Canine Renal Tubular Cells Research Articles

Investigation of effect of tectorigenin (O-methylated isoflavone) on Ca2+ signal transduction and cytotoxic responses in canine renal tubular cells.

Tectorigenin, a traditional Chinese medicine, is isolated from the flower of plants such as Pueraria thomsonii Benth. It is an O-methylated isoflavone, a type of flavonoid. Previous studies have shown that tectorigenin evoked various physiological responses in different models, but the effect of tectorigenin on cytosolic-free Ca2+ levels ([Ca2+]i) and cytotoxicity in renal tubular cells is unknown. Our research explored if tectorigenin changed Ca2+ signal transduction and viability in Madin-Darby Canine Kidney (MDCK) renal tubular cells. [Ca2+]iin suspended cells were measured by applying the fluorescent Ca2+-sensitive probe fura-2. Viability was explored by using water-soluble tetrazolium-1 as a fluorescent dye. Tectorigenin at concentrations of 5-50 μM induced [Ca2+]irises. Ca2+ removal reduced the signal by approximately 20%. Tectorigenin (50 μM) induced Mn2+ influx suggesting of Ca2+ entry. Tectorigenin-induced Ca2+ entry was inhibited by 10% by three inhibitors of store-operated Ca2+ channels, namely, nifedipine, econazole, and SKF96365. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin inhibited 83% of tectorigenin-evoked [Ca2+]irises. Conversely, treatment with tectorigenin abolished thapsigargin-evoked [Ca2+]irises. Inhibition of phospholipase C with U73122 inhibited 50% of tectorigenin-induced [Ca2+]irises. Tectorigenin at concentrations between 10 and 60 μM killed cells in a concentration-dependent fashion. Chelation of cytosolic Ca2+ with 1,2-bis (2-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid/acetoxy methyl did not reverse tectorigenin's cytotoxicity. Our data suggest that, in MDCK cells, tectorigenin evoked [Ca2+]irises and induced cell death that was not associated with [Ca2+]irises. Therefore, tectorigenin may be a Ca2+-independent cytotoxic agent for kidney cells.

Naproxen-induced Ca2+ movement and death in MDCK canine renal tubular cells.

Naproxen is an anti-inflammatory drug that affects cellular calcium ion (Ca(2+)) homeostasis and viability in different cells. This study explored the effect of naproxen on [Ca(2+)](i) and viability in Madin-Darby canine kidney cells (MDCK) canine renal tubular cells. At concentrations between 50 μM and 300 μM, naproxen induced [Ca(2+)](i) rises in a concentration-dependent manner. This Ca(2+) signal was reduced partly when extracellular Ca(2+) was removed. The Ca(2+) signal was inhibited by a Ca(2+) channel blocker nifedipine but not by store-operated Ca(2+) channel inhibitors (econazole and SKF96365), a protein kinase C (PKC) activator phorbol 12-myristate 13-acetate, and a PKC inhibitor GF109203X. In Ca(2+)-free medium, pretreatment with 2,5-di-tert-butylhydroquinone or thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+) pumps, partly inhibited naproxen-induced Ca(2+) signal. Inhibition of phospholipase C with U73122 did not alter naproxen-evoked [Ca(2+)](i) rises. At concentrations between 15 μM and 30 μM, naproxen killed cells in a concentration-dependent manner, which was not reversed by prechelating cytosolic Ca(2+) with the acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl. Annexin V/propidium iodide staining data suggest that naproxen induced apoptosis. Together, in MDCK renal tubular cells, naproxen induced [Ca(2+)](i) rises by inducing Ca(2+) release from multiple stores that included the endoplasmic reticulum and Ca(2+) entry via nifedipine-sensitive Ca(2+) channels. Naproxen induced cell death that involved apoptosis.

Ca2+ movement and apoptosis induced by deltamethrin in Madin–Darby canine kidney canine renal tubular cells

