scholarly journals Potential and Limits of Kidney Cells for Evaluation of Renal Excretion

2021 ◽  
Vol 14 (9) ◽  
pp. 908
Author(s):  
Christian Lechner ◽  
Ursula Mönning ◽  
Andreas Reichel ◽  
Gert Fricker
Keyword(s):  
Proximal Tubule ◽  
Cell Lines ◽  
Renal Excretion ◽  
Expression Patterns ◽  
Transport Proteins ◽  
Primary Cells ◽  
Isolated Cells ◽  
Proximal Tubule Cells ◽  
Tubule Cells

A large number of therapeutic drugs, herbal components and their metabolites are excreted by the kidneys. Therefore, generally applied models for estimating renal excretion, including freshly isolated rat proximal tubule cells, cultured tubule cells and immortalized kidney cell lines MDCKII, NRK-52E, IHKE-1 and Caki-1, were investigated regarding their predictive potential for active renal transport. Cultured proximal tubule cells showed an epithelial cell-like morphology and formed tight monolayers. However, mRNA expression analyses and immunohistochemical studies revealed patterns of tight junction proteins that were notably different from freshly isolated cells and distinct from those in vivo. High levels of mannitol permeation were found in NRK-52E, IHKE-1 and Caki-1 cells, suggesting that they are not suitable for bidirectional transport studies. Cultured cells and freshly isolated cells also differed in proximal tubule markers and transport proteins, indicating that cultured primary cells were in a state of dedifferentiation. Cell lines MDCKII, NRK-52E, IHKE-1 and Caki-1 did not accurately reflect the characteristics of proximal tubules. The expression patterns of marker and transport proteins differed from freshly isolated primary cells. In summary, each of these models has profound disadvantages to consider when adopting them reliable models for the in vivo situation. Thus, they should not be used alone but only in combination.

Stretch- and volume-activated channels in isolated proximal tubule cells

1992 ◽  
Vol 262 (5) ◽  
pp. F857-F870 ◽  
Author(s):  
D. Filipovic ◽  
H. Sackin
Keyword(s):  
Membrane Potential ◽  
Proximal Tubule ◽  
Cell Volume ◽  
Regulatory Volume Decrease ◽  
Cell Swelling ◽  
Isolated Cells ◽  
Open Probability ◽  
Proximal Tubule Cells ◽  
Open Time ◽  
Tubule Cells

Apical and basolateral channels were studied in isolated proximal tubule cells of Necturus kidney. Many of these isolated cells maintained their polarity, with clearly delineated apical and basolateral regions. A 20-pS stretch-activated (SA) cation-selective channel was identified at the apical side of these cells. This channel was permeable to Ca, K, and Na but was not significantly gated by either membrane potential or cytosolic Ca. Negative pipette pressure (15 cmH2O) increased the open probability (Po) of this channel from 0.04 +/- 0.02 to 0.26 +/- 0.08 (n = 6). Two types of Ca-independent, mechanosensitive, K-selective (SAK) channels were identified at the basolateral surface of polarized proximal tubule cells, i.e., a 30-pS long-open time (50 +/- 7 ms) channel (n = 9), and a 46-pS short-open time (1.3 +/- 0.7 ms) channel (n = 10). Pipette suction (-12 cmH2O) increased the Po of the short-open time channels from 0.008 to 0.015 and increased the Po of the long-open time channel from 0.03 to 0.19. The effect of swelling was studied with isolated cells suspended at the tip of patch pipettes. A 50% dilution of the bath doubled cell volume, hyperpolarized the membrane potential by 11 +/- 0.7 mV, and increased the Po of the basolateral SAK channels. This was followed by a spontaneous regulatory volume decrease (RVD), repolarization of the membrane potential, and a decrease in Po. In contrast, isosmotic (bath side) replacement of an impermeant anion (methanesulfonate) with a permeant anion (Cl) doubled cell volume in 5 min but without a subsequent RVD. This sustained swelling hyperpolarized the cell potential by 5.5 +/- 0.7 mV (n = 16) and increased the Po of short-open time channel by a factor of 2.3 from 0.03 +/- 0.01 to 0.07 +/- 0.02 (n = 6). The increase in Po was primarily produced by a reduction in the interburst closed time, which decreased from 142 +/- 43 ms in K methanesulfonate to 36 +/- 11 ms in KCl solutions. These results are consistent with the hypothesis that cell swelling activates Ca-independent K channels at the basolateral membrane of renal proximal tubule. Efflux of K through these channels may partially mediate renal cell volume regulation.

