l -ornithine activates Ca2+ signaling to exert its protective function on human proximal tubular cells

Journal Pre-proof l-ornithine activates Ca2+ signaling to exert its protective function on human proximal tubular cells

Samuel Shin, Farai C. Gombedza, Bidhan C. Bandyopadhyay PII:

S0898-6568(19)30280-3

DOI:

https://doi.org/10.1016/j.cellsig.2019.109484

Reference:

CLS 109484

To appear in:

Cellular Signalling

Received date:

9 September 2019

Revised date:

21 November 2019

Accepted date:

21 November 2019

Please cite this article as: S. Shin, F.C. Gombedza and B.C. Bandyopadhyay, l-ornithine activates Ca2+ signaling to exert its protective function on human proximal tubular cells, Cellular Signalling(2018), https://doi.org/10.1016/j.cellsig.2019.109484

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© 2018 Published by Elsevier.

Journal Pre-proof L-ornithine activates Ca2+ signaling to exert its protective function on human proximal tubular cells Samuel Shin1§, Farai C. Gombedza1§, and Bidhan C. Bandyopadhyay1,2,3 *

1

Calcium Signaling Laboratory, Research Service, Veterans Affairs Medical Center, 50 Irving

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Street, NW, Washington DC, 20422 Division of Renal Diseases & Hypertension, Department of Medicine, The George Washington

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Department of Biomedical Engineering, The Catholic University of America, 620 Michigan

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Avenue NE, Washington DC, 20064, USA.

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University, Washington DC, 20037 USA

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Keywords: Proximal tubular cells, L-amino acids, Ca2+-sensing Receptor, Ca2+ signaling, Oxidative stress, cell death protection.

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Running Title: L-Ornithine protects against apoptosis

§

These authors contributed equally to this study.

*To whom correspondence should be addressed: Bidhan C. Bandyopadhyay, PhD. Chief, Calcium Signaling Laboratory, 151 Research Service, Veterans Affairs Medical Center, 50 Irving Street, NW, Washington, DC 20422, Phone: (202) 745-8622, Fax: (202) 462-2006; E-mail: [email protected]

1

Journal Pre-proof 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

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is abundantly present in PT luminal fluid and can activate calcium-sensing receptor (CaSR), a

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GPCR, expressed in the PT luminal membrane. We used human kidney 2 (HK-2) cells, a PT

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cells line, and performed Ca2+ imaging and electrophysiological experiments to show that Lornithine has a concentration-dependent effect on CaSR activation. We further demonstrate that

canonical

(TRPC)

dependent

receptor-operated

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potential

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the operation of CaSR activated Ca2+ signaling in HK-2 cells mediated by the transient receptor Ca2+

entry

(ROCE)

using

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pharmacological and siRNA inhibitors. Since PT cells are vulnerable to ROS, we simulated such deleterious effects using genetically encoded peroxide-induced ROS production (HypeRed

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indicator) to show that the L-ornithine-induced ROCE mediated [Ca2+]i signaling protects from

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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.

Journal Pre-proof

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[Ca2+]i: Intracellular Ca2+ 2-APB: 2-aminoethoxydiphenyl borate CaSR: Ca2+ sensing receptor DMEM: Dulbecco’s Modified Eagle Medium ECL: Enhanced chemiluminescence ERK: Extracellular signal-regulated kinase FBS: Fetal Bovine Serum Fura-2-AM: Fura-2-acetoxymethylester HK-2: human kidney 2 IP3 : Inositol triphosphate JNK: c-Jun NH2-terminal kinase (JNK) L-Orn: L-ornithine MAPK: Mitogen-activated protein kinases PT: Proximal tubule ROCE: Receptor operated Ca2+ entry ROS: Reactive oxygen species SOCE: Store-operated Ca2+ entry TBS-T: TBS-Tween TRPC: Transient receptor potential canonical

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Abbreviations

Journal Pre-proof 1 INTRODUCTION The proximal tubule (PT) accounts for 50-60% of inorganic solute reabsorption, and reabsorption of more than 90% of all sugars and amino acids (Gonzalez-Vicente & Garvin, 2017).