This study explored the effect of deltamethrin, a pesticide, on free Ca2+ concentration [Ca2+]i, viability, and apoptosis in Madin–Darby canine kidney (MDCK) canine renal tubular cells. Deltamethrin at concentrations between 10μM and 40μM evoked [Ca2+]i rises in a concentration-dependent manner. The Ca2+ entry was inhibited by nifedipine, econazole, phorbol 12-myristate 13-acetate, and SKF96365. Treatment with the endoplasmic reticulum Ca2+ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) in a Ca2+-free medium abolished deltamethrin-induced [Ca2+]i rise. Treatment with deltamethrin also abolished BHQ-induced [Ca2+]i rise. Inhibition of phospholipase C (PLC) activity with U73122 abolished deltamethrin-evoked [Ca2+]i rise. Deltamethrin killed cells at 30–60μM in a concentration-dependent manner. The cytotoxic effect of deltamethrin was not reversed by prechelating cytosolic Ca2+ with the acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. Annexin V/propidium iodide staining data suggest that 30–50μM deltamethrin induced apoptosis. Together, in MDCK renal tubular cells, deltamethrin induced [Ca2+]i rises that involved Ca2+ entry through protein kinase C-mediated store-operated Ca2+ channels, and PLC-dependent Ca2+ release from the endoplasmic reticulum. Deltamethrin also induced Ca2+-independent cell death that might involve apoptosis.

Effect of phenethyl isothiocyanate on Ca2+ movement and viability in MDCK canine renal tubular cells

The effect of the natural compound phenethyl isothiocyanate (PEITC) on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in MDCK renal cells is unknown. This study explored whether PEITC changed [Ca(2+)](i) in MDCK cells using the Ca(2+)-sensitive fluorescent dye fura-2. PEITC at 200-700 μM increased [Ca(2+)](i) in a concentration-dependent manner. The signal was reduced by removing extracellular Ca(2+). PEITC-induced Ca(2+) influx was inhibited by nifedipine, econazole, SK&F 96365 and protein kinase C modulators. In Ca(2+)-free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) inhibited PEITC-induced rise in [Ca(2+)](i). Incubation with PEITC also inhibited TG or BHQ-induced rise in [Ca(2+)](i). Inhibition of phospholipase C with U73122 abolished PEITC-induced rise in [Ca(2+)](i). At 15-75 μM, PEITC decreased viability. The cytotoxic effect of PEITC was enhanced by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester. Annexin V-FITC data suggest that 20 and 50 μM PEITC induced apoptosis. At 10 and 15 μM, PEITC did not increase reactive oxygen species (ROS) production. Together, in renal tubular cells, PEITC-induced rise in [Ca(2+)](i) by inducing phospholipase C-dependent Ca(2+) release from endoplasmic reticulum and Ca(2+) entry via store-operated Ca(2+) channels. PEITC induced apoptosis in a concentration-dependent, ROS/Ca(2+)-independent manner.

Effect of bisphenol A on Ca2+ fluxes and viability in Madin-Darby canine renal tubular cells

The effect of the environmental contaminant, bisphenol A, on cytosolic free Ca2+ concentrations ([Ca2+]i) in Madin-Darby canine kidney (MDCK) cells is unclear. This study explored whether bisphenol A changed basal [Ca2+]i levels in suspended MDCK cells by using fura-2 as a Ca2+-sensitive fluorescent dye. Bisphenol A, at concentrations between 50 and 300 µM, increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced, partly, by removing extracellular Ca2+. Bisphenol A induced Mn2+ influx, leading to quenching of fura-2 fluorescence, suggesting Ca2+ influx. This Ca2+ influx was inhibited by phospholipase A2 inhibitor aristolochic acid, store-operated Ca2+ channel blockers nifedipine and SK&F96365, and protein kinase C inhibitor GF109203X. In Ca2+-free medium, pretreatment with the mitochondrial uncoupler, carbonylcyanide m-chlorophenylhydrazone (CCCP), and the endoplasmic reticulum Ca2+ pump inhibitors, thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ), inhibited bisphenol A–induced Ca2+ release. Conversely, pretreatment with bisphenol A abolished thapsigargin (or BHQ)- and CCCP-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 abolished bisphenol-induced [Ca2+]i rise. Bisphenol A caused a concentration-dependent decrease in cell viability via apoptosis in a Ca2+-independent manner. Collectively, in MDCK cells, bisphenol A induced [Ca2+]i rises by causing phospholipase C–dependent Ca2+ release from the endoplasmic reticulum and mitochondria and Ca2+ influx via phospholipase A2–, protein kinase C–sensitive, store-operated Ca2+ channels.