scholarly journals Antenatal betamethasone attenuates the angiotensin-(1–7)-Mas receptor-nitric oxide axis in isolated proximal tubule cells

2017 ◽  
Vol 312 (6) ◽  
pp. F1056-F1062 ◽  
Author(s):  
Yixin Su ◽  
Jianli Bi ◽  
Victor M. Pulgar ◽  
Mark C. Chappell ◽  
James C. Rose
Keyword(s):  
Nitric Oxide ◽  
Proximal Tubule ◽  
Cell Model ◽  
Primary Cultures ◽  
Specific Effect ◽  
Renal Tubules ◽  
Proximal Tubule Cells ◽  
Mas Receptor ◽  
Tubule Cells

We previously reported a sex-specific effect of antenatal treatment with betamethasone (Beta) on sodium (Na+) excretion in adult sheep whereby treated males but not females had an attenuated natriuretic response to angiotensin-(1–7) [Ang-(1–7)]. The present study determined the Na+ uptake and nitric oxide (NO) response to low-dose Ang-(1–7) (1 pM) in renal proximal tubule cells (RPTC) from adult male and female sheep antenatally exposed to Beta or vehicle. Data were expressed as percentage of basal uptake or area under the curve for Na+ or percentage of control for NO. Male Beta RPTC exhibited greater Na+ uptake than male vehicle cells (433 ± 28 vs. 330 ± 26%; P < 0.05); however, Beta exposure had no effect on Na+ uptake in the female cells (255 ± 16 vs. 255 ± 14%; P > 0.05). Ang-(1–7) significantly inhibited Na+ uptake in RPTC from vehicle male (214 ± 11%) and from both vehicle (190 ± 14%) and Beta (209 ± 11%) females but failed to attenuate Na+ uptake in Beta male cells. Beta exposure also abolished stimulation of NO by Ang-(1–7) in male but not female RPTC. Both the Na+ and NO responses to Ang-(1–7) were blocked by Mas receptor antagonist d-Ala7-Ang-(1–7). We conclude that the tubular Ang-(1–7)-Mas-NO pathway is attenuated in males and not females by antenatal Beta exposure. Moreover, since primary cultures of RPTC retain both the sex and Beta-induced phenotype of the adult kidney in vivo they appear to be an appropriate cell model to examine the effects of fetal programming on Na+ handling by the renal tubules.

scholarly journals MO331LINEAGE TRACING OF REGENERATING PROXIMAL TUBULE CELLS (STC) BY SINGLE CELL PROFILING IN ACUTE KIDNEY INJURY

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Eleni Stamellou ◽  
Mingbo Cheng ◽  
Viktor Sterzer ◽  
Katja Leuchtle ◽  
Thiago Strieder ◽  
...  
Keyword(s):  
Acute Kidney Injury ◽  
Proximal Tubule ◽  
Single Cell ◽  
Transgenic Mouse ◽  
Ischemia Reperfusion ◽  
Expression Patterns ◽  
Kidney Injury ◽  
Multiple Time ◽  
Proximal Tubule Cells ◽  
Tubule Cells

Abstract Background and Aims Acute tubular injury accounts for the most common intrinsic cause for acute kidney injury (AKI). The scattered tubular cell (STC) phenotype was discovered as a uniform reaction of tubule cells triggered by injury. Our group was the first to identify an inducible transgenic mouse (PEC-rtTA-mouse) specifically labeling STCs with eGFP. Analysis of the transcriptional factors and associated signaling pathways might reveal the function and role of STCs in AKI. Method Here, we performed single-cell RNA sequencing of unilateral ischemia-reperfusion murine model of AKI 8, 24, 48 hours and 6 and 12 days after AKI induction. Results Genes expressing proximal tubular proteins and transporters were markedly downregulated during transition into the STC phenotype upon injury; but expression recovered over time and upon resolution and tubular cells re-differentiated into proximal tubule cells. This provides evidence for the first time that the STC phenotype is a transient and reversible phenotype triggered by injury. Among cells in the STC phenotype, we could identify 2 sub-clusters; a highly proliferating sub-cluster that in the cell cycle analysis showed the highest proportion of cycling cells. The second eGFP-positive cluster appeared very early after AKI and expressed a distinct set of genes (defined by 7 anchor genes). Some of the highly up-regulated genes are known markers of STCs hence confirming the specificity of our transgenic mouse line. Conclusion Our study provides gene expression patterns specifically in STCs upon injury and repair at multiple time points and suggests that the STC phenotype is a transient and reversible phenotype triggered by injury.