To support these critical active transport machinery, PT cells have a massive demand for

ATP, which is generated through large amount of glucose oxidation, producing higher metabolic activity. Such process results in excessive reactive oxygen species (ROS) production due to

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enhanced mitochondrial respiration activity, compounding the enhanced ROS production in the

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PT cells. However, PT cells are unable to synthesize antioxidant, glutathione; thus, making the

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PT segment of the nephron particularly vulnerable to oxidative damage (Visarius, Putt, Schare,

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Pegouske, & Lash, 1995). Consequently, enhanced ROS generation and reduced production in antioxidants enhance the susceptibility of PT cells to oxidative damage. Despite all these

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conditions, the PT cells show resiliency, and we therefore propose that these cells must possess

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some inherent protective mechanisms to overcome those injuries from oxidative damage. Renal tubular cells are exposed to ROS inducers like peroxides that can induce Ca2+

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influx through plasma membrane Ca2+ entry channels (Bowles, Graier, & Sturek, 2001) to

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maintain the intracellular Ca2+ ([Ca2+]i), which can further influence cellular process such as proliferation and apoptosis (Schreiber, 2005; Belkacemi, Bedard, Simoneau, & Lafond, 2005). Our recent study shows that prolonged Ca2+ entry induces sustained [Ca2+]i rise, causing apoptosis in porcine PT cells (Yiu, Ibeh, Roy, & Bandyopadhyay, 2017), unlike normal PT cells, which mediates a transient Ca2+ entry (Ibeh et al., 2019). Almost 90% of all renal amino acids are reabsorbed though PT (Curthoys & Moe, 2014). Due to a near complete reabsorption of filtered amino acids, PT luminal fluid contains a large amount of L-amino acids like small intestine. L-ornithine, a non-proteinogenic amino acid, has

Journal Pre-proof been found in the highest concentration in the PT luminal fluid compared to other parts of the nephron (van de Poll, Soeters, Deutz, Fearon, & Dejong, 2004).

We propose that such

abundance of L-ornithine in PT luminal fluid could activate the Ca2+-sensing receptor (CaSR), a G-protein coupled receptor, predominantly expressed in the luminal region of the PT. Previous studies

have

examined

the

CaSR-mediated

protective role of L-amino

acids against

inflammation, oxidative stress, and apoptosis (Katayama & Mine, 2007; Mine & Zhang, 2015;

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Gu et al., 2018). Therefore, we tested the specific physiological role of L-ornithine in human PT

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cells, by examining the effect on Ca2+ signaling and its possible downstream function. We show

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here that L-ornithine exerts its effect on Ca2+ signaling through receptor-operated Ca2+ entry

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Reagents and chemicals

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2 METHODS AND MATERIALS

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(ROCE) pathway, which can reverse the ROS mediated downstream effect on cell death.

NPS-2143 (catalog #: 3626) and SKF-96365 (catalog #: 1147) were purchased from Tocris

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Bioscience (Minneapolis, MN). Calcium chloride, monosodium phosphate, disodium phosphate,

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sodium oxalate, hydrogen peroxide solution, L-ornithine monohydrate (catalog #: O2375), Pyr6 (catalog #: SML1241), Pyr10 (catalog #: SML1243), and 2-aminoethoxydiphenyl borate (2APB; catalog #: D9754) were purchased from Sigma-Aldrich (St Louis, MO). Dulbecco’s Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS; catalog #: 26140079), penicillin and streptomycin and Fura-2-acetoxymethylester (Fura-2-AM) were purchased from Invitrogen (Carlsbad, CA). All the chemicals used were analytical grade.

Cell culture

Journal Pre-proof Human kidney 2 (HK-2) ), a proximal tubular cell line derived from normal kidney were purchased

from ATCC

(https://www.atcc.org/Products/All/CRL-2190.aspx),

for which we

maintained Biosafety classification 2 (based on U.S. Public Health Service Guidelines). HK-2 cells were cultured using DMEM. All the materials were purchased from Invitrogen. DMEM media

supplemented

with

10%

fetal

bovine

serum,

2mM

glutamine

and

1%

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penicillin/streptomycin were used for cell culture at 37°C in 5% CO 2 /95% air. Preparation of crystals

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Crystals were prepared as described previously (Cini et al., 2001; Sun, Xu, & Ouyang, 2017).

