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.

Effect of lumen pH on cell pH and cell potential in rabbit proximal tubules

1989 ◽  
Vol 256 (6) ◽  
pp. F1075-F1083 ◽  
Author(s):  
M. Kuwahara ◽  
K. Ishibashi ◽  
R. Krapf ◽  
F. C. Rector ◽  
C. A. Berry
Keyword(s):  
Cell Membrane ◽  
Apical Cell ◽  
Disulfonic Acid ◽  
Secondary Effects ◽  
Cell Potential ◽  
Apical Cell Membrane ◽  
Cell Ph ◽  
Luminal Ph ◽  
Convoluted Tubules ◽  
K Permeability

To determine the effect of luminal pH on cell pH and basolateral cell membrane potential difference (Vbl) of rabbit proximal convoluted tubules, Vbl was measured by conventional microelectrodes and intracellular pH was measured microfluorometrically. Lowering lumen pH acidified the cell and depolarized Vbl. Three factors contributed to depolarization of Vbl. Lowering lumen pH decreased apical cell membrane potassium permeability (PK) as indicated by the following: 1) at lumen pH 7.4 raising lumen [K] depolarized Vbl; 2) lowering lumen pH eliminated the depolarization of Vbl induced by increasing lumen [K]. An additional effect was suggested by the following: lumen Ba2+ blunted, but did not eliminate, the Vbl response to lowering lumen pH. An effect on basolateral K permeability (PK) via its effect on cell pH was suggested by the fact that lowering lumen pH dramatically reduced the depolarization induced by increasing bath [K]. Lowering lumen pH might influence Vbl by inhibiting H+-HCO3- transport. Addition of 1 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) to the bath solution hyperpolarized Vbl and enhanced the depolarization induced by lowering luminal pH. At luminal pH 6.0 SITS had no effect, suggesting elimination of H+ secretion. Addition of 1 mM luminal amiloride had no effect on Vbl or the response of Vbl to lowering luminal pH, but in the presence of amiloride SITS still hyperpolarized Vbl, suggesting amiloride-insensitive electrogenic H+ secretion. These results suggest that lumen pH-dependent depolarization of Vbl is due to 1) a decrease in apical PK; 2) cell acidification with secondary effects on basolateral PK; and 3) a decrease in apical electrogenic H+ transport.

scholarly journals Expression and Role of Parathyroid Hormone-Related Protein in Human Renal Proximal Tubule Cells during Recovery from ATP Depletion

1999 ◽  
Vol 10 (2) ◽  
pp. 238-244
Author(s):  
ADOLFO GARCÍA-OCAÑA ◽  
SUSAN C. GALBRAITH ◽  
SCOTT K. VAN WHY ◽  
KAI YANG ◽  
LINA GOLOVYAN ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Recovery Period ◽  
Ischemic Injury ◽  
Atp Depletion ◽  
Proximal Tubule Cell ◽  
Pthrp Expression ◽  
Human Proximal Tubule Cell

Abstract. Parathyroid hormone (PTH)-related protein (PTHrP) is widely expressed in normal fetal and adult tissues and regulates growth and differentiation in a number of organ systems. Although various renal cell types produce PTHrP, and PTHrP expression in rat proximal renal tubules is upregulated in response to ischemic injury in vivo, the role of PTHrP in the kidney is unknown. To study the effects of injury on PTHrP expression and its consequences in more detail, the immortalized human proximal tubule cell line HK-2 was used in an in vitro model of ATP depletion to mimic in vivo renal ischemic injury. These cells secrete PTHrP into conditioned medium and express the type I PTH/PTHrP receptor. Treatment of confluent HK-2 cells for 2 h with substrate-free, glucose-free medium containing the mitochondrial inhibitor antimycin A (1 μM) resulted in 75% depletion of cellular ATP. After an additional 2 h in glucose-containing medium, cellular ATP levels recovered to approximately 75% of baseline levels. PTHrP mRNA levels, as measured in RNase protection assays, peaked at 2 h into the recovery period (at four times baseline expression). The increase in PTHrP mRNA expression was correlated with an increase in PTHrP protein content in HK-2 cells at 2 to 6 h into the recovery period. Heat shock protein-70 mRNA expression was not detectable under baseline conditions but likewise peaked at 2 h into the recovery period. Treatment of HK-2 cells during the recovery period after injury with an anti-PTHrP(1-36) antibody (at a dilution of 1:250) resulted in significant reductions in cell number and uptake of [3H]thymidine, compared with nonimmune serum at the same titer. Similar results were observed in uninjured HK-2 cells. It is concluded that this in vitro model of ATP depletion in a human proximal tubule cell line reproduces the pattern of gene expression previously observed in vivo in rat kidney after ischemic injury and that PTHrP plays a mitogenic role in the proliferative response after energy depletion.

