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. 2020 Mar;67:109484.
doi: 10.1016/j.cellsig.2019.109484. Epub 2019 Nov 23.

l-ornithine activates Ca 2+ signaling to exert its protective function on human proximal tubular cells

Samuel Shin  1 , Farai C Gombedza  1 , Bidhan C Bandyopadhyay  2
Affiliations
Free PMC article

l-ornithine activates Ca 2+ signaling to exert its protective function on human proximal tubular cells

Samuel Shin et al. Cell Signal. 2020 Mar.
Free PMC article

Abstract

Oxidative stress and reactive oxygen species (ROS) generation can be influenced by G-protein coupled receptor (GPCR)-mediated regulation of intracellular Ca2+ ([Ca2+]i) signaling. ROS production are much higher in proximal tubular (PT) cells; in addition, the lack of antioxidants enhances the vulnerability to oxidative damage. Despite such predispositions, PT cells show resiliency, and therefore must possess some inherent mechanism to protect from oxidative damage. While the mechanism in unknown, we tested the effect of l-ornithine, since it is abundantly present in PT luminal fluid and can activate Ca2+-sensing receptor (CaSR), a GPCR, expressed in the PT luminal membrane. We used human kidney 2 (HK2) cells, a PT cell line, and performed Ca2+ imaging and electrophysiological experiments to show that l-ornithine has a concentration-dependent effect on CaSR activation. We further demonstrate that the operation of CaSR activated Ca2+ signaling in HK-2 cells mediated by the transient receptor potential canonical (TRPC) dependent receptor-operated Ca2+ entry (ROCE) using pharmacological and siRNA inhibitors. Since PT cells are vulnerable to ROS, we simulated such deleterious effects using genetically encoded peroxide-induced ROS production (HyperRed indicator) to show that the l-ornithine-induced ROCE mediated [Ca2+]i signaling protects from ROS production. Furthermore, we performed cell viability, necrosis and apoptosis assays, and mitochondrial oxidative gene expression to establish that presence of l-ornithine rescued the ROS-induced damage in HK-2 cells. Moreover, l-ornithine-activation of CaSR can reverse ROS production and apoptosis via mitogen-activated protein kinase p38 activation. Such nephroprotective role of l-ornithine can be useful as the translational option for reversing kidney diseases involving PT cell damage due to oxidative stress or crystal nephropathies.

Keywords: Ca(2+) signaling; Ca(2+)-sensing receptor; Cell death protection; Oxidative stress; Proximal tubular cells; l-Amino acids.

Conflict of interest statement

CONFLICT OF INTERESTS

The authors declare that there is no conflict of interests.

