Micropuncture study of proximal tubule pH in avian kidney

1987 ◽  
Vol 253 (4) ◽  
pp. R587-R591 ◽  
Author(s):  
G. Laverty ◽  
M. Alberici
Keyword(s):  
Proximal Tubule ◽  
Proximal Tubules ◽  
Ph Gradient ◽  
Chloride Diffusion ◽  
Ph Gradients ◽  
Single Lumen ◽  
Micropuncture Study ◽  
Gradient Based ◽  
Luminal Ph

Transepithelial potentials (PD) and pH gradients were measured by in vivo micropuncture in superficial proximal tubules of anesthetized European starlings. The average PD for 46 tubules was 2.24 +/- 1.17 mV (mean +/- SD), lumen negative. Only a single lumen-positive potential was recorded, even though late proximal segments are accessible to micropuncture. Proximal luminal pH, measured with single-barrel pH-sensitive microelectrodes, averaged 7.62 +/- 0.26. The pH of peritubular blood, also measured with microelectrodes, was 7.58 +/- 0.15. Correction of the luminal pH for the average PD resulted in a value of 7.58. A few measurements made with double-barrel microelectrodes indicated a pH in proximal tubules of 7.65 +/- 0.08 with a PD of -3.32 +/- 1.47 mV. Thus there appears to be no transepithelial pH gradient across these tubules. On the other hand, pH measurements of cortical collecting ducts averaged 6.40 +/- 0.37, indicating significant acidification in more distal segments. Starlings made acutely acidotic by HCl infusion had significantly reduced arterial and renal cortical pH values, but there was still no significant pH gradient across the proximal tubule. In conclusion, these superficial "reptilian-type" proximal tubules do not appear to have the capacity to maintain a pH gradient. Based on this and on the lack of lumen-positive chloride diffusion potentials, there appears to be no early proximal preferential bicarbonate reabsorption by these nephrons.

Megalin/gp330 mediates uptake of albumin in renal proximal tubule

1996 ◽  
Vol 271 (4) ◽  
pp. F900-F907 ◽  
Author(s):  
S. Cui ◽  
P. J. Verroust ◽  
S. K. Moestrup ◽  
E. I. Christensen
Keyword(s):  
Bovine Serum Albumin ◽  
Ligand Binding ◽  
Serum Albumin ◽  
Proximal Tubule ◽  
Proximal Tubules ◽  
Rat Serum ◽  
Gold Particles ◽  
Receptor Associated Protein ◽  
Rat Serum Albumin

Serum albumin filtered in renal glomeruli is reabsorbed very efficiently in the proximal tubule by endocytosis. The present study was undertaken to determine whether megalin/gp330 binds and mediates endocytosis of albumin. Rat serum albumin (RSA) labeled with 125I and colloidal gold particles labeled with bovine serum albumin (BSA) were microinfused into rat surface proximal tubules in vivo, and tubular uptake was determined in the presence or absence of different substances known to interfere with ligand binding to megalin. Binding of 125I-BSA and 125I-RSA to purified megalin was also determined directly using Sepharose columns. The results revealed that the tubular uptake of 125I-labeled RSA was significantly inhibited by receptor-associated protein (RAP), which reduced the uptake by > 50% and by cold RSA. The uptake of BSA gold by the proximal tubule was very intensive. BSA gold was found in small and large endocytic vacuoles, dense apical tubules, and in lysosomes. The uptake was reduced by RAP to 17%, by EDTA to 19%, by BSA to 16%, by megalin to 35%, by cytochrome c to 49%, and, together with gentamicin, there was virtually no uptake. Megalin-Sepharose columns bound 125I-labeled BSA as well as 125I-RSA, the binding was inhibited by RAP and EDTA, and analysis of the eluate revealed the bound tracer to be albumin. In conclusion, the present study demonstrates that megalin is a mediator of albumin reabsorption in renal proximal tubules.

