conditions identical to those used for electrical measurements. Cai increased from a basal level (153 +/- 36 nM) to a peak level (588 +/- 53 nM) approximately 3 min after the addition of ouabain. When a Ca-free superfusate was used, ouabain did not increase Cai or decrease NPo, although the decrease in i was similar to that observed in Ca-containing solutions. Similar increases in Cai were elicited by the Ca ionophore ionomycin (5 microM) in the presence of 0.1 mM extracellular Ca. This maneuver also resulted in a decrease in NPo which was similar to that observed in the presence of ouabain. Ouabain had no observable effect on cell pH.(ABSTRACT TRUNCATED AT 250 WORDS)
Urea induces Egr-1 and c-fos expression in renal epithelial cells
Authors: Cohen D. M. , Gullans S. R.
Published: December 2017

The membrane-permeant solute urea, in concentrations present in the mammalian renal medulla, increased expression at the mRNA level of two immediate-early gene (IEG) transcription factors, Egr-1 and c-fos, in a time- and dose-dependent fashion in confluent growth-suppressed Madin-Darby canine kidney (MDCK) cells. This upregulation occurred in the absence of both cytotoxicity and an inhibition of protein synthesis, two potential nonspecific inducers of IEG expression. These findings were of interest because we have previously shown that hyperosmotic stress induced by the functionally membrane-impermeant solute NaCl increased expression of these IEG, whereas hyperosmotic stress induced by the membrane-permeant solute glycerol failed to do so. The urea-induced increase in Egr-1 mRNA expression was not secondary to enhanced message stability as determined by actinomycin D experiments and is therefore likely a consequence of urea-induced transcriptional activation. Augmented Egr-1 expression in response to urea treatment was also observed in another renal epithelial cell line, LLC-PK1, but not in other cell types examined. Therefore cells of renal epithelial origin may be uniquely capable of responding to hyperosmotic urea with increased expression of IEG transcription factors, and this increase is likely transcriptionally mediated.
EGF and PGE2 inhibit rabbit CCD Na+ transport by different mechanisms: PGE2 inhibits Na(+)-K+ pump
Authors: Warden D. H. , Stokes J. B.
Published: December 2017

The rabbit cortical collecting duct absorbs Na+ by a transport system comprised of an apical membrane Na+ channel and a basolateral membrane Na(+)-K(+)-adenosinetriphosphatase. The rate of Na+ absorption across this epithelium is acutely inhibited by several hormones and autacoids including epidermal growth factor (EGF) and prostaglandin E2 (PGE2). We used electrophysiological analysis to determine which Na+ transport mechanism is primarily regulated in response to EGF and PGE2. We used concentrations of EGF and PGE2 that inhibited Na+ absorption to a comparable degree. We assessed the effects of these agents on Na+ transport primarily by the calculated equivalent current; the validity of this indicator was verified using simultaneous tracer flux measurements. EGF and PGE2 had different effects on the intracellular electrophysiological parameters. EGF (in the presence of a cyclooxygenase inhibitor) hyperpolarized the apical membrane voltage in a manner analogous to the Na(+)-channel blocker amiloride, reduced the transepithelial conductance, and increased the fractional resistance of the apical membrane. In comparison, PGE2 depolarized the apical membrane voltage in a manner analogous to the Na(+)-K+ pump inhibitor ouabain, and caused no significant changes in transepithelial conductance or apical membrane conductance. The finding that EGF hyperpolarized the apical membrane indicates that this agent attenuates Na+ absorption by reducing apical Na+ entry due to a decrease in the magnitude of the apical membrane Na+ conductance. In contrast, the electrophysiological changes produced by PGE2 indicate primary inhibition of the basolateral Na(+)-K+ pump following PGE2 treatment.
Effects of ET-1 on water and chloride transport in cortical collecting ducts of the rat
Authors: Tomita K. , Nonoguchi H. , Terada Y. , Marumo F.
Published: December 2017

Endothelin-1 (ET-1) is known as a vasoconstrictor peptide. However, recent reports suggested the effects on the transport of renal tubule. We previously reported that ET-1 inhibited arginine vasopressin (AVP)-dependent adenosine 3',5'-cyclic monophosphate in rat collecting ducts. Physiologically, ET-1 reversibly and significantly inhibited AVP-stimulated water permeability in inner medullary collecting duct (IMCD). We therefore investigated the effects on water and electrolyte transport in rat cortical collecting ducts (CCD), where Na and Cl are actively reabsorbed more than in IMCD. Pathogen-free male Sprague-Dawley rats weighing 80-120 g were used after treatment with deoxycorticosterone pivalate for 1-2 wk. Isolated CCD were microperfused in vitro. The Cl concentration was measured by a continuous-flow ultra-microcolorimeter, and the raffinose concentration was measured as a volume marker by a continuous-flow ultra-microfluorometer. In the presence of 10(-9) M AVP, 10(-8) M ET-1 significantly inhibited fluid absorption (nl.mm-1 x min-1) from 0.25 +/- 0.02 to 0.15 +/- 0.05 (mean +/- SE, n = 6, P < 0.01), Cl absorption (pmol.mm-1 x min-1) from 30. 6 +/- 2.8 to 14.9 +/- 4.0 (P < 0.01), and potential difference (mV) from -5.4 +/- 1.3 to -4.0 +/- 1.2 (P < 0.01). Similar results were obtained in the lower concentration of 10(-10) M AVP and 10(-10) M ET-1. As for the osmotic water permeability (microns/s), 10(-8) M ET-1 significantly inhibited this from 320.1 +/- 50.9 to 202.1 +/- 42.2 (n = 7, P < 0.01) in the presence of 10(-9) M AVP.(ABSTRACT TRUNCATED AT 250 WORDS)
Effects of formate and oxalate on chloride absorption in rat distal tubule
Authors: Wang T. , Agulian S. K. , Giebisch G. , Aronson P. S.
Published: December 2017