Independent Effects of the Broccoli-Derived Compound Sulforaphane on Ca(superscript 2+) Influx and Apoptosis in Madin-Darby Canine Renal Tubular Cells

This study explored whether sulforaphane changed basal [Ca²⁺]i levels in suspended Madin-Darby canine kidney (MDCK) cells by using fura-2 as a Ca²⁺-sensitive fluorescent dye. Sulforaphane at concentrations between 2.5-10 microM increased [Ca²⁺]i in a concentration-dependent manner. This Ca²⁺ influx was inhibited by phospholipase A2 inhibitor aristolochic acid but not by Ca²⁺ channel blockers such as nifedipine, nimodipine, nicardipine, diltiazem, verapamil, econazole and SK&F96365. The Ca²⁺ signal was abolished by removing extracellular Ca²⁺. In Ca²⁺-free medium, pretreatment with sulforaphane did not alter the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin-induced Ca²⁺ release suggesting sulforaphane did not induce slow Ca²⁺ release from endoplasmic reticulum. At concentrations between 1 and 20 microM, sulforaphane induced concentration-dependent decrease in cell viability which was not affected by pre-chelation of cytosolic Ca²⁺ with BAPTA/AM. Flow cytometry data suggest that 20 (but not 5 and 10) microM sulforaphane induced significant increase in sub G1 phase indicating involvement of apoptosis. Collectively, in MDCK cells, sulforaphane induced [Ca²⁺]i rises by causing Ca²⁺ entry through phospholipase A2-sensitive pathways without inducing Ca²⁺ release from the endoplasmic reticulum. Sulforaphane also induced Ca²⁺-independent cell death that might involve apoptosis.

Effect of m-3M3FBS on Ca2+ movement in Madin-Darby canine renal tubular cells

The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS), a presumed phospholipase C (PLC) activator, on cytosolic free Ca(2+) concentrations ([Ca( 2+)](i)) in Madin-Darby canine kidney (MDCK) cells is unclear. This study explored whether m-3M3FBS changed basal [Ca(2+)](i) levels in suspended MDCK cells using fura-2 as a Ca(2+)-sensitive fluorescent dye. M-3M3FBS at concentrations between 0.1 and 20 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was decreased by removing extracellular Ca(2+). M-3M3FBS-induced Ca(2+) influx was inhibited by the store-operated Ca(2+) channel blockers nifedipine, econazole, and SK&F96365, and by the phospholipase A2 inhibitor aristolochic acid. In Ca(2+)-free medium, 20-microM m-3M3FBS pretreatment abolished the [Ca(2+)](i) rise induced by the endoplasmic reticulum Ca(2+) pump inhibitors thapsigargin (TG) and cyclopiazonic acid (CPA). Conversely, pretreatment with TG or CPA partly reduced m-3M3FBS-induced [Ca(2+)](i) rise. The inhibition of PLC with U73122 did not alter m-3M3FBS-induced [Ca(2+)](i) rise. Collectively, in MDCK cells, m-3M3FBS induced [Ca(2+)](i) rises by causing PLC-independent Ca(2+) release from the endoplasmic reticulum and Ca(2+) influx via store-operated Ca(2+) channels and other unidentified Ca(2+) channels.

Effect of capsaicin on Ca2+ fluxes in Madin‐Darby canine renal tubular cells

AbstractThe effect of capsaicin, a transient receptor potential vanniloid‐1 (TRPV1) receptor agonist, on cytosolic free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells is unclear. This study explored whether capsaicin changed basal [Ca2+]i levels in suspended MDCK cells by using fura‐2 as a Ca2+‐sensitive fluorescent dye. Capsaicin at concentrations between 10–100 µM increased [Ca2+]i in a concentration‐dependent manner. The Ca2+ signal was reduced by 80% by removing extracellular Ca2+. Capsacin induced Mn2+ influx, leading to quench of fura‐2 fluorescence suggesting Ca2+ influx. This Ca2+ influx was inhibited by phospholipase A2 inhibitor aristolochic acid and the non‐selective Ca2+ entry blocker La3+, but not by store‐operated Ca2+ channel blockers nifedipine, econazole, and SK&F96365, and protein kinase C/A modulators. In Ca2+‐free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished capsaicin‐induced Ca2+ release. Conversely, pretreatment with capsaicin partly reduced thapsigargin‐induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not alter capsaicin‐induced [Ca2+]i rise. The TRPV1 receptor antagonist capsazepine also induced significant Ca2+ entry and Ca2+ release. Collectively, in MDCK cells, capsaicin induced [Ca2+]i rises by causing phospholipase C‐independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2‐regulated, La3+‐sensitive Ca2+ channels in a manner dissociated from stimulation of TRPV1 receptors. Drug Dev Res, 2009. © 2009 Wiley‐Liss, Inc.