Figures

Figure 1.
Figure 1.
A. Mean Fura-2 Fluorescent traces of HK-2 cells in 1.2 mM [Ca2+]o solution upon application of L-ornithine (L-Orn) to elicit concentration-dependent response (0 mM, 1.0 mM, 3.0 mM, and 10 mM). Bar diagram depicts peak ratiometric [Ca2+]i after L-Orn application. B. Whole-cell patch clamp current of HK-2 cell upon concentration dependent application of L-Orn (100, 500, and 1000 uM) with voltage sweep from −100 to +100 mV. Bar diagram depicts peak current at +100 mV. Fura-2 fluorescent traces of transfected HK-2 cells with C. 0 nM, D. 10 nM, or E. 20 nM CaSR siRNA bathed in 0.5 mM [Ca2+]o solution with application of 10 mM L-Orn followed by 2.0 mM [Ca2+]o. F. Bar diagram depicts peak [Ca2+]i entry for C-D. Two-tailed t-test statistical analysis performed for A, B, and F. *, p<0.05; **, p<0.01.
Figure 2.
Figure 2.
Mean Fura-2 fluorescent traces of HK-2 cells bathed in 0.5 mM [Ca2+]o solution incubated in A. 1 μM NPS-2143 (NPS), B. 1 μM SKF-96163 (SKF), or C. 12 μM 2-APB, with application of 10 mM L-ornithine (L-Orn) followed by 2.0 mM [Ca2+]o solution. D. Bar diagram depicts peak Ca2+ entry for A, B, and C. Average whole-cell patch clamp current of HK-2 cell upon application of L-Orn followed by inhibition with E. NPS, F. SKF, or G. 2-APB, with voltage sweep from −100 to +100 mV. Bar diagram for each current graph depicts peak current at +100 mV. Two-tailed t-test statistical analysis performed for D, E, F, and G. +, added activator/inhibitor. *, p<0.05; **, p<0.01.
Figure 3.
Figure 3.
A. Mean Fura-2 fluorescent traces of HK-2 cells bathed in 0.5 mM [Ca2+]o solution incubated in 3 μM Pyr6 or 3 μM Pyr10, with application of 10 mM L-ornithine (L-Orn) followed by 2.0 mM [Ca2+]o solution. Bar diagram depicts peak Ca2+ entry for A. Average whole-cell patch clamp current of HK-2 cell upon application of L-Orn followed by inhibition with B. Pyr6, or C. Pyr10, with voltage sweep from −100 to +100 mV. Bar diagram for each current graph depicts peak current at +100 mV. +, added activator/inhibitor. Two-tailed t-test statistical analysis performed for A, B, and C. *, p<0.05; **, p<0.01.
Figure 4.
Figure 4.
A. HK-2 cells were transfected with HyperRed (ROS indicator) and treated with/without L-ornithine (L-Orn) for 24 hours. B. Representative bar graph of data from A. C: Mean fluorescence traces of Fura-2 loaded HK-2 cells. Cells were bathed in 1.2 mM Ca2+ SES buffer and indicated agents were added. C. Control conditions: 50 μM of H2O2 and 2 mM Ca2+ were added at their indicated positions. A substantial and prolonged Ca2+ entry was observed. HK-2 cells were incubated with 1 mM L-Orn or 1 mM L-Orn and 1 μM Pyr10 for 1 hour and 50 μM H2O2 and 2 mM Ca2+ were added at their indicated positions. E. Representative bar graph in mean±s.e.m. for B and D depict [Ca2+]i peak entry. +, added activator/inhibitor. Statistical analysis for unpaired two-tailed t-test performed for B and D; *, p < 0.05; **, p < 0.01.
Figure 5.
Figure 5.
L-ornithine (L-Orn) protects HK-2 cells from mixed crystal or H2O2 induced apoptosis and necrosis. HK-2 cells were treated or not treated with L-ornithine for an hour prior to incubating cells with or without CaP/CaOx mixed crystals or H2O2. Cells were stained with A. DAPI, B. PI, or C. Annexin staining to assess cell death. White arrows indicate defragmentation of the nuclear DNA. +, added activator/inhibitor. Bar diagrams represent mean+s.e.m. Statistical analysis performed on A-C with two-tailed t-test. *, p < 0.05; **, p < 0.01. Scale bars, 100 μM.
Figure 6.
Figure 6.
HK-2 cells were pretreated with or without L-ornithine (L-Orn; 10 μM) before inducing ROS with H2O2 (500 μM). A. Cell viability assay was performed on these cells. B. RT-PCR was performed to assess the gene expression of BAX, BCL2, and GAPDH. BAX to BCL2 ratio, normalized to GAPDH. −, absent; +, present. Representative bar graphs of A-B in mean+s.e.m. Statistical analysis for unpaired two-tailed t-test performed for A-B; *, p < 0.05; **, p < 0.01.
Figure 7.
Figure 7.
L-ornithine (L-Orn) in HK-2 cells inducing p38 phosphorylation. A. Western blot analysis of p-p38 in HK-2 cells treated with L-Orn 10 or 100 uM for 30 to 60 min. B. Bar diagram represents A in mean+s.e.m. C. Western blot analysis of p-p38 in HK-2 cells treated with L-Orn, H2O2, and/or Pyr10 for 1 hr. −, absent; +, present. D. Bar diagram represents A in mean+s.e.m. Statistical analysis performed on B and D with two-tailed t-test. *, p < 0.05; **, p < 0.01.
Figure 8.
Figure 8.
Schematic diagram of L-ornithine activated ROCE pathway protecting PT cell. L-ornithine (L-Orn) allosterically activates the CaSR and triggers the ROCE Ca2+ signaling pathway. The resulting Ca2+-entry prevents necrosis or apoptosis by inhibiting the downstream mechanism of ROS formation, induction of oxidative stress, and expressions of BAX and p-P38 pro-apoptotic pathway. CaSR: Ca2+-sensing receptor; Gp: G-protein; ER: endoplasmic reticulum; PLC: Phospholipase C; DAG: diacylglycerol.
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