Na+ -Dependent and -Independent Amino Acid Transport Systems in Immortalized Human Kidney Epithelial Cells Derived from the Proximal Tubule

Hirsch, Jochen R.; Gonska, Tanja; Waldegger, Siegfried; Läng, Florian; Schlatter, Eberhard

doi:10.1159/000025843

Cited by 8 publications

(9 citation statements)

References 24 publications

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“…Previous studies showed the presence of different amino acid transport systems in IHKE-1 cells (22,23). In the current experiments, addition of 1 mM alanine to the bath solution led to depolarizations of V m of IHKE-1 cells by 17.6 Ϯ 0.7 mV (n ϭ 59), indicating its electrogenic transport.…”

Section: Resultssupporting

confidence: 56%

“…Increasing extracellular K ϩ -concentration from 3.6 mM to 18.6 mM depolarized cells by 3.4 Ϯ 0.1 mV (n ϭ 101). The addition of 3 mM Ba 2ϩ , a blocker of K ϩ -channels, depolarized V m by 11.3 Ϯ 0.7 mV (n ϭ 14), again indicating the presence of a resting K ϩ conductance in these cells (23). Removal of extracellular Na ϩ led to a marked hyperpolarization due to the presence of a nonselective cation conductance, which also determines the relatively depolarized resting voltage of these cells (23,25).…”

Section: Resultsmentioning

confidence: 96%

“…With the same experimental model, we performed for the first time electrophysiologic studies with the slow whole-cell patch clamp technique with immortalized human kidney epithelial cells derived from the proximal tubule (IHKE-1 cells) (23,25) and for which various morphologic (24,25), biochemical (20), and functional (22-24) characteristics of proximal tubule cells have been shown by several laboratories. This method allows real time and paired observations of CDME effects on V m itself and on electrogenic transport systems such as the Na ϩ -coupled alanine and phosphate transporter and indirectly via changes in cellular pH of the Na ϩ /H ϩ exchanger.…”

Section: Discussionmentioning

confidence: 99%

“…IHKE-1 cells possess different Na ϩ -dependent amino acid transport systems in their apical membrane (22,23). We first investigated the effect of CDME on the Na ϩ -alanine cotransporter, which leads to significant depolarization in these cells (23).…”

Section: Discussionmentioning

confidence: 99%

“…Several studies on IHKE-1 cells have shown that these cells are typical proximal tubule cells. They express specific key enzymes (21), different Na ϩ -dependent and -independent amino acid transporters (22,23), and organic cation transporters (24) and they are able to regulate transport by natriuretic peptides (25) or to take up albumin (26). Here we present evidence for the existence of proximal tubule-specific Na ϩ /H ϩ -exchanger (NHE-3) and the Na ϩ -phosphate transporter (NaPi-IIa) in the apical membrane.…”

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confidence: 99%

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Inhibition of Na+-Dependent Transporters in Cystine-Loaded Human Renal Cells

Çetinkaya¹,

Schlatter²,

Hirsch³

et al. 2002

Journal of the American Society of Nephrology

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Abstract. Cystinosis is the most common cause of the renal Fanconi syndrome in children, leading to severe electrolyte disturbances and growth failure. A defective lysosomal transporter, cystinosin, results in intralysosomal accumulation of cystine. Loading cells with cystine dimethyl ester (CDME) is the only available model for this disease. This model was used to present electrophysiologic studies on immortalized human kidney epithelial (IHKE-1) cells that had been derived from the proximal tubule with the slow whole-cell patch clamp technique. Basal membrane voltages (V m ) of IHKE-1 cells were Ϫ30.7 Ϯ 0.4 mV (n ϭ 151). CDME concentration-dependently altered V m with an initial depolarization (2.7 Ϯ 0.2 mV; n ϭ 76; 1 mM CDME) followed by a more pronounced hyperpolarization (Ϫ9.9 Ϯ 1.0 mV; n ϭ 49). Three Na ϩ -dependent transporters were examined. Alanine (1 mM) depolarized IHKE-1 cells by 17.6 Ϯ 0.7 mV (n ϭ 59), and phosphate (1.8 mM) depolarized by 9.7 Ϯ 1.1 mV (n ϭ 18).Acidification of IHKE-1 cells with propionate (20 mM) resulted in a depolarization of V m by 7.1 Ϯ 0.3 mV (n ϭ 21) followed by a repolarization by 2.9 Ϯ 0.3 mV/min (n ϭ 17), reflecting Na ϩ /H ϩ -exchanger activity. Acute addition of 1 mM CDME did not alter the alanine-and propionate-induced changes in V m , but it reduced the phosphate-induced depolarization by 37 Ϯ 9% (n ϭ 10). Incubation with 1 mM CDME reduced the activity of all three transporters. Depolarizations by alanine and phosphate and the repolarization after propionate were inhibited by 57 Ϯ 4% (n ϭ 30), 45 Ϯ 9% (n ϭ 9), and 78 Ϯ 15% (n ϭ 8), respectively. In conclusion, this study demonstrates that CDME acutely alters V m of IHKE-1 cells and that at least three Na ϩ -dependent transporters are inhibited, the Na ϩ -phosphate cotransporter most sensitively. This might suggest that phosphate depletion and dissipation of the Na ϩ -gradient are involved in the development of the Fanconi syndrome of cystinosis.