Cell and luminal pH in the proximal tubule of Necturus kidney

1984 ◽  
Vol 247 (6) ◽  
pp. F932-F938 ◽  
Author(s):  
G. Planelles ◽  
A. Kurkdjian ◽  
T. Anagnostopoulos
Keyword(s):  
Proximal Tubule ◽  
Apical Cell ◽  
Proximal Tubule Cell ◽  
Peritubular Capillary ◽  
Initial Portion ◽  
Apical Cell Membrane ◽  
Blood Ph ◽  
Cell Ph ◽  
Luminal Ph

Double-barreled, selective microelectrodes filled with liquid ion exchanger were used to determine proximal tubule cell pH (pHcell), luminal pH (pHlum), and peritubular capillary blood pH (pHbl.pt) in Necturus kidney in vivo. The average pHbl.pt of 16 animals was 7.64 +/- 0.3; pHcell was 7.36 +/- 0.02 (n = 50), and pHlum was 7.50 +/- 0.05 (n = 16). Because of the variability in pHbl.pt from one animal to another, we studied the blood/cell/lumen pH differences. We sequentially measured with a single microelectrode pHcell and pHlum, and then pHbl.pt in an adjacent peritubular capillary. In 25 such paired determinations, the average pHbl.pt - pHcell difference was 0.28 +/- 0.03, cell acid, and the pHbl.pt - pHlum difference was 0.14 +/- 0.02, lumen acid. The pHcell in this series was significantly more acid than the pHlum (by 0.14 +/- 0.02), but in a few instances the pH gradient across the apical cell membrane was inversed. All measurements were performed in the initial portion of the proximal tubule. We conclude that 1) proximal cell pH is acid with regard to peritubular blood pH, 2) the proximal tubule of Necturus kidney is capable of establishing a small transepithelial pH difference (lumen acid), and 3) pHcell is generally more acid then pHlum.

Permeability properties of rat renal lysosomes

1994 ◽  
Vol 266 (1) ◽  
pp. C121-C133 ◽  
Author(s):  
A. I. Piqueras ◽  
M. Somers ◽  
T. G. Hammond ◽  
K. Strange ◽  
H. W. Harris ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Lipid Bilayer ◽  
Gradient Centrifugation ◽  
Water Flux ◽  
Water Channel ◽  
Water Channels ◽  
Water Proton ◽  
Ph Gradients ◽  
Osmotic Water

Although lysosomes maintain large pH gradients and may be subjected to significant osmotic gradients in vivo, little is known about their passive permeability properties. In recent studies, vacuolar H(+)-adenosine-triphosphatases (ATPases), such as those found in lysosomes, have been suggested to act as water channels. In addition, the erythrocyte and proximal tubule water channel CHIP28 is present on the plasma membrane of proximal tubule cells and may undergo endocytosis so that it is incorporated in lysosomes. We therefore examined water, proton, and small nonelectrolyte permeabilities in freshly purified lysosomes from rat renal proximal tubule. Lysosomes were purified by differential and Percoll gradient centrifugation. The preparation contained only lysosomes when examined by electron microscopy. Moreover, analysis by flow cytometry showed virtually all particles to be positive for acid phosphatase and cathepsin B activities. Permeabilities were measured on a stopped-flow fluorimeter by monitoring the self-quenching or pH-sensitive quenching of entrapped fluorescein derivatives. Osmotic water permeability (Pf) averaged 0.011 +/- 0.003 cm/s (n = 6), a value similar to that of biological membranes containing water channels. However, Pf was insensitive to the organic mercurial reagent p-chloromercuribenzene-sulfonate and to HgCl2 and exhibited an activation energy of 10.8 +/- 0.8 kcal/mol. These results indicate that water flux in lysosomes occurred via the lipid bilayer, and not via water channels. Addition of ATP led to lysosomal acidification (proton flux = 4.6 +/- 0.8 x 10(-11) mmol H+.s-1.cm-2), which was completely inhibited by 0.1 microM bafilomycin. Pf was insensitive to this agent as was the passive proton permeability (0.36 +/- 0.18 cm/s, n = 4). Permeabilities to small nonelectrolytes varied in proportion to the oil-water partition coefficient, confirming the applicability of Overton's rule to lysosomes. We conclude that proximal tubular lysosomes exhibit high Pf, which occurs via the lipid bilayer and not via vacuolar H(+)-ATPase.