A reinvestigation of the function of the mammalian urinary bladder

1977 ◽  
Vol 232 (3) ◽  
pp. F187-F195 ◽  
Author(s):  
S. A. Lewis
Keyword(s):  
Urinary Bladder ◽  
Membrane Surface ◽  
Apical Cell ◽  
Membrane Damage ◽  
In Vitro Experiments ◽  
Apical Cell Membrane ◽  
Cell Membrane Damage ◽  
Using Data

The function of adult mammalian urinary bladder is evaluated in light of recent in vitro experiments. The discrepancy between in vivo and in vitro experimental results is examined and a possible solution proposed. Techniques for eliminating edge damage and measuring apical membrane surface area are described. A new chamber design for microelectrode studies is illustrated. The possibility of apical cell membrane damage caused by microelectrodes is critically examined and tested using the polyene antibiotic Nystatin. Using data from transepithelial and microelectrode experiments, a model for net Na+ transport across the bladder is proposed and then critically analyzed. The possible clinical implications of the in vitro experiments are briefly discussed.

scholarly journals Differential kidney proximal tubule cell responses to protein overload by albumin and its ligands

2020 ◽  
Vol 318 (3) ◽  
pp. F851-F859 ◽  
Author(s):  
Kimberly R. Long ◽  
Youssef Rbaibi ◽  
Megan L. Gliozzi ◽  
Qidong Ren ◽  
Ora A. Weisz
Keyword(s):  
Proximal Tubule ◽  
Cell Function ◽  
Degradation Kinetics ◽  
Cellular Responses ◽  
Endocytic Pathway ◽  
Proximal Tubule Cell ◽  
Albumin Production ◽  
High Concentrations ◽  
Well Differentiated

Albuminuria is frequently associated with proximal tubule (PT) cytotoxicity that can feed back to cause glomerular damage and exacerbate kidney disease. PT cells express megalin and cubilin receptors that bind to and internalize albumin over a broad concentration range. How the exposure to high concentrations of albumin leads to PT cytotoxicity remains unclear. Fatty acids and other ligands bound to albumin are known to trigger production of reactive oxygen species (ROS) that impair PT function. Alternatively or in addition, uptake of high concentrations of albumin may overload the endocytic pathway and elicit downstream responses. Here, we used a well-differentiated PT cell culture model with high endocytic capacity to dissect the effects of albumin versus its ligands on endocytic uptake and degradation of albumin, production of ROS, and cell viability. Cellular responses differed dramatically, depending on the preparation of albumin tested. Knockdown of megalin or cubilin failed to prevent ROS production mediated by albumin ligands, suggesting that receptor-mediated internalization of albumin was not necessary to trigger cellular responses to albumin ligands. Moreover, albumin induced cytotoxic responses when added to the basolateral surface of PT cells. Whereas overnight incubation with high concentrations of fatty acid-free albumin had no overt effects on cell function or viability, lysosomal degradation kinetics were slowed upon longer exposure, consistent with overload of the PT endocytic/degradative pathway. Together, the results of our study demonstrate that the PT responds independently to albumin and to its ligands and suggest that the consequences of albumin overload in vivo may be dependent on metabolic state.