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Section: Resultssupporting

confidence: 56%

Section: Resultsmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Inhibition of Na+-Dependent Transporters in Cystine-Loaded Human Renal Cells

Çetinkaya¹,

Schlatter²,

Hirsch³

et al. 2002

Journal of the American Society of Nephrology

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Chloroacetaldehyde- and acrolein-induced death of human proximal tubule cells

et al. 2005

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Ifosfamide (ifo) is a commonly used drug in chemotherapy. It is metabolized to acrolein (acro) and chloroacetaldehyde (CAA), which are thought to be responsible for renal side effects. We studied the effects of ifo and cyclophosphamide (cyclo) as well as their metabolites, acro and CAA, on cellular protein content, necrosis, apoptosis and cytosolic calcium concentration using a human proximal tubule cell line. The protein content decreased during acro or CAA administration (15 to 300 micromol/l), but not during ifo or cyclo exposure over a time period of up to 72 h. Mild apoptosis was induced only by high acro (150, 300 micromol/l) and low CAA concentrations (15, 75 micromol/l) and only in a narrow time window (24 h). Necrosis was increased after exposure to acro or CAA at all concentrations. CAA was more potent than acro. Ifo and cyclo did not induce necrosis or apoptosis. Glutathione abolished CAA-induced cell death. Cytosolic calcium concentrations increased after acro or CAA administration and showed an oscillating pattern. Cytosolic Ca(2+) chelation did not prevent necrosis. We conclude that neither ifo nor cyclo induce cell damage, but that their metabolites acro and CAA induce cell death. This cell death occurs mainly by necrosis and not by apoptosis.

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Regulation of renal amino acid (AA) transport by hormones, drugs and xenobiotics - a review

2003

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Major advances have recently been made in our understanding of the mechanisms and functions of amino acid transport in mammalian cells: - from the whole organism to transporter molecular structure. In this article, we present a brief overview of current knowledge concerning amino acid transporters, followed by a detailed discussion of the relevance of this new information to our broader understanding of the physiological regulation of amino acid handling in the kidney. We focus especially on the influence of hormones and xenobiotics on renal amino acid transport systems. In a growing number of cases, it now seems possible to correlate the effects of hormones, drugs, and xenobiotics with the capacity of renal amino acid transporters. This topic is of clinical relevance for the treatment of many amino acid reabsorption disorders. For example, under suitable conditions glucocorticoids and thyroid hormones stimulate renal reabsorption of amino acids and might therefore be of benefit in the treatment of different kinds of aminoaciduria. Hormonal regulation also underlies the postnatal development of renal amino acid reabsorption capacity, which can be stimulated to mature earlier after exogenous administration of e.g. glucocorticoids. In contrast, many compounds (e.g. heavy metal complexes) selectively damage renal amino acid transporters resulting in urinary amino acid loss. These types of phenomena (stimulation or inhibition of amino acid transporters in the kidney) are reviewed from the perspectives of our new molecular understanding of transport processes and of clinical relevance.

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Na+ -Dependent and -Independent Amino Acid Transport Systems in Immortalized Human Kidney Epithelial Cells Derived from the Proximal Tubule

Cited by 8 publications

References 24 publications

Inhibition of Na+-Dependent Transporters in Cystine-Loaded Human Renal Cells

Inhibition of Na+-Dependent Transporters in Cystine-Loaded Human Renal Cells

Chloroacetaldehyde- and acrolein-induced death of human proximal tubule cells

Regulation of renal amino acid (AA) transport by hormones, drugs and xenobiotics - a review

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