Ammonia entry along rat proximal tubule in vivo: effects of luminal pH and flow rate

1987 ◽  
Vol 253 (4) ◽  
pp. F760-F766 ◽  
Author(s):  
E. E. Simon ◽  
L. L. Hamm
Keyword(s):  
Proximal Tubule ◽  
Flow Rate ◽  
Ammonia Concentration ◽  
Bicarbonate Concentration ◽  
Perfusion Rate ◽  
Total Ammonia ◽  
Luminal Ph ◽  
In Vivo Effects ◽  
Rat Proximal Tubule

The roles of luminal pH and flow rate in determining ammonia entry along the rat proximal tubule were examined using in vivo microperfusion. With perfusion rate constant at 15 nl/min, perfusate bicarbonate concentration was varied. Collected fluid ammonia concentration correlated with collected fluid bicarbonate concentration, consistent with nonionic diffusion (r = 0.726; P less than 0.001). Hence ammonia entry was dependent on luminal pH. With perfusate bicarbonate constant at 5 or 25 mM, perfusion rate was varied. In all groups, there was little change in collected fluid ammonia concentration with flow rate. Thus ammonia entry was also highly dependent on flow rate. With paired collections using a 25 mM bicarbonate perfusate, collected fluid bicarbonate was higher at a 30 nl/min perfusion rate than at 15 nl/min (16.8+/- 1.1 vs. 10.3+/- 1.1 mM), whereas total ammonia concentrations were similar (0.54+/- 0.1 and 0.55+/- 0.1). Thus the NH3 concentration was higher at 30 than at 15 nl/min (6.1+/- 1.2 vs. 3.4+/- 0.5 microM; P less than 0.025), a result not predicted by simple nonionic diffusion. Thus these studies demonstrate the importance of nonionic diffusion in determining ammonia entry along the proximal tubule. However, the results also demonstrate that flow rate importantly determines ammonia entry in vivo in a manner not predicted by simple nonionic diffusion of NH3. This augmentation of ammonia entry with increasing flow rate may involve flow-dependent alterations in ammonia synthesis or transport of NH+4.

Gluconeogenesis from glutamine and lactate in the isolated humanrenal proximal tubule: longitudinal heterogeneity and lack of response to adrenaline

2001 ◽  
Vol 360 (2) ◽  
pp. 371-377 ◽  
Author(s):  
Agnès CONJARD ◽  
Mireille MARTIN ◽  
Jérôme GUITTON ◽  
Gabriel BAVEREL ◽  
Bernard FERRIER
Keyword(s):  
Proximal Tubule ◽  
Human Kidney ◽  
Glucose Production ◽  
Proximal Tubules ◽  
Renal Gluconeogenesis ◽  
Adrenaline Infusion ◽  
Gluconeogenic Substrates ◽  
Stimulation Of