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They were concocted by mixing solutions of 2.4 mM CaCl2 , 0.9 mM Na2 HPO 4 and 5.8 mM

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NaH2 PO 4 ; mixing solutions of 2.4 mM CaCl2 , 1.0 mM Na2 C2 O4 ; and combining them. Crystals

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were prepared in 1X Hanks' Balanced Salt Solution (HBSS), pH 7.4 to a concentration of 800μg/ml. The crystals were shaken for half an hour at RT and subsequently centrifuged at

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Crystal internalization

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crystal internalization.

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10,000 rpm for 5 min. The resultant crystal pellets were 2x washed with HBSS and then used for

HK-2 cells were seeded and grown to 80% confluency in complete media. Serum-free and DMEM media was used for application of crystals (800μg/ml) and incubated for 4 h at 37 °C in 5%

CO 2 .,

Complete

media

(DMEM

with

10%

FBS,

2mM

glutamine

and

1%

penicillin/streptomycin) was added after 4 h, and continually kept for an additional 20 h. Transfection of siRNA We used our previously validated (Yiu, Ibeh, Roy, & Bandyopadhyay, 2017) specific siRNA to CaSR following the protocol described in McNaughton et al. (sc-44373; 10nM; Santa Cruz

Journal Pre-proof Biotechnology, Santa Cruz, CA) and the scrambled siRNA-A (Santa Cruz Biotechnology, Santa Cruz, CA; sc-37007; as negative control) to target the CaSR expression. Transfections were performed

using Lipofectamine-2000

Reagent (Thermo Fisher Scientific, Waltham, MA)

according to manufacturer’s instructions.

[Ca2+] i Fura-2 measurements by time-lapse fluorescence

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Ratiometric [Ca2+]i measurements were conducted as previously described (Bandyopadhyay,

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Swaim, Sarkar, Liu, & Ambudkar, 2012; Yiu, Ibeh, Roy, & Bandyopadhyay, 2017). Cells were

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loaded with Fura-2 and placed on an IX81 motorized inverted microscope equipped with an IX2-

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UCB control box (Olympus USA, Center Valley, PA). Measurements were performed in a humidified microincubator with gas mixture of 95% air and 5% CO 2 at constant temperature set

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to 37°C. Fura-2 fluorescence was recorded at emission peak absorbance of 500 nm wavelength

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with excitation peak absorbance that continuously shifted 340 nm and 380 nm wavelengths. To obtain the time-dependent changes, time-lapse recordings were set at different time intervals

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(300–500 s) for taking images at 1 s intervals, and then averaged the recording data from at least

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50–150 cells to select the region of interest, whereas the background fluorescence was automatically subtracted prior obtaining 340/380 ratio calculation and graphing). Analysis was performed offline using Slidebook™ software and further analyzed using statistical analysis by Origin 6.1. Electrophysiology Whole-patch clamp single cell ion channel recordings were performed as described previously (Yiu, Ibeh, Roy, & Bandyopadhyay, 2017; Ibeh et al., 2019) following the method stated earlier (Bandyopadhyay, Pingle, & Ahern, 2011). Cells were bathed in external solution containing (in

Journal Pre-proof mM): 140 NaCl, 4 KCl, 1 MgCl2 , 2 CaCl2 , 5 D-glucose, and 10 HEPES (NaOH, pH 7.4). We used intracellular solution, which contains (in mM): 50 CsCl, 10 NaCl, 60 CsF, 20 EGTA, and 10 HEPES (CsOH, pH 7.2). Whole-cell recordings were obtained with EPC-10 digitally controlled amplifier and Patchmaster software (HEKA, Lambrecht, Germany). I-V relationships were measured every 3 secs by applying voltage ramps (300 ms) from -100 mV to +100 mV from a holding potential of -80 mV. Data were acquired at 5.00 kHz and filtered with 2.873 kHz.

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After establishing the whole-cell configuration, the membrane resistance was >500 MΩ. All

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experiments were done at a constant room temperature set at 25 o C.