Melittin-induced [Ca2+]i increases and subsequent death in canine renal tubular cells

The effect of melittin on cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and viability is largely unknown. This study examined whether melittin alters Ca(2+) levels and causes Ca(2+)-dependent cell death in Madin-Darby canine kidney (MDCK) cells. [Ca(2+)](i) and cell death were measured using the fluorescent dyes fura-2 and WST-1 respectively. Melittin at concentrations above 0.5 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced by 75% by removing extracellular Ca(2+). The melittin-induced Ca(2+) influx was also implicated by melittin-caused Mn(2+) influx. After pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), melittin-induced Ca(2+) release was inhibited; and conversely, melittin pretreatment abolished thapsigargin-induced Ca(2+) release. At concentrations of 0.5-20 microM, melittin killed cells in a concentration-dependent manner. The cytotoxic effect of 0.5 microM melittin was nearly completely reversed by prechelating cytosolic Ca(2+) with BAPTA. Melittin at 0.5-2 microM caused apoptosis as assessed by flow cytometry of propidium iodide staining. Collectively, in MDCK cells, melittin induced a [Ca(2+)](i) rise by causing Ca(2+) release from endoplasmic reticulum and Ca(2+) influx from extracellular space. Furthermore, melittin can cause Ca(2+)-dependent cytotoxicity in a concentration-dependent manner.

Effect of Anandamide on Cytosolic Ca2+ Levels and Proliferation in Canine Renal Tubular Cells

The effect of the endogenous cannabinoid anandamide on cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and proliferation is largely unknown. This study examined whether anandamide altered Ca(2+) levels and caused Ca(2+)-dependent cell death in Madin-Darby canine kidney (MDCK) cells. [Ca(2+)](i) and cell death were measured using the fluorescent dyes fura-2 and WST-1 respectively. Anandamide at concentrations above 5 muM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced by 78% by removing extracellular Ca(2+). The anandamide-induced Ca(2+) influx was insensitive to L-type Ca(2+) channel blockers and the cannabinoid receptor antagonist AM 251, but was inhibited differently by aristolochic acid, WIN 55,212-2 (a cannabinoid receptor agonist), phorbol ester, GF 109203X and forskolin. After pretreatment with thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), anandamide-induced Ca(2+) release was inhibited. Inhibition of phospholipase C with U73122 did not change anandamide-induced Ca(2+) release. At concentrations of 100 muM and 200 muM, anandamide killed 50% and 95% cells, respectively. The cytotoxic effect of 100 muM anandamide was completely reversed by pre-chelating cytosolic Ca(2+) with BAPTA. Collectively, in MDCK cells, anandamide induced [Ca(2+)](i) rises by causing Ca(2+) release from endoplasmic reticulum and Ca(2+) influx from extracellular space. Furthermore, anandamide can cause Ca(2+)-dependent cytotoxicity in a concentration-dependent manner.

Effect of Y-24180 on Ca 2+ movement and proliferation in renal tubular cells

In canine renal tubular cells, the effect of Y-24180, a presumed specific platelet activating factor (PAF) receptor antagonist, on intracellular Ca 2+ concentration ([Ca 2+] i) was measured by using fura-2 as a Ca 2+-sensitive fluorescent probe. Y-24180 (0.1–10 μM) caused a rapid and sustained [Ca 2+] i rise in a concentration-dependent manner. The [Ca 2+] i rise was prevented by 30% by removal of extracellular Ca 2+, but was not changed by dihydropyridines, verapamil and diltiazem. Y-24180-induced Ca 2+ influx was confirmed by Mn 2+-influx induced quench of fura-2 fluorescence. In Ca 2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca 2+-ATPase, caused a monophasic [Ca 2+] i rise, after which the increasing effect of 5 μM Y-24180 on [Ca 2+] i was abolished; conversely, depletion of Ca 2+ stores with 5 μM Y-24180 abolished thapsigargin-induced [Ca 2+] i rise. U73122, an inhibitor of phoispholipase C, inhibited ATP-, but not Y-24180-induced [Ca 2+] i rise. Overnight treatment with Y-24180 did not alter cell proliferation rate. Collectively, these results suggest that Y-24180 acts as a potent, but not cytotoxic, Ca 2+ mobilizer in renal tubular cells, by stimulating both extracellular Ca 2+ influx and intracellular Ca 2+ release. Since alterations in Ca 2+ movement may interfere many cellular signaling processes unrelated to modulation of PAF receptors, caution must be applied in using this chemical as a selective PAF receptor antagonist.