Recent studies in vivo have suggested that, in humans in the postabsorptive state, the kidneys contribute a significant fraction of systemic gluconeogenesis, and that the stimulation of renal gluconeogenesis may fully explain the increase in systemic gluconeogenesis during adrenaline infusion. Given the potential importance of human renal gluconeogenesis in various physiological and pathophysiological situations, we have conducted a study in vitro to further characterize this metabolic process and its regulation. For this, successive segments (S1, S2 and S3) of human proximal tubules were dissected and incubated with physiological concentrations of glutamine or lactate, two potential gluconeogenic substrates that are taken up by the human kidney in vivo, and glucose production was measured. The effects of adrenaline, noradrenaline and cAMP, a well established stimulator of gluconeogenesis in animal kidney tubules, were also studied in suspensions of human renal proximal tubules. The results indicate that the three successive segments have about the same capacity to synthesize glucose from glutamine; by contrast, the S2 and S3 segments synthesize more glucose from lactate than the S1 segment. In the S2 and S3 segments, lactate appears to be a better gluconeogenic precursor than glutamine. The addition of cAMP, but not of adrenaline or noradrenaline, led to the stimulation of gluconeogenesis from lactate and glutamine by human proximal tubules. These results indicate that, in the human kidney in vivo, lactate might be the main gluconeogenic precursor, and that the stimulation of renal gluconeogenesis observed in vivo upon adrenaline infusion may result from an indirect action on the renal proximal tubule.

In vivo proximal tubular fluid-to-plasma chloride concentration gradient in the rabbit

1982 ◽  
Vol 242 (6) ◽  
pp. F575-F579
Author(s):  
R. C. Vari ◽  
C. E. Ott
Keyword(s):  
Proximal Tubule ◽  
Concentration Gradient ◽  
Concentration Ratio ◽  
Mammalian Species ◽  
Chloride Concentration ◽  
Proximal Tubules ◽  
Plasma Chloride ◽  
Tubule Fluid

It has been reported that the concentration of chloride in the proximal tubule is greater than that in plasma in several mammalian species. Much of the theory concerning fluid and electrolyte reabsorption in the proximal tubule is based on data taken from in vitro isolated proximal tubules of the rabbit nephron. This study measured in vivo the rabbit proximal tubule fluid-to-ultrafiltrate chloride concentration ratio [(TF/UF)Cl] and its relationship to proximal tubule length as estimated by the tubule fluid-to-plasma inulin concentration ratio [(TF/P)In]. From six rabbits, 19 random proximal tubules were micropunctured and analyzed for inulin and chloride concentrations, the latter being measured by microelectrometric titration. Plasma ultrafiltrate was determined by correcting plasma chloride concentration for protein concentration. The average single nephron filtration rate was 20.2 +/- 0.8 nl/min. The (TF/UF)Cl ratio was 1.10 +/- 0.03, which was significantly different from unity. Furthermore, regression analysis yielded no significant correlation between the (TF/UF(Cl and (TF/P)In ratio. This study demonstrates that a tubule lumen-to-plasma chloride concentration gradient exists in the in vivo proximal tubule of the rabbit that is apparently established early and is not correlated with proximal tubule length.

scholarly journals Activation of dopamine D1-like receptors induces acute internalization of the renal Na+/phosphate cotransporter NaPi-IIa in mouse kidney and OK cells

2005 ◽  
Vol 288 (4) ◽  
pp. F740-F747 ◽  
Author(s):  
Desa Bacic ◽  
Paola Capuano ◽  
Michel Baum ◽  
Jianning Zhang ◽  
Gerti Stange ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Brush Border Membrane ◽  
Fluorescent Protein ◽  
Mouse Kidney ◽  
Proximal Tubules ◽  
Opossum Kidney ◽  
Kidney Slices ◽  
Opossum Kidney Cells ◽  
Green Fluorescent