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DAPI staining

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Apoptotic nuclei were detected using DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich;

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catalog #: D9542; Li et al, 2001). Cell culture and crystal induction followed the same procedure as described earlier (Yiu, Ibeh, Roy, & Bandyopadhyay, 2017). Cells were fixed, after crystal

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induction, with 3% paraformaldehyde. Permeabilization (using 0.01% Triton X-100 in 0.1%

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BSA) was continued for 2 min, cells were then washed with 1×PBS. DAPI (1 μg/ml in 1× PBS pH 7.4) stains were applied to the cells for 10 min at room temperature while keeping in the

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dark. Cells were then washed (2X) with 1× PBS and visualized under fluorescence microscope (Axiovision, Carl Zeiss). Cell death percentage was calculated: # of cells indicative of cell death / the total # of cells * 100. Cell viability assay Cell viability assay was performed as described (Strober, 1997). Briefly, HK-2 cells were seeded and grown to 80% confluency then incubated with ROS inducer H2 O2 of various concentrations. Following crystal induction, cells were trypsinized and stained with trypan blue stain (SigmaAldrich; catalog #: T8154). Live (unstained) and dead (stained) cells were counted using a

Journal Pre-proof hemocytometer and standard procedure. Cell viability was calculated relative to control (no crystal) condition. Cell counts were performed in triplicates from 3 to 4 separate experiments. Annexin V/PI staining for apoptosis necrosis assay Apoptotic and necrotic cells were assessed using Alexa Fluro 488 Annexin V/Dead Cell apoptosis kit (Thermo Fisher Scientific; catalog #: V13241) according to manufacturer’s

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instructions and as previously described (Koopman et al., 1994). 1x binding buffer was used to wash the HK-2 cells and then was incubated with Annexin V/PI for 15 minutes at room

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RNA extraction and RT-PCR

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laser-scan fluorescence microscope (Carl Zeiss).

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temperature. Cells were then washed twice with the 1x binding and imaged with Zeiss LSM710

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Total RNA’s were isolated from HK-2 cells using TRIzol reagent (Invitrogen) as previously described (Bandyopadhyay, Pingle, & Ahern, 2011). DNase treatments, cDNA synthesis was

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performed using cDNA synthesis kit (Promega, Madison, WI; catalog #: A5000) for reverse

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transcription and then amplify the genes using gene-specific primers (Table 1) using the master

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mix PCR amplification reagent (Promega). All primers were purchased from either Invitrogen or Integrated DNA Technologies (Coralville, IA). Thermocycler (Bio-Rad, Hercules, CA), were set to PCR conditions as previously described (Yiu, Ibeh, Roy, & Bandyopadhyay, 2017). Table 1. List of primers used for RT-PCR. Primer Sequence (sense, antisense) BCL-2 5’-ATG GAC GGG TCC GGG GAG-3’ 5’-GGT GCC GGT TCA GGT ACT CA-3’ BAX-1 5’-ATG GAC GGG TCC GGG GAG-3’ 5’-ATC CAG CCC AAC AGC CGC-3’ GAPDH 5’-ATC GTG GGG CGC CCC AGG CAC-3’ 5’-CTC CTT AAT GTC ACG CAC GAT TTC-3’

Protein Analysis and Western Blotting

Journal Pre-proof Protein preparation, SDS-PAGE (using 4 –12% gels; Invitrogen), transferred from the gel onto an Immobilon-PSQ transfer membrane (Millipore Corp, Bedford, MA) were performed at room temperature as previously described (Yiu, Ibeh, Roy, & Bandyopadhyay, 2017; Ibeh et al., 2019). Antibody labeling was detected by enhanced chemiluminescence (ECL) using primary antibodies: mouse monoclonal antibodies against phospho-p38 (Cell Signaling Technology, Santa Cruz, CA; catalog #: 9216; 1:500 dilution), mouse monoclonal antibodies against p38

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(Santa Cruz Biotechnology, Danvers, MA; catalog #: sc-81621; 1:500 dilution) and a mouse

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monoclonal antibody against GAPDH (Santa Cruz Biotechnology; sc-32233; 1:2500 dilution).

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Anti-mouse secondary antibodies from Sigma-Aldrich and ECL kit from Pierce (Invitrogen)

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were used.

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Statistical analysis

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Experimental data were plotted, and the curve-fitting was performed using Origin 6.1. The data were expressed as means +/- S.E.M. Statistical comparisons were performed using Student’s

3 RESULTS

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< 0.05.