Effect of olvanil (N-vanillyl-cis-9-octadecenoamide) on cytosolic Ca 2+ increase in renal tubular cells

In canine renal tubular cells, effect of olvanil, a presumed cannabinoid and vanilloid receptor modulator, on intracellular Ca 2+ concentration ([Ca 2+] i) was measured by using fura-2. Olvanil (5–100 μM) caused a rapid and sustained [Ca 2+] i rise in a concentration-dependent manner. Olvanil-induced [Ca 2+] i rise was prevented by 70 and 90% by removal of extracellular Ca 2+ and La 3+, respectively, but was not changed by dihydropyridines, verapamil and diltiazem. In Ca 2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca 2+-ATPase, caused a monophasic [Ca 2+] i rise, after which the increasing effect of olvanil on [Ca 2+] i was abolished; also, pretreatment with olvanil partly reduced thapsigargin-induced [Ca 2+] i rise. U73122, an inhibitor of phoispholipase C, abrogated ATP-, but partly inhibited olvanil-, induced [Ca 2+] i rise. Two cannabinoid receptor antagonists (AM251 and AM281; 5 μM) and a vanilloid receptor antagonist (capsazepine; 100 μM) did not alter olvanil (50 μM)-induced [Ca 2+] i rise. These results suggest that olvanil rapidly increases [Ca 2+] i in renal tubular cells, by stimulating both extracellular Ca 2+ influx and intracellular Ca 2+ release via mechanism(s) independent of stimulation of cannabinoid and vanilloid receptors.

CP55,940 increases intracellular Ca 2+ levels in Madin-Darby canine kidney cells

The effect of CP55,940, a presumed CB1/CB2 cannabinoid receptor agonist, on intracellular free Ca 2+ levels ([Ca 2+] i) in Madin-Darby canine kidney cells was examined by using the fluorescent dye fura-2 as a Ca 2+ indicator. CP55,940 (2–50 μM) increased [Ca 2+] i concentration-dependently with an EC 50 of 8 μM. The [Ca 2+] i signal comprised an initial rise and a sustained phase. Extracellular Ca 2+ removal decreased the maximum [Ca 2+] i signals by 32±12%. CP55,940 (20 μM)-induced [Ca 2+] i signal was not altered by 5 μM of two cannabinoid receptor antagonists, AM-251 and AM-281. CP55,940 (20 μM)-induced [Ca 2+] i increase in Ca 2+-free medium was inhibited by 86±3% by pretreatment with 1 μM thapsigargin, an endoplasmic reticulum Ca 2+ pump inhibitor. Conversely, pretreatment with 20 μM CP55,940 in Ca 2+-free medium for 6 min abolished thapsigargin-induced [Ca 2+] i increases. CP55,940 (20 μM)-induced intracellular Ca 2+ release was not inhibited when inositol 1,4,5-trisphosphate formation was abolished by suppressing phospholipase C with 2 μM U73122. Collectively, this study shows that CP,55940 induced significant [Ca 2+] i increases in canine renal tubular cells by releasing stored Ca 2+ from the thapsigargin-sensitive pools in an inositol 1,4,5-trisphosphate-independent manner, and also by causing extracellular Ca 2+ entry. The CP55,940's action appears to be dissociated from stimulation of cannabinoid receptors.

CNP is present in canine renal tubular cells and secreted by cultured opossum kidney cells.

C-type natriuretic peptide (CNP) is a vasoactive and antimitogenic peptide that is structurally similar but genetically distinct from atrial natriuretic peptide. While first discovered in the brain, CNP has been shown to be produced by endothelial cells and may function in a paracrine and autocrine fashion in the control of vascular tone. Recently, CNP immunoreactivity and B-type natriuretic peptide receptors (NPR-B), for which CNP is a specific ligand, have been identified in the kidney. The present study was designed to determine whether renal epithelial cells produce and secrete CNP and whether CNP immunoreactivity is present in canine kidney. Opossum kidney (OK) cells that express proximal tubular cell characteristics were incubated for 6 h in fetal calf serum-free Dulbecco's modified Eagle's medium (DMEM). CNP immunoreactivity was measured in the preincubation and 6-h conditioned media by radioimmunoassay (RIA) using a specific antibody to CNP-22. Furthermore the molecular form of this CNP-like protein was determined by reverse-phase high-performance liquid chromatography (HPLC), and intracellular localization of the CNP immunoreactivity was determined by immunohistochemical staining. CNP immunoreactivity was also determined in renal tissue from dogs subjected to saline or endothelin infusion. Six-hour incubation in DMEM resulted in accumulation of CNP immunoreactivity (baseline below detection level vs. 6 h = 117.3 +/- 8.3 pg/ml, P < 0.001). Intracellular CNP concentration determined after sonication was 1.9 +/- 0.2 micrograms/g protein, and immunohistochemical staining for CNP was markedly positive in the cytoplasm.(ABSTRACT TRUNCATED AT 250 WORDS)