The Na+/phosphate cotransporter NaPi-IIa (SLC34A1) is the major transporter mediating the reabsorption of Pi in the proximal tubule. Expression and activity of NaPi-IIa is regulated by several factors, including parathyroid hormone, dopamine, metabolic acidosis, and dietary Pi intake. Dopamine induces natriuresis and phosphaturia in vivo, and its actions on several Na+-transporting systems such as NHE3 and Na+-K+-ATPase have been investigated in detail. Using freshly isolated mouse kidney slices, perfused proximal tubules, and cultured renal epithelial cells, we examined the acute effects of dopamine on NaPi-IIa expression and localization. Incubation of isolated kidney slices with the selective D1-like receptor agonists fenoldopam (10 μM) and SKF-38393 (10 μM) for 1 h induced NaPi-IIa internalization and reduced expression of NaPi-IIa in the brush border membrane (BBM). The D2-like selective agonist quinpirole (1 μM) had no effect. The D1 and D2 agonists did not affect the renal Na+/sulfate cotransporter NaSi in the BBM of the proximal tubule. Studies with isolated perfused proximal tubules demonstrated that activation of luminal, but not basolateral, D1-like receptors caused NaPi-IIa internalization. In kidney slices, inhibition of PKC (1 μM chelerythrine) or ERK1/2 (20 μM PD-098089) pathways did not prevent the fenoldopam-induced internalization. Inhibition with the PKA blocker H-89 (10 μM) abolished the effect of fenoldopam. Immunoblot demonstrated a reduction of NaPi-IIa protein in BBMs from kidney slices treated with fenoldopam. Incubation of opossum kidney cells transfected with NaPi-IIa-green fluorescent protein chimera shifted fluorescence from the apical membrane to an intracellular pool. In summary, dopamine induces internalization of NaPi-IIa by activation of luminal D1-like receptors, an effect that is mediated by PKA.

scholarly journals An evaluation of the antioxidant protein α1-microglobulin as a renal tubular cytoprotectant

2016 ◽  
Vol 311 (3) ◽  
pp. F640-F651 ◽  
Author(s):  
Richard A. Zager ◽  
Ali C. M. Johnson ◽  
Kirsten Frostad
Keyword(s):  
Proximal Tubule ◽  
Proximal Tubules ◽  
Monocyte Chemoattractant Protein 1 ◽  
Monocyte Chemoattractant ◽  
Neutrophil Gelatinase Associated Lipocalin ◽  
Monocyte Chemoattractant Protein ◽  
Antioxidant Protein ◽  
Neutrophil Gelatinase

α1-Microglobulin (A1M) is a low-molecular-weight heme-binding antioxidant protein that is readily filtered by the glomerulus and reabsorbed by proximal tubules. Given these properties, recombinant A1M (rA1M) has been proposed as a renal antioxidant and therapeutic agent. However, little direct evidence to support this hypothesis exists. Hence, we have sought “proof of concept” in this regard. Cultured proximal tubule (HK-2) cells or isolated mouse proximal tubule segments were challenged with a variety of prooxidant insults: 1) hemin, 2) myoglobin; 3) “catalytic” iron, 4) H2O2/Fenton reagents, 5) a Ca2+ ionophore, 6) antimycin A, or 7) hypoxia (with or without rA1M treatment). HK-2 injury was gauged by the percent lactate dehydrogenase release and 4,5-(dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide uptake. In vivo protection was sought in rA1M-treated mice subjected to 1) graded myohemoglobinura (2, 4, 8, or 9 ml/kg glycerol injection), 2) purified myoglobinemia/uria, or 3) endotoxemia. In vivo injury was assessed by blood urea nitrogen, creatinine, and the expression of redox-sensitive genes (heme oxygenase-1, neutrophil gelatinase-associated lipocalin, and monocyte chemoattractant protein-1 mRNAs). Although rA1M totally blocked in vitro hemin toxicity, equimolar albumin (another heme binder) or 10% serum induced equal protection. rA1M failed to mitigate any nonhemin forms of either in vitro or in vivo injury. A1M appeared to be rapidly degraded within proximal tubules (by Western blot analysis). Surprisingly, rA1M exerted select injury-promoting effects (increased in vitro catalytic iron/antimycin toxicities and increased in vivo monocyte chemoattractant protein-1/neutrophil gelatinase-associated lipocalin mRNA expression after glycerol or endotoxin injection). We conclude that rA1M has questionable utility as a renal antioxidant/cytoprotective agent, particularly in the presence of larger amounts of competitive free heme (e.g., albumin) binders.

pH gradient-dependent increased Na+-H+ antiport capacity of the rabbit remnant kidney