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unpaired t-test (two-tailed) in Origin 6.1. Statistically significant comparisons were accepted at P

3.1 L-ornithine activates CaSR in HK-2 cells Although the activation mechanism of CaSR by aromatic L-amino acids such as L-phenylalanine or L-tryptophan have been extensively studied (Conigrave, Quinn, & Brown, 2000), the details of L-ornithine activation is unknown. We wanted to see whether L-ornithine can activate the CaSR using Ca2+ imaging experiments in HK-2 cells, and found that L-ornithine elicits an increase in [Ca2+]i in a concentration-dependent (0-10 mM) fashion (Figure 1A; 0 mM and 1

Journal Pre-proof mM: P = 0.045, 1 mM and 3 mM: P = 0.64, 3 mM and 10 mM: P = 0.0079). To further confirm our finding, we performed whole-cell patch clamp experiment on those HK-2 cells and applied increasing concentrations of L-ornithine to look at the TRPC Ca2+ channel activity. Our results show that the L-ornithine induces a TRPC-like outwardly rectifying current with increasing concentrations (100 µM and 500 µM: P = 0.003, 500 µM and 1 mM: P = 0.001, 100 µM and 1000 µM: P < 0.001), suggesting that L-ornithine can induce GPCR-mediated Ca2+ entry in

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human PT cells (Figure 1B). To show the gene specific effect of CaSR, we did the targeted

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knockdown of CaSR gene using specific siRNA to CaSR (Yiu, Ibeh, Roy, & Bandyopadhyay,

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2017), and performed Ca2+ imaging experiments. Our data show that CaSR-siRNA has a concentration-dependent (10-20 nM) effect in HK-2 cells to attenuate the Ca2+ entry. Moreover,

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our results show that transfecting the CaSR-siRNA decreases the Ca2+ entry response by half,

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while doubling the siRNA amount decreases the entry even further (P < 0.001), though not as

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apparently as transfecting with half the amount (P < 0.001; Figure 1C-F). Taken together, these results show that L-ornithine can activate the CaSR in a concentration-dependent manner and

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exert its effect on Ca2+ signaling.

3.2 L-ornithine activates TRPC channel mediated Ca2+entry pathway in HK-2 cells While L-ornithine is a basic natural amino acid that can serve as a ligand for CaSR (Zhang et al., 2019), the downstream Ca2+-signaling pathway is largely unknown. Role of L-ornithine has been suggested in pancreatic acinar cells in mediating Ca2+ signaling and ROS generation (Chvanov et al., 2015), however, its mechanism of action in kidney cells has not been studied. Therefore, to examine the mechanism that L-ornithine action in these cells we used various pharmacological inhibitors which targets CaSR and its downstream Ca2+ signaling pathway(s). First, we used

Journal Pre-proof NPS-2143, a specific pharmacological blocker (Nemeth, 2002), to target CaSR for measurement of CaSR-activated Ca2+ influx. Our data show that NPS-2143 inhibited more than a two-thirds of the Ca2+ entry response (Figure 2A) compared to the control (Figure 1C; P = 0.003, not shown). We previously demonstrated that the Ca2+ signaling response by L-amino acids has been shown to activate a CaSR/TRPC channel-mediated mechanism in PT cells (Ibeh et al. 2019). To assess if the TRPC channel plays a role in mediating the Ca 2+ signaling, we used a known TRPC

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channel inhibitor, SKF-96365 (Figure 2B; Beech, Xu, McHugh, & Flemming, 2003). Compared

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to the response in control cells (Figure 1C), the inhibition exerted by SKF-96365 (P = 0.006, not

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shown), is almost equal to the amount of inhibitory response caused by NPS-2143 (P = 0.986), suggesting that L-ornithine mediated mostly TRPC channel-induced Ca2+ entry (Figure 2B).

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However, there is significant leftover of Ca2+ entry response (Figure 2B), which could be due to

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participation of other ER-store dependent mechanism. To determine what extent L-ornithine

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involves the IP3 -mediated pathway, we used an inhibitor for IP3 -induced Ca2+ release, 2-APB (Kukkonen, Lund, & Akerman, 2001), which produced inhibition of nearly half of the Ca2+

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response induced by L-ornithine (Figure 2C; P = 0.042, not shown), indicating the participation