Na+/H+ exchanger activity and phosphorylation in temperature-sensitive immortalized proximal tubule cell lines derived from the spontaneously hypertensive rat

2000 ◽  
Vol 98 (4) ◽  
pp. 409-418 ◽  
Author(s):  
Leong L. NG ◽  
Sonja JENNINGS ◽  
Joan E. DAVIES ◽  
Paulene A. QUINN
Keyword(s):  
Proximal Tubule ◽  
Cell Lines ◽  
Western Blotting ◽  
Spontaneously Hypertensive Rat ◽  
Proximal Tubule Cell ◽  
Permissive Temperature ◽  
Proximal Tubule Cells ◽  
Spontaneously Hypertensive ◽  
Hypertensive Rat ◽  
Tubule Cells

Freshly isolated proximal tubules from the spontaneously hypertensive rat (SHR) demonstrate elevated Na+/H+ exchanger (NHE) activity, but the underlying mechanism is unclear. Because of the difficulties in preparing sufficient numbers of proximal tubule cells for detailed biochemical studies, we have generated cell lines from SHR and Wistar–Kyoto rat (WKY) proximal tubule cells. Cell lines were obtained by transforming the cells with an origin-defective mutant of simian virus 40 encoding a heat-labile T antigen (tsA58 transformant). Such cells proliferate at the permissive temperature of 33 °C, but growth is abolished at the restrictive temperature of 39 °C. The predominant NHE isoform expressed was isoform 1, as determined by sensitivity to HOE-694 (3-methylsulphonyl-4-piperidinobenzoyl guanidine) and Western blotting using specific polyclonal antisera to NHE-1. NHE-3 protein was also present. Northern blots of poly(A) mRNA extracts of the cell lines revealed a low abundance of transcripts for NHE-2, -3 and -4, with no systematic difference between the lines. Although the intracellular pH was similar in the SHR and WKY lines, HOE-694-sensitive H+ efflux due to NHE-1 was substantially elevated in SHR lines compared with WKY lines (95.0±2.8 and 39.9±5.7 mmol·min-1·l-1 respectively; P < 0.001; n = 6). H+ efflux due to non-Na+-dependent mechanisms were similar in lines from the two strains. Western blotting revealed that NHE-1 density was also very similar in SHR and WKY lines, and subcellular fractionation of homogenates indicated that NHE-1 was localized predominantly to plasma membranes. Thus the turnover number of NHE-1 was increased. Immunoprecipitation of 32P-labelled phosphoproteins from these lines demonstrated an approximately 2-fold higher degree of phosphorylation of NHE-1 in SHR compared with WKY lines. These cell lines form a useful model for defining the biochemical mechanisms leading to the NHE-1 phenotype in the SHR kidney, in addition to investigations of other SHR phenotypic markers.

scholarly journals The role of Sgk-1 in the upregulation of transport proteins by PPAR-  agonists in human proximal tubule cells

2008 ◽  
Vol 24 (4) ◽  
pp. 1130-1141 ◽  
Author(s):  
S. Saad ◽  
D. J. Agapiou ◽  
X.-M. Chen ◽  
V. Stevens ◽  
C. A. Pollock
Keyword(s):  
Proximal Tubule ◽  
Transport Proteins ◽  
Proximal Tubule Cells ◽  
Ppar Agonists ◽  
Tubule Cells ◽  
Human Proximal Tubule

Improved culture conditions stimulate gluconeogenesis in primary cultures of renal proximal tubule cells

1995 ◽  
Vol 268 (4) ◽  
pp. C1053-C1061 ◽  
Author(s):  
G. Nowak ◽  
R. G. Schnellmann
Keyword(s):  
Proximal Tubule ◽  
Primary Cultures ◽  
Lactate Production ◽  
Glucose Production ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Lactate Consumption ◽  
Renal Proximal Tubule Cells

Unlike renal proximal tubule cells (RPTC) in vivo, RPTC cultured in standard conditions are hypoxic, glycolytic, and not gluconeogenic. This study investigated the effects of glucose and lactate on glycolysis and gluconeogenesis in rabbit RPTC cultured in conditions of increased oxygen supply (Shake). Confluent Shake cultures grown in the presence of glucose exhibited increased oxygen consumption and decreased glycolysis compared with stationary (Still) cultures. Addition of 5 mM lactate to a 5 mM glucose medium decreased net glucose consumption and glucose oxidation in Shake cultures by 34 and 50%, respectively, and resulted in net lactate consumption. Addition of 5 mM lactate to a glucose-free medium resulted in a threefold increase in net glucose production (0.024 +/- 0.003 vs. 0.074 +/- 0.013 mumol.mg protein-1.day-1) in Shake cultures. Net glucose production further increased to 0.430 +/- 0.020 and 1.640 +/- 0.040 mumol.mg protein-1.day-1 when glucose reuptake was inhibited by 1 mM phloridzin or 1 mM phloridzin + 1 mM phloretin, respectively. These results show that, under conditions of improved oxygenation and in the presence of lactate and physiological levels of glucose and insulin, RPTC aerobic metabolism increases and glucose metabolism changes from glycolysis and net lactate production to gluconeogenesis and net lactate consumption.