We previously demonstrated that formate and oxalate stimulate Cl-transport in the rat proximal tubule in accord with a model involving NaCl uptake across the luminal membrane via Cl-/organic anion exchange in parallel with Na+/H+ exchange and organic acid recycling. The purpose of the present study was to test whether similar mechanisms contribute to Cl- transport in the rat distal tubule. In distal tubules microperfused in situ with an isotonic solution, addition of 0.5 mM formate to the luminal perfusate increased Cl- (JCl) and fluid (Jv) absorption by 77 and 93%, respectively. Addition of 5 microM oxalate increased JCl and Jv by 52 and 108%, respectively. In distal tubules perfused with a hypotonic solution, formate stimulated JCl and Jv by 85 and 98%, respectively, and oxalate stimulated JCl and Jv by 80 and 115%, respectively. Addition of 0.5 mM acetate caused no significant change in JCl and Jv. The stimulation of JCl and Jv by formate was largely abolished by addition of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (100 microM) or ethylisopropyl-amiloride (10 microM) to the luminal perfusate. Chlorothiazide (0.5 mM) inhibited baseline JCl and Jv but caused no inhibition of the increments in Jv and JCl induced by formate. Formate stimulation of JCl and Jv was confined to early segments of the distal tubule. We conclude that formate and oxalate markedly enhance JCl and Jv in the early distal tubule by a thiazide-insensitive mechanism involving NaCl entry across the apical membrane by Cl-/organic anion exchange in parallel with Na+/H+ exchange.
Na+ pump inhibition downregulates an ATP-sensitive K+ channel in rabbit proximal convoluted tubule
Authors: Hurst A. M. , Beck J. S. , Laprade R. , Lapointe J. Y.
Published: December 2017

In several epithelial and nonepithelial tissues a functional link between the basolateral Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) and a basolateral K+ conductance has been established. However, the nature of this link is unclear. We have previously identified a K+ channel on the basolateral membrane of the proximal convoluted tubule perfused in vitro, the activity of which is increased by stimulation of Na+ transport [J. S. Beck, A. M. Hurst, J.-Y. Lapointe, and R. Laprade. Am. J. Physiol. 264 (Renal Fluid Electrolyte Physiol. 33): F496-F501, 1993]. In the present study we investigate whether basolateral membrane K+ channel activity is tightly coupled to Na(+)-K(+)-ATPase activity. In cell-attached patches (150 mM K+ pipette), following stimulation of channel activity by addition of Na(+)-cotransported solutes to the tubule lumen, mean channel open probability (NPo) was reduced from 0.35 +/- 0.09 to 0.14 +/- 0.06 (n = 7, P < 0.05) by blocking the Na(+)-K(+)-ATPase with 100 microM strophanthidin. In excised patches the channel was reversibly blocked by 2 mM ATP from the cytosolic face of the patch, such that NPo fell to 20.1 +/- 7.0% (n = 5, P < 0.001) of control and recovered to 52.2 +/- 11.2% (n = 5, P < 0.05) after washout of ATP. Diazoxide, a putative opener of ATP-sensitive K+ channels, when added to the bathing solution of an unstimulated tubule (microperfused in the absence of Na(+)-cotransported solutes), increased NPo from 0.046 +/- 0.035 to 0.44 +/- 0.2 (n = 6, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Acidification adaptation in cultured inner medullary collecting duct cells
Authors: Mankus R. , Schwartz J. H. , Alexander E. A.
Published: December 2017

Chronic acid feeding stimulates the rat inner medullary collecting duct (IMCD) to increase acid secretion in vivo (acidification adaptation), but the mechanism for this phenomenon is unknown. Our purpose was to determine whether IMCD cells undergo adaptation in vitro and to explore the mechanism of this response. Confluent cultured rat IMCD cells were exposed to incubation media supplemented with 10(-7) M deoxycorticosterone acetate, pH 7.0 [acid incubated (AI)] or 7.7 [control (C)], for 48 h, and cell pH (pHi) was determined using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Resting pHi was 7.46 +/- 0.05 for AI and 7.25 +/- 0.04 for C (P < 0.05). N-ethylmaleimide-sensitive pHi recovery after an acute acid pulse was 0.030 +/- 0.002 for AI and 0.020 +/- 0.002 pH U/min for C (P < 0.05). However, when AI and C cells were incubated with 7 x 10(-6) M cycloheximide, the increment in pHi and enhanced proton pump activity was abolished. In addition, exocytic function, as measured by Lucifer yellow release, was increased significantly in AI cells. In summary, incubation of IMCD cells in acid medium stimulates acidification adaptation by a mechanism dependent on new protein synthesis.
K infusion corrects thick ascending limb Cl reabsorption in K-depleted rats by an aldosterone-independent mechanism
Authors: McKay A. J. , Peterson L. N.
Published: December 2017

Others have provided evidence that thick ascending limb (TAL) NaCl reabsorption is aldosterone dependent in adrenalectomized animals. In rats fed a K-free diet, plasma K concentration ([K]) is reduced and plasma aldosterone concentration [Aldo] is decreased. Because aldosterone release is regulated by extracellular fluid (ECF) [K], the purpose of the present study was to determine whether aldosterone deficiency mediates inhibition of TAL NaCl transport in K-depleted rats (K-Dep). Cl reabsorption was measured in functionally isolated loop segments microperfused in vivo (22 nl/min) using a modified perfusate that minimizes proximal nephron reabsorption. The results of our studies show that the defect in TAL Cl reabsorption in K-Dep rats is quantitatively significant and can be rapidly reversed by the acute systemic infusion of K. However, acute administration of aldosterone, in the presence of sustained K-Dep, failed to reverse the impairment in TAL Cl reabsorption in K-Dep rats. Furthermore, acute infusion of K, in the presence of an aldosterone antagonist, in K-Dep rats rapidly corrected the defect in TAL Cl reabsorption. Additional studies showed that despite normalization of ECF [K] by acute K infusion in K-Dep rats, plasma [Aldo] failed to increase. In contrast, acute infusion of KCl in control rats increased plasma [Aldo] by 46%, but Cl reabsorption was unchanged. In summary, these results provide conclusive evidence that the correction of defective TAL Cl reabsorption in response to the acute administration of K in K-Dep rats occurs via an aldosterone-independent mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)
Regulation of renal epithelial cell endocytosis of calcium oxalate monohydrate crystals
Authors: Lieske J. C. , Toback F. G.
Published: December 2017

The earliest events in the formation of kidney stones are unknown. The most common crystal in kidney stones, calcium oxalate monohydrate (COM), when added to cultures of monkey kidney epithelial cells (BSC-1 line), was internalized by 19% of the cells after 30 min. COM crystal endocytosis was enhanced by serum, ADP, and epidermal growth factor, which are potent mitogens for these cells. Endocytosis of COM crystals was inhibited by diverse molecules including Tamm-Horsfall glycoprotein (THP), the tetrapeptide arginine-glycine-aspartic acid-serine, fibronectin, transforming growth factor-beta 2, and heparin. The capacity of THP, fibronectin, or heparin to inhibit endocytosis was mediated by an interaction of these molecules with cells, not by coating the crystals. Thus renal epithelial cell endocytosis of COM crystals can be regulated by diverse molecules including THP, the most common protein found in human urine. Crystal endocytosis and subsequent cellular responses could be important pathogenic steps in nephrolithiasis.
Effect of morphine on uptake of immunoglobulin G complexes by mesangial cells and macrophages
Authors: Singhal P. C. , Pan C. , Gibbons N.
Published: December 2017

Focal glomerular sclerosis is the predominant renal lesion in heroin addicts. We studied whether morphine, a metabolite of heroin, could directly affect the uptake of immunoglobulin G (IgG) complexes by cultured mesangial cells (MC) and macrophages (MP). Pre-incubation of morphine (10(-6) M) decreased uptake of IgG complexes [morphine, 66,577 +/- 6,248 vs. control, 95,735 +/- 5,227 counts.min-1 (cpm).mg protein-1; P < 0.02] by MC. Morphine (10(-4)-10(-6) M) also inhibited (P < 0.01) uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate-labeled low-density lipoprotein by MC. MP pretreated with morphine (10(-6) M) also showed significantly (P < 0.02) lower uptake of IgG complexes (control 94,959 +/- 4,980 vs. morphine, 58,360 +/- 11,608 cpm/mg protein). Binding studies carried out at 4 degrees C on MC and MP indicated that morphine did not modulate surface binding of IgG complexes. Naloxone, a morphine antagonist, also produced a rather decreased (P < 0.05) uptake of IgG complexes by both MP and MC and did not inhibit the effect of morphine on the uptake of IgG complexes. In vivo studies indicated that morphine-treated rats had a higher (P < 0.05) accumulation of aggregated human IgG complexes (125I-labeled ahIgG) in their glomeruli when compared with untreated rats (control rats, 256,929 +/- 40,008 cpm/g protein vs. experimental rats, 398,317 + 51,512 cpm/g). Increased accumulation of 125I-ahIgG in the glomeruli from morphine-treated rats may either be related to increased delivery of 125I-ahIgG into the mesangium or be a result of decreased drainage of 125I-ahIgG from the mesangium.(ABSTRACT TRUNCATED AT 250 WORDS)
Mechanism of ischemia-enhanced aminoglycoside binding and uptake by proximal tubule cells
Authors: Molitoris B. A. , Meyer C. , Dahl R. , Geerdes A.
Published: December 2017

Preceding ischemia or concurrent hypotension is known to enhance aminoglycoside nephrotoxicity; however, the underlying mechanisms responsible have not been determined. To investigate the effect of preceding mild ischemia on cellular gentamicin handling, brush-border membrane vesicle binding and in vivo cellular gentamicin uptake were quantified using [3H]gentamicin as a tracer. Fifteen minutes of ischemia resulted in a marked increase in apical membrane gentamicin binding (2.8 +/- 0.4 vs. 4.9 +/- 0.8 nmol/mg protein, P < 0.01). This increase was associated with an increased number of binding sites (3.7 +/- 0.3 vs. 9.1 +/- 2.3 nmol/mg protein, P < 0.01) and a reduced binding affinity (11.8 +/- 2.2 vs. 27.7 +/- 10.4 microM, P < 0.01). This increase in gentamicin binding was accompanied by alterations in apical membrane phospholipids including a doubling of phosphatidylinositol (PI) levels (13.8 +/- 0.4 vs. 27.5 +/- 3.1 nmol/mg protein, P < 0.01). Furthermore, treatment of apical membrane vesicles with PI-specific phospholipase C markedly reduced the difference in gentamicin binding between paired control and ischemic membrane fractions. Increased gentamicin binding was associated with increased in vivo uptake of gentamicin by S1/S2 and S3 cells. Outer cortical uptake of gentamicin increased from 2.18 +/- 0.39 to 2.68 +/- 0.27 nmol/mg protein (P < 0.01) after 15 min of ischemia and 4 h of reperfusion. Juxtamedullary uptake also increased from 1.39 +/- 0.31 to 1.75 +/- 0.12 nmol/mg protein (P < 0.01). Immunocytochemical techniques, utilizing immunogold labeling, showed gentamicin was taken up via the receptor-mediated endocytic pathway by S1/S2 and S3 cells. After ischemic injury gentamicin was localized in abnormal intracellular accumulations in S3 but not S1 or S2 cells. Taken together, these data indicate ischemia results in a marked increase in apical gentamicin binding due to increases in apical PI content. This is associated with increased internalization by S1/S2 and S3 cells and abnormal intracellular compartmentalization of gentamicin within S3 cells.
Alpha 1-VIII collagen is expressed in the rat glomerulus and in resident glomerular cells
Authors: Rosenblum N. D. , Briscoe D. M. , Karnovsky M. J. , Olsen B. R.
Published: December 2017

Current knowledge regarding the molecular composition of extracellular matrices in the glomerulus does not explain how these components interact to form stable three-dimensional structures. The recent recognition that short-chain collagens such as type VIII collagen function as molecular bridges in some nonrenal tissues has raised the possibility that such molecules may serve a similar function in the glomerulus. We have recently shown that cultured rat mesangial cells synthesize and secrete several short-chain collagenous proteins, one of which has properties similar to alpha 1-VIII collagen. In the present study we have isolated a rat mesangial cell alpha 1-VIII collagen cDNA clone, the sequence of which is 81% homologous to mouse alpha 1-VIII collagen. We used this cDNA to determine that alpha 1-VIII collagen mRNA is expressed in rat renal cortex and in cultured glomerular mesangial, epithelial, and endothelial cells. Additionally, we demonstrated that alpha 1-VIII collagen is secreted by cultured mesangial cells as an 80-kDa translation product. By immunocytochemistry, alpha 1-VIII collagen localized to the media of large intrarenal arteries and to the capillary loops and the mesangium of normal rat kidney. These results indicate that type VIII collagen is a normal constituent of the rat glomerulus as well as large intrarenal arteries. We speculate that type VIII collagen may function in part to determine the three-dimensional organization of the subendothelial and mesangial matrices.
Acute and early effects of aldosterone on Na-K-ATPase activity in Madin-Darby canine kidney epithelial cells
Authors: Shahedi M. , Laborde K. , Bussieres L. , Sachs C.
Published: December 2017

The time course and mechanism of early effects of aldosterone on renal Na-K-adenosinetriphosphatase (Na-K-ATPase) activity and number of units were studied in MDCK cells. Aldosterone induced a time- and dose-dependent stimulation of Na-K-ATPase activity. The stimulatory effect of aldosterone on activity and number of pump units increased progressively and was inhibited by spironolactone. In presence of cycloheximide, the stimulatory effect of aldosterone on activity and number of catalytic sites persisted to the same extent until 30 min and decreased by 20% after 60 min. In these cells, dimethylamiloride addition during preincubation abolished the aldosterone-induced stimulation in Na-K-ATPase activity up to 60 min. In contrast, furosemide addition did not alter the effect of aldosterone on Na-K-ATPase activity. The present study demonstrates an early effect of aldosterone on Na-K-ATPase activity that can be separated into the following two successive periods: 1) increase in pump number due to insertion of presynthetized units secondary to Na entry through an amiloride-sensitive apical pathway; and 2) an increase in pump number by de novo protein synthesis.
cAMP-independent, G protein-linked inhibition of Na+/H+ exchange in renal brush border by D1 dopamine agonists
Authors: Felder C. C. , Albrecht F. E. , Campbell T. , Eisner G. M. , Jose P. A.
Published: December 2017

When D1 dopamine agonists are incubated with renal cortical tissue, Na+/H+ exchange activity is inhibited, presumably due to D1 receptor-mediated stimulation of adenylyl cyclase and subsequent increase in protein kinase A activity. Although the role of adenosine 3',5'-cyclic monophosphate (cAMP) and cAMP-dependent protein kinase in the regulation of Na+/H+ exchange activity is well established, receptors functionally coupled to adenylyl cyclase can regulate Na+/H+ exchange activity independently of changes of cAMP accumulation. The current studies were designed to determine whether D1 agonists can inhibit Na+/H+ exchange activity independently of changes of cAMP accumulation and also to determine the role of G proteins in this process. The D1 agonist, fenoldopam, inhibited Na+/H+ exchange activity in a time-related and concentration-dependent manner. The 50% inhibitory concentration was 5-34 microM. Occupation of the renal D1 receptor mediates this action, since the D1 antagonist, SKF 83742, partially blocks the effect. This action, however, was independent of adenylyl cyclase, protein kinase A, and protein kinase C activity. Inhibition of adenylyl cyclase with dideoxyadenosine or inhibition of protein kinase A and C with the isoquinolines N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (H-4) and 1-(5-isoquinolinesfulfonyl)-2-methylpiperazine (H-7) did not block the effect of fenoldopam on the exchanger. The action of fenoldopam is not due to an amiloride-like action on the exchanger, because kinetic analysis of the inhibitory action was noncompetitive and the effect of fenoldopam was time dependent. The process involved G proteins, since guanosine 5'-O-(2-thiodiphosphate) prevented while guanosine 5'-O-(3-thiotriphosphate) increased the inhibitory effect of fenoldopam.
Arginine synthesis in mouse and rabbit nephron: localization and functional significance
Authors: Levillain O. , Hus-Citharel A. , Morel F. , Bankir L.
Published: December 2017

In the rat kidney, arginine (Arg) synthesis is restricted to the proximal tubule with a decreasing intensity from its convoluted (PCT) to its straight part (PST). The present study was designed to investigate the pattern of Arg synthesis along the nephron in other mammals, the mouse and rabbit. Microdissected representative nephron segments were incubated with 0.1 mM L-[ureido-14C]citrulline in a sealed chamber. Addition of arginase and urease to the incubation medium led to the hydrolysis of Arg into ornithine, NH3, and 14CO2. The latter was trapped in KOH and counted (results are in fmol Arg.min-1.mm tubular length-1). As in the rat, the main site of Arg synthesis in both species was found to be the PCT (mouse, 191; and rabbit, 57). A lower production was observed in rabbit and mouse PST and in rabbit distal segments. Along the PCT (from 1st to 4th mm after the glomerulus), a steep decrease is observed in mouse (595 and 37, respectively) but not in rabbit (57 and 23). The fate of the newly synthesized Arg probably depends on its site of production. Intracellular arginase activity is known to be present in the cortical (C) and medullary (OS) PST, in both mouse and rabbit. In rabbit only, arginase activity is also found in the PCT. We observed that a large part of Arg was further hydrolyzed into urea and ornithine in CPST and OSPST of mouse (66 and 80%, respectively) and rabbit (40 and 70%) but not in rabbit PCT (8%). Thus Arg produced by PCT in both species is probably released in the cortical blood, whereas Arg produced in PST may serve locally to produce urea and ornithine, and the latter could be used for polyamine synthesis.
Human glomeruli express TIMP-1 mRNA and TIMP-2 protein and mRNA
Authors: Carome M. A. , Striker L. J. , Peten E. P. , Moore J. , Yang C. W. , Stetler-Stevenson W. G. , Striker G. E.
Published: December 2017

Alterations in the balance between synthesis and degradation of extracellular matrix may result in glomerulosclerosis. The interaction between metalloproteinases and their inhibitors presumably modulates the rate of glomerular matrix degradation. We examined the gene expression of tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 in human glomeruli and TIMP-2 protein in tissue sections. Kidney tissue was obtained from adults undergoing nephrectomy for renal tumor (n = 9) or biopsy for nephrosis and renal failure (n = 1). Glomeruli were microdissected and subjected to reverse transcription. TIMP cDNAs were quantitated by competitive polymerase chain reaction assays. Five nephrectomy specimens had normal glomeruli and four had diffuse glomerulosclerosis. TIMP-1 and TIMP-2 cDNA levels, detected in glomeruli from all patients, were increased fourfold and threefold, respectively, in patients with glomerulosclerosis. The elevated TIMP cDNA levels could not be attributed to an increased number of glomerular cells. TIMP-2 protein was detected within normal and sclerotic glomeruli. In conclusion, both TIMP genes were expressed in normal glomeruli, and their level of expression was increased in glomerulosclerosis associated with renal carcinoma, suggesting that expression of these inhibitors may correlate with the development of sclerosis.
Metanephric transforming growth factor-beta 1 regulates nephrogenesis in vitro
Authors: Rogers S. A. , Ryan G. , Purchio A. F. , Hammerman M. R.
Published: December 2017

Development of the metanephric kidney during embryogenesis is regulated by a number of polypeptide growth factors of renal origin. We have defined previously a role for insulin-like growth factors (IGF) I and II and for transforming growth factor (TGF)-alpha. To delineate the effect of TGF-beta 1, on renal organogenesis, we cultured metanephroi surgically dissected from 13-day-old rat embryos in serum-free chemically defined media. TGF-beta 1 mRNA was present in kidneys from 13-day-old rat embryos, and positive immunostaining for TGF-beta 1 could be demonstrated in cultured metanephroi. However, TGF-beta bioactivity could not be detected in media obtained from the metanephroi. Addition of 10(-9) M TGF-beta 1 to cultures inhibited tubulogenesis, but had no effect on synthesis of IGF-I or -II. Addition of anti-TGF-beta 1 antibodies to cultures accelerated tubulogenesis within the metanephric blastema. These findings establish the potential for TGF-beta 1 production within the rat metanephros during development in vivo. It is possible that this peptide exerts a negative control on the process of tubulogenesis within metanephric blastema and in this manner acts to shape the architecture of mature kidney.
The growth hormone insulin-like growth factor axis in kidney revisited
Authors: Hammerman M. R. , Miller S. B.
Published: December 2017

Studies characterizing actions of growth hormone (GH) and insulin-like growth factors (IGF) in kidneys of adult and developing animals and humans have provided a good deal of insight into the functions of these peptides. Although certain of the actions may be mediated directly by GH, most appear to result from effects of GH to increase levels of circulating IGF or IGF produced in kidney. In addition to GH, epidermal growth factor (EGF) enhances the renal synthesis of IGF-I. Enhancement of renal IGF-I expression is GH independent in compensatory hypertrophy. Stimulation of kidney IGF-I production also occurs in diabetes mellitus. Renal IGF-I production is elevated in these settings in the absence of changes in circulating IGF-I, consistent with a causative role of renal IGF-I for the accompanying increased glomerular filtration rate and kidney growth. Actions of IGF in kidney are initiated following binding of peptides to specific receptors. Receptor number may be altered during compensatory growth and in diabetes mellitus. In addition to IGF, several IGF binding proteins (IGFBP) are produced in kidney and are likely to both inhibit and enhance the actions of IGF in different circumstances through sequestration of peptides and regulation of peptide interactions with their receptors. Administration of IGF-I to rats following acute ischemic injury hastens the recovery of normal renal function and accelerates the regeneration of the damaged proximal tubular epithelium. IGF-I increases the glomerular filtration rate in humans with normal and reduced functional kidney mass. These findings establish the potential for use of this peptide as a therapeutic agent in the settings of acute and chronic renal failure.
Source of kidney determines blood pressure in young renal transplanted rats
Authors: Kopf D. , Waldherr R. , Rettig R.
Published: December 2017

Recipients of a renal graft from adult stroke-prone spontaneously hypertensive rat (SHRSP) but not normotensive Wistar-Kyoto rat (WKY) donors have been shown to develop posttransplantation hypertension. To investigate whether hypertension would also travel with the kidneys from young prehypertensive SHRSP and whether the age-related increase in blood pressure in F1 hybrids (F1H) bred from SHRSP and WKY parents was attenuated by early bilateral nephrectomy and transplantation of a WKY kidney, we transplanted kidneys from 14 male SHRSP and 16 WKY donors aged 35-42 days to young male F1H. Recipients had both native kidneys removed. Fifteen unilaterally nephrectomized nontransplanted F1H served as controls. At the time when kidneys were harvested for transplantation, systolic blood pressures in young SHRSP and WKY donors were not significantly different (114 +/- 5 vs. 113 +/- 3 mmHg). After transplantation, recipients of an SHRSP kidney rapidly developed posttransplantation hypertension with systolic blood pressures above 180 mmHg at 3 mo after transplantation. In contrast, systolic blood pressure in recipients of a WKY kidney rose only slightly with increasing age and remained significantly lower than in non-transplanted controls. Transplanted rats exhibited normal weight gain, and renal function (glomerular filtration rate, renal blood flow) in grafted kidneys was well preserved; plasma renin activity was reduced compared with nontransplanted controls. These data demonstrate that 1) hypertension can be transmitted to normotensive recipients by transplantation of a kidney from young prehypertensive SHRSP donors and 2) the age-related increase in arterial pressure in F1H is attenuated by early bilateral nephrectomy and transplantation of a WKY kidney.(ABSTRACT TRUNCATED AT 250 WORDS)
Direct visualization of renin-cell distribution in preglomerular vascular trees dissected from rat kidney
Authors: Casellas D. , Dupont M. , Kaskel F. J. , Inagami T. , Moore L. C.
Published: December 2017

Three methods to visualize directly the distribution of granulated renin-positive cells in vascular trees microdissected from rat kidney were developed. Kidneys were removed from anesthesized rats, hemisectioned, macerated in HCl, and soaked in distilled water for 24-48 h. Cortical preglomerular vascular trees consisting of arcuate and cortical radial arteries and afferent arterioles were microdissected with the aid of a stereomicroscope. Granulated cells can be visualized in three ways. First, under transmitted or incident light observation, granulated cells are readily distinguished from the surrounding smooth muscle cells, because of marked differences in the refractive properties of these two cell types. Second, quinacrine, a fluorescent, intravital stain selective for dense-core granules, can be administered (2 mg/kg iv) to the rat 1 h before nephrectomy. When illuminated with 440-nm light, granulated cells fluorescence strongly at 510 nm. Third, specific immunostaining for renin can be obtained with a polyclonal anti-rat renin antibody and avidin-biotin immunoperoxidase staining in vascular trees subjected to cell permeabilization with Triton. These new techniques permit the direct visualization of the distribution of granulated renin-positive cells in preglomerular vessels under conditions in which the vascular architecture is largely preserved.
Osmotically sensitive renin release from permeabilized juxtaglomerular cells
Authors: Jensen B. L. , Skott O.
Published: December 2017

Renin secretion from juxtaglomerular (JG) cells is sensitive to external osmolality in a way that has been suggested to depend either on cellular volume or on effects on secretory granules. To distinguish between these possibilities, a technique for permeabilization of JG cell membranes was developed. Rat glomeruli with attached JG cells were isolated and permeabilized by 20 microM digitonin for 12 min and followed by continuous exposure to 2 microM digitonin. Experiments on proximal tubules showed that cellular volume was unaffected by changes in external sucrose concentration after a similar permeabilization procedure. With permeabilized JG cells the following changes in osmolality were tested (in mM sucrose): +90 (n = 6), +60 (n = 5), +30 (n = 6), +15 (n = 6), -15 (n = 5), -30 (n = 6), -60 (n = 6), and -90 (n = 6). With nonpermeabilized cells similar experiments were done with changes of +90 (n = 7), +30 (n = 4), -30 (n = 4), and -90 (n = 6) mM sucrose. Increases in osmolality caused inhibition of renin release, whereas decreases stimulated secretion. Within +/- 10% variations in osmolality there were no differences between the responses in permeabilized and intact cells, whereas the responses with larger changes were less in the permeabilized cells. Increase or decrease in urea concentration by 30 mM did not affect renin release. Thus water fluxes can influence renin release by a mechanism that is independent of cell volume.
Sch-28080 inhibits bafilomycin-sensitive H+ secretion in turtle bladder independently of luminal [K+]
Authors: Kohn O. F. , Mitchell P. P. , Steinmetz P. R.
Published: December 2017

To explore the possible contribution of an H-K-adenosine-triphosphatase (H-K-ATPase) to H+ secretion (JH) in the isolated turtle bladder, we measured electrogenic JH (JeH) as short-circuit current and total JH (JTH) by pH stat titration in the presence of ouabain at different ambient K+ concentration ([K+]) and during luminal addition of a known gastric H-K-ATPase inhibitor, Schering (Sch)-28080. JH was not reduced by decreasing ambient [K+] to undetectable or very low levels (< 0.05 mM by atomic absorption) and luminal BaCl2 addition to further reduce local [K+] at the apical membrane. These K(+)-removal studies indicate that H+ transport is not coupled to countertransport of K+. JTH did not exceed JeH at any point: in K(+)-free solutions JTH was 0.73 +/- 0.05, and JeH was 0.95 +/- 0.08 mumol/h; in standard (3.5 mM) K+ solutions JTH was 0.72 +/- 0.05 and JeH 0.98 +/- 0.06 mumol/h; in high (118 mM) K+ solutions JTH was 0.65 +/- 0.07 and JeH 0.94 +/- 0.08 mumol/h. Sch-28080 caused a rapid inhibition of JH, with similar half-maximal inhibitory concentrations (IC50) in K(+)-free, standard [K+], and high [K+] solutions. Bafilomycin inhibited JeH and JTH with an IC50 of approximately 100 nM. The observed non-potassium-competitive inhibition of JH by Sch-28080 and the bafilomycin sensitivity distinguish the H-ATPase of the turtle bladder from the gastric H-K-ATPase. The rapidity of the inhibition by Sch-28080 suggests that it acts at an accessible luminal site of the ATPase.
Liberators of NO exert a dual effect on renin secretion from isolated mouse renal juxtaglomerular cells
Authors: Schricker K. , Kurtz A.
Published: December 2017

This study aimed to examine the role of nitric oxide (NO) in the regulation of renin secretion from renal juxtaglomerular (JG) cells. Using primary cultures of mouse renal JG cells, we found that sodium nitroprusside (SNP) and 3-morpholino-sydnonimin-hydrochloride (SIN-1), two structurally different liberators of NO, led to a transient inhibition during the first hour followed by a marked dose-dependent stimulation of renin secretion lasting for an additional 20 h. This stimulatory effect was blunted by methylene blue (50 microM) and was reversible within minutes after removal of the NO liberators. SNP and SIN-1 also stimulated guanylate cyclase activity in the cultures with a maximum within the first hour of incubation. Increasing intracellular guanosine 3',5'-cyclic monophosphate levels by 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (100 microM) or by atrial natriuretic peptide (10 nM) decreased basal renin secretion but did not inhibit the effect of SNP. The stimulatory effect of SNP was not related to adenosine 3',5'-cyclic monophosphate levels in the JG cells and was blunted after chelation of extracellular calcium by 2 mM ethylene glycol-bis(beta-amino-ethyl ether)-N,N,N'N'-tetraacetic acid. Taken together, our findings suggest that liberators of NO have two effects on renin secretion from isolated JG cells: an inhibitory effect mediated by stimulation of soluble guanylate cyclase activity and a stimulatory effect mediated by an as yet unknown pathway that requires extracellular calcium.
Vasopressin- and cAMP-induced changes in ultrastructure of isolated perfused inner medullary collecting ducts
Authors: Nielsen S. , Muller J. , Knepper M. A.
Published: December 2017

Studies were performed to correlate arginine vasopressin (AVP)-induced changes in epithelial ultrastructure with changes in osmotic water permeability in isolated perfused rat terminal inner medullary collecting ducts (tIMCD). The tubules were perfused in three time periods, i.e., a 40-min basal period, a 40-min period with 0.1 nM AVP in the bath, and a 60-min withdrawal period. In each phase, the osmotic water permeability (Pf) was measured, and the perfused tubules were fixed for electron microscopy. AVP caused a four- to eightfold increase in Pf and induced several ultrastructural changes as follows: increased cell height of IMCD cells, expansion of the intercellular spaces, formation of large vacuoles, and increased coated pit density in the apical plasma membrane [from 0.6 +/- 0.2 (n = 6) to 2.9 +/- 0.3 (n = 7) pits/100 microns membrane length]. During AVP withdrawal, Pf decreased toward the basal value in association with partial reversal of the ultrastructural changes including a decrease in coated pit density to 1.0 +/- 0.2 (n = 4). Stimulation with 8-bromoadenosine 3',5'-cyclic monophosphate (8-bromo-cAMP) (0.1 mM) produced similar changes in Pf. Coated pit density increased to 2.1 +/- 0.4 (n = 4) after cAMP stimulation and after cAMP withdrawal decreased to 1.2 +/- 0.2 (n = 6). In contrast to stimulation with AVP, cAMP stimulation did not result in dilated intercellular spaces or formation of large vacuoles. The only ultrastructural feature that directly correlated with the water permeability was the density of coated pits in the apical membrane. Organelles involved in the endocytic pathway were studied with cationized ferritin or albumin-gold in the luminal perfusate. At the end of 40 min basal perfusion or AVP stimulation, luminal tracer was found almost exclusively in large multivesicular bodies (MVB). Tubules perfused with tracer during AVP withdrawal demonstrated rapid tracer accumulation in small vesicles and small MVB within 3-5 min, a time point corresponding to the rapid phase of Pf decrease. Later (30-60 min) the label was mainly confined to large MVB. Occasionally during AVP stimulation or withdrawal, small coated vesicles and smooth vesicles with coated extensions were noted to contain tracer. The data demonstrate AVP-mediated coated pit formation and cellular changes and show very rapid internalization of apical membrane after AVP withdrawal.
Osmolarity-stimulated urea transport in rat terminal IMCD: role of intracellular calcium
Authors: Gillin A. G. , Star R. A. , Sands J. M.
Published: December 2017

We showed previously that both increasing osmolality by adding NaCl or manitol (hyperosmolarity) or adding vasopressin can stimulate urea permeability in rat terminal inner medullary collecting ducts (IMCD). Vasopressin acts via adenosine 3',5'-cyclic monophosphate (cAMP), but the mechanism by which hyperosmolarity acts is unknown. To study the mechanism, we determined the effect of varying osmolality (with NaCl) on two potential second messenger systems, i.e., cAMP and intracellular calcium. There was no significant difference in cAMP production among tubules incubated at 290, 490, 690, or 890 mosmol/kg. In contrast, cAMP did increase significantly after vasopressin (10(-8) M) addition. Intracellular calcium increased significantly when osmolality was increased from 290 to 490 mosmol/kg in the absence of vasopressin. To examine whether changes in intracellular calcium affect urea permeability, we added thapsigargin (and removed bath calcium) while maintaining osmolality at 290 mosmol/kg. Both intracellular calcium and urea permeability increased significantly. Next, we buffered intracellular calcium by pretreatment with the acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, 50 microM). BAPTA completely blocked the increase in urea permeability occurring when osmolality was increased from 290 to 490 mosmol/kg, but did not block the increase in urea permeability occurring when vasopressin (10(-8) M) was added. In summary, 1) hyperosmolarity increases intracellular calcium, but has no effect on cAMP accumulation; 2) thapsigargin increases intracellular calcium and urea permeability; and 3) BAPTA blocks the hyperosmolarity-stimulated increase in urea permeability, but not vasopressin-stimulated urea permeability.(ABSTRACT TRUNCATED AT 250 WORDS)
Endothelial modulation of renal interlobar arteries from pregnant rats
Authors: Griggs K. C. , Conrad K. P. , Mackey K. , McLaughlin M. K.
Published: December 2017

The purpose of this study was to determine whether there are gestational effects on 1) the response of resistance arteries from the renal vasculature to phenylephrine and 2) the endothelial modulation of these arteries. Interlobar arteries (200-300 microns ID) were isolated from the kidneys of virgin and pregnant rats at 18-20 days of gestation (term, 22 +/- 1 days) and studied in a pressurized arteriograph system. Intact arteries from virgin and pregnant rats did not differ in sensitivity to phenylephrine. Arteries without endothelium from both groups were more sensitive to phenylephrine than arteries with endothelium. Sensitivity was increased 3.4-fold by endothelial removal in arteries from virgin rats and 1.5-fold in the pregnant group. Concentration-response relationships to phenylephrine were determined in arteries with endothelium and then repeated in the presence of 2.5 x 10(-4) M N omega-nitro-L-arginine (L-NNA), an inhibitor of nitric oxide synthase. All arteries were more sensitive to phenylephrine in the presence of L-NNA, with an average increase of 3.2-fold for the arteries from virgin rats and 1.6-fold from pregnant rats. These results indicate that the increased sensitivity to phenylephrine is primarily due to elimination of endothelium-derived relaxing factor (EDRF) and that basal EDRF activity is decreased during late gestation. To determine whether stimulated endothelium-dependent relaxation is enhanced in pregnancy, arteries with endothelium were constricted with phenylephrine to 50% of their maximum and relaxed to increasing concentrations of methacholine.(ABSTRACT TRUNCATED AT 250 WORDS)
Thyroid hormone stimulates the Na(+)-PO4 symporter but not the Na(+)-SO4 symporter in renal brush border
Authors: Beers K. W. , Dousa T. P.
Published: December 2017

In our previous studies we established that thyroid hormones [L-thyroxine (T4) or 3,5,3'-triiodothyronine (T3)] elicit an increase in Na(+)-Pi symport in rat and mouse renal brush-border membrane (BBM) vesicles (BBMV), but the Na(+)-coupled symports of other solutes were not influenced. However, a recent report [H. S. Tenenhouse, J. Lee, and N. Harvey. Am. J. Physiol. 261 (Renal Fluid Electrolyte Physiol. 30): F420-F426, 1991]claimed that T3 increases to a similar degree both Na(+)-Pi symport and Na(+)-SO4 symport in murine renal BBM. Adult male rats were fed either normal (0.7% Pi; NPD) or high-phosphate (1.4% Pi; HPD) diet and received T3 (0.2 mg/100 g body wt ip) for 3 days before the kidneys were removed, BBMV were prepared, and the transport rates were determined. Although the Na(+)-Pi symport significantly increased (delta = +35%) in both NPD and HPD rats treated with T3, the Na(+)-35SO4 symport and Na(+)-D-[3H]glucose symports were not influenced by T3. Furthermore, treatment of NPD-fed mice with T3 using a similar protocol as rats resulted in a significant increase (delta = +26%) of Na(+)-(Pi)4 symport, but did not alter Na(+)-SO4 symport or Na(+)-glucose symport. Our findings thus document that T3 regulates selectively the Na(+)-Pi symporter in BBM without having any effect on other major divalent anions, such as SO4.
Thyroid hormone stimulates Na(+)-K(+)-ATPase gene expression in cultured rat mesangial cells
Authors: Ohara T. , Ikeda U. , Muto S. , Oguchi A. , Tsuruya Y. , Yamamoto K. , Kawakami K. , Shimada K. , Asano Y.
Published: December 2017

Thyroid hormone 3,3',5-triiodothyronine (T3) is an important regulator of Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity in a variety of mammalian target tissues. In the present study, we examined the expression of Na(+)-K(+)-ATPase alpha- and beta-subunit mRNAs by T3 in cultured rat mesangial cells. Northern blot analysis of total RNA isolated from cultured rat mesangial cells revealed the expression of mRNAs encoding Na(+)-K(+)-ATPase alpha 1- and beta 1-subunits; mRNAs encoding alpha 2- and alpha 3-subunits were undetectable. Exposure of mesangial cells to T3 (10(-8) M) caused a threefold increase in the alpha 1-mRNA expression, which was first detected at 6 h and sustained for at least 48 h. The beta 1-mRNA expression was gradually increased by T3, with a maximum fourfold elevation at 48 h. The 50% effective concentration (EC50) for the alpha 1- and beta 1-mRNA induction by T3 was approximately 10(-10) M. The half-life of alpha 1-mRNA analyzed by actinomycin D chase was approximately 3 h and was not affected by T3. The augmented alpha 1-mRNA expression by T3 was associated with a 2.4-fold increase in the alpha 1-subunit protein accumulation and a 1.6-fold increase in Na(+)-K(+)-ATPase activity. These data suggest that thyroid hormone stimulates Na(+)-K(+)-ATPase gene expression, protein accumulation, and enzyme activity in rat mesangial cells.