1985 ◽  
Vol 249 (1) ◽  
pp. F90-F98 ◽  
Author(s):  
E. P. Nord ◽  
A. Hafezi ◽  
J. D. Kaunitz ◽  
W. Trizna ◽  
L. G. Fine
Keyword(s):  
Proximal Tubule ◽  
Membrane Vesicles ◽  
Limited Range ◽  
Ph Gradient ◽  
Equilibrium Conditions ◽  
Luminal Membrane ◽  
Experimental Protocols ◽  
Acid Base Status ◽  
Remnant Kidney

The adaptative response of the renal proximal tubule to a reduction of renal mass was studied in brush border membrane vesicles prepared from the solitary remnant kidney (RK) of subtotally nephrectomized rabbits. The in vivo acid-base status of RK and sham-operated controls (SK) was similar. In the absence of organic solutes, Na+ flux across the membrane demonstrated features of Na+-H+ antiport, i.e., stimulation by a transmembrane pH gradient and inhibition by amiloride. Kinetic parameters for Na+-H+ antiport were derived with different experimental protocols. In the presence of an opposing H+ gradient and over a limited range of Na+ concentration, JNamax was enhanced 65% in RK vesicles compared with SK vesicles and KtNa was unchanged. The enhanced JNamax was not apparent under H+ equilibrium conditions, Comparable values for JNamax and KtNa were obtained by studying RK vesicles at external Na+ concentrations of 0-200 mM and resolving uptake into a substrate component, representing Na+-H+ antiport, and a nonsaturable diffusive component. The apparent Na+ permeability (P'Na) of RK vesicles was identical to the P'Na of normal kidney vesicles, under both H+ gradient and H+ equilibrium conditions. H+ permeability, measured with acridine orange, was also the same in RK and SK vesicles. These studies demonstrate that in the remnant kidney model of chronic renal insufficiency there is an increase in the JNamax of the Na+-H+ antiporter in the luminal membrane of the proximal tubule that is revealed only under transmembrane H+ gradient conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Degradation and transport of AVP by proximal tubule

1987 ◽  
Vol 253 (6) ◽  
pp. F1120-F1128
Author(s):  
F. A. Carone ◽  
E. I. Christensen ◽  
G. Flouret
Keyword(s):  
Proximal Tubule ◽  
High Performance ◽  
Reduced Glutathione ◽  
Proximal Tubules ◽  
Electron Microscope Autoradiography ◽  
Lysosomal Fraction ◽  
Renal Brush Border Membranes ◽  
Renal Brush Border

High-performance liquid chromatography (HPLC) analysis revealed that [3,4,5-3H-Phe3,Arg8]vasopressin ([3H]AVP) was not degraded by isolated renal brush-border membranes or by a cortical lysosomal fraction in vitro; however, in the presence of 1 mM reduced glutathione, [3H]AVP was degraded by both preparations. Renal cortical homogenates in vitro and luminal peptidases of proximal tubule in vivo degraded [3H]AVP and in both instances yielded phenylalanine, hexapeptide AVP 1-6, heptapeptide AVP 1-7, octapeptide AVP 1-8, and two uncharacterized products X and Y. These data suggest that filtered AVP is reduced in the proximal tubule by a reduced glutathione-dependent transhydrogenase and subsequently cleaved to [3H]Phe by tubular aminopeptidases. Following microinfusion of [3H]AVP into proximal tubules, 15.7% of the label was absorbed. Five and fifteen minutes after infusion of [3H]AVP, sequestration of total label in proximal tubules was 4.5 and 2.1%, respectively, and quantitative electron microscope autoradiography revealed accumulation of grains over apical endocytic vacuoles and lysosomes consistent with endocytic uptake and rapid lysosomal degradation of AVP and/or a large metabolite. Thus, enzymatic cleavage of AVP by luminal and lysosomal peptidases in proximal tubules could involve disulfide bond, C-terminal, and N-terminal loci.

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