A1 adenosine receptor knockout mice are protected against acute radiocontrast nephropathy in vivo

2006 ◽  
Vol 290 (6) ◽  
pp. F1367-F1375 ◽  
Author(s):  
H. Thomas Lee ◽  
Michael Jan ◽  
Soo Chan Bae ◽  
Jin Deok Joo ◽  
Farida R. Goubaeva ◽  
...  
Keyword(s):  
Renal Failure ◽  
Acute Renal Failure ◽  
Proximal Tubule ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cell ◽  
Proximal Tubule Cells ◽  
Proximal Tubular ◽  
Tubule Cells

The role of renal A1 adenosine receptors (A1AR) in the pathogenesis of radiocontrast nephropathy is controversial. We aimed to further elucidate the role of A1AR in the pathogenesis of radiocontrast nephropathy and determine whether renal proximal tubule A1AR contribute to the radiocontrast nephropathy. To induce radiocontrast nephropathy, A1AR wild-type (WT) or knockout (KO) mice were injected with a nonionic radiocontrast (iohexol, 1.5–3 g iodine/kg). Some A1WT mice were pretreated with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; a selective A1AR antagonist) before iohexol injection. A1AR contribute to the pathogenesis of radiocontrast nephropathy in vivo as the A1WT mice developed significantly worse acute renal failure, more renal cortex vacuolization, and had lower survival 24 h after iohexol treatment compared with the A1KO mice. DPCPX pretreatment also protected the A1WT mice against radiocontrast-induced acute renal failure. No differences in renal cortical apoptosis or inflammation were observed between A1WT and A1KO mice. To determine whether the proximal tubular A1AR mediate the direct renal cytotoxicity of radiocontrast, we treated proximal tubules in culture with iohexol with or without 2-chloro- N6-cyclopentyladenosine (a selective A1AR agonist) or DPCPX pretreatment. We also subjected cultured proximal tubule cells overexpressing A1AR or lacking A1AR to radiocontrast injury. Iohexol caused a direct dose-dependent reduction in proximal tubule cell viability as well as proliferation. Neither the A1AR agonist nor the antagonist treatment affected proximal tubule viability or proliferation. Moreover, overexpression or lack of A1AR failed to impact the iohexol toxicity on proximal tubule cells. Therefore, we conclude that radiocontrast causes acute renal failure via mechanisms dependent on A1AR; however, renal proximal tubule A1AR do not contribute to the direct tubular toxicity of radiocontrast.

Reabsorptive and secretory 5-methyltetrahydrofolate transport pathways in cultured human proximal tubule cells

1997 ◽  
Vol 272 (3) ◽  
pp. F380-F388 ◽  
Author(s):  
K. M. Morshed ◽  
K. E. McMartin
Keyword(s):  
Proximal Tubule ◽  
Cell Polarity ◽  
Cellular Uptake ◽  
Intracellular Transport ◽  
Transport Pathways ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Folate Homeostasis ◽  
Human Proximal Tubule

Decreases in plasma folate levels leading to folate deficiency can result from increased urinary loss of folate, due to changes in either the renal reabsorption or secretion of folate. Hence, human proximal tubule (HPT) cells were cultured on microporous membranes to separate apical (AP) and basolateral (BL) domains and to assess the transport of 5-methyltetrahydrofolate from AP-to-BL (i.e., reabsorptive) and BL-to-AP (secretory) directions. Cellular uptake of alpha-methylglucoside occurred specifically from the AP direction, and transport of p-aminohippurate occurred more readily from the BL direction, demonstrating cell polarity similar to that in vivo. Under tight monolayer conditions, binding of folate to the AP membrane occurred more readily from the AP direction, although AP binding also occurred from the BL chamber. Intracellular transport occurred equally from both AP and BL directions. When loaded from either direction, folate was effluxed from HPT cells into both AP and BL chambers. About 20-30% of the internalized substrate was converted to nonfolate catabolites. Thus HPT cells readily take up folate via both the AP and BL membranes, metabolize it intracellularly and secrete the products across both membranes. These studies suggest that renal folate homeostasis is regulated bidirectionally.

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