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.

Evidence for PAH extraction from superficial cortical efferent vessel plasma

1983 ◽  
Vol 245 (5) ◽  
pp. F577-F583
Author(s):  
S. W. Weinstein ◽  
R. Klose ◽  
A. M. Kumar
Keyword(s):  
Proximal Tubule ◽  
Tubular Secretion ◽  
Proximal Convoluted Tubule ◽  
Peritubular Capillary ◽  
Blood Samples ◽  
Rapid Extraction ◽  
Peritubular Capillaries ◽  
Pars Recta ◽  
Proximal Tubular

Consistent with its anatomical association with the proximal tubule we have previously shown that superficial cortical efferent vessel blood contains an admixture of early and late proximal tubular reabsorbate. Since tubular secretion of p-aminohippurate (PAH) occurs predominantly in the late proximal tubule, extraction of this compound should occur preferentially from efferent vessel blood. As a result, the midportion of the proximal convoluted tubule supplied by the more downstream peritubular capillaries would receive blood containing a disproportionately reduced concentration of PAH. To study this, proximal and distal tubular fluid and efferent vessel blood samples were collected from rats. The data confirm that preferential secretion of PAH occurs in the pars recta and demonstrate that PAH is extracted from efferent vessel plasma by the pars recta. This in turn preferentially reduces PAH concentration in early postglomerular blood before it reaches the peritubular capillary network. We speculate that PAH and similar substances secreted by the pars recta are short-circuited by rapid extraction from early postglomerular blood, reducing their delivery to the mid-proximal convoluted tubule. Such circumstances must be considered in any analysis of organic compound secretion by the in vivo proximal tubule.

In Vitro to In Vivo Concordance of Toxicity Using the Human Proximal Tubule Cell Line HK-2

2020 ◽  
Vol 39 (5) ◽  
pp. 452-464
Author(s):  
Miriam E. Mossoba ◽  
Robert L. Sprando
Keyword(s):  
Proximal Tubule ◽  
Cell Line ◽  
Cell Injury ◽  
Culture Media ◽  
Sugar Transport ◽  
Molecular Characteristics ◽  
Proximal Tubule Cell ◽  
Tubule Cell

The renal proximal tubule cell line, human kidney 2 (HK-2), recapitulates many of the functional cellular and molecular characteristics of differentiated primary proximal tubule cells. These features include anchorage dependence, gluconeogenesis capability, and sodium-dependent sugar transport. In order to ascertain how well HK-2 cells can reliably reveal the toxicological profile of compounds having a potential to cause proximal tubule injury in vivo, we sought to evaluate the effects of known proximal tubule toxicants using the HK-2 cell line. We selected 20 pure nephrotoxic compounds that included chemotherapeutic drugs, antibiotics, and heavy metal-containing compounds and evaluated their ability to induce HK-2 cell injury relative to 10 innocuous pure compounds or cell culture media alone. We performed a comprehensive set of in vitro cellular toxicological assays to evaluate cell viability, oxidative stress, mitochondrial integrity, and a specific biomarker of renal injury, Kidney Injury Molecule 1. For each of our selected compounds, we were able to establish a reproducible profile of toxicological outcomes. We compared our results to those described in peer-reviewed publications to understand how well the HK-2 cellular model agrees with overall in vivo rat or human toxicological outcomes. This study begins to address the question of how well in vitro data generated with HK-2 cells can mirror in vivo animal and human outcomes.

Effect of metabolic acidosis on proximal tubular total CO2 absorption

1985 ◽  
Vol 249 (1) ◽  
pp. F62-F68 ◽  
Author(s):  
R. T. Kunau ◽  
J. I. Hart ◽  
K. A. Walker
Keyword(s):  
Metabolic Acidosis ◽  
Proximal Tubule ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Co2 Absorption ◽  
Arterial Blood ◽  
Blood Ph ◽  
In Series ◽  
Proximal Tubular

In vivo microperfusion studies of the proximal convoluted tubule of the rat were performed to determine the effect of metabolic acidosis on total CO2 (tCO2) absorption. In series I, tubular perfusion was performed in control and acidotic rats in a manner by which similar mean total CO2 concentrations in the proximal tubule were maintained. Comparable ranges of perfusion rate were studied in both groups. Following 3 days of HCl ingestion, plasma tCO2 was 20.0 +/- 0.9 mM in the acidotic rats whereas it was 29.6 +/- 0.53 mM in control rats. The arterial blood pH values were 7.25 +/- 0.02 vs. 7.43 +/- 0.01. Starting tCO2 perfusate concentrations were identical in both groups, 29.3 and 29.7 mM, as were the concentrations at the end of the perfused segments, 21.2 and 21.9 mM. The absorption of tCO2 (JtCO2, pmol X mm-1 X min-1) was significantly greater in the acidotic rats than in the controls, 576 +/- 39 vs. 256 +/- 21. At all perfusion rates studied, proximal tubular JtCO2 was higher in the acidotic than in the control rats. In series II, similar lengths of the late proximal tubule were perfused at the same rate in control and acidotic rats. Again, JtCO2 was higher in the acidotic rats, 352 +/- 19 vs. 198 +/- 13. The results indicate that at comparable luminal tCO2 concentration and tubular fluid flow rates, tCO2 absorption is significantly increased in the acidotic state. Although other mechanisms cannot be excluded, the finding of an increase in proximal tCO2 absorption in the acidotic rats is in agreement with the presence of an accelerated Na+/H+ exchange rate in brush border membrane vesicles obtained from the renal cortex of animals with metabolic acidosis.

Megalin binds and internalizes angiotensin II

2005 ◽  
Vol 288 (2) ◽  
pp. F420-F427 ◽  
Author(s):  
Romer Gonzalez-Villalobos ◽  
R. Bryan Klassen ◽  
Patricia L. Allen ◽  
L. G. Navar ◽  
Timothy G. Hammond
Keyword(s):  
Proximal Tubule ◽  
Membrane Vesicles ◽  
Peptide Inhibitors ◽  
Yolk Sac ◽  
Proximal Tubule Cell ◽  
Ang Ii ◽  
Glomerular Filtrate ◽  
Thyroid Colloid

Megalin is an abundant membrane protein heavily involved in receptor-mediated endocytosis. The major functions of megalin in vivo remain incompletely defined as megalin typically faces specialized milieus such as glomerular filtrate, airways, epididymal fluid, thyroid colloid, and yolk sac fluid, which lack many of its known ligands. In the course of studies on ANG II internalization, we were surprised when only part of the uptake of labeled ANG II into immortalized yolk sac cells (BN-16 cells) was blocked by specific peptide inhibitors and direct competitors of the angiotensin type 1 receptor. This led us to test if megalin was a receptor for ANG II. Four lines of direct evidence demonstrate that megalin and, to a lesser extent, its chaperone protein cubilin are receptors for ANG II. First, in BN-16 cells anti-megalin and anti-cubilin antisera interfere with ANG II uptake. Second, also in BN-16 cells, pure ANG II competes for uptake of a known megalin ligand. Third, in proximal tubule cell brush-border membrane vesicles extracted from mice, anti-megalin antisera interfere with ANG II binding. Fourth, purified megalin binds ANG II directly in surface plasmon resonance experiments. The finding that megalin is a receptor for ANG II suggests a major new function for the megalin pathway in vivo. These results also indicate that ANG II internalization in some tissues is megalin dependent and that megalin may play a role in regulating proximal tubule ANG II levels.

Sign in / Sign up

Export Citation Format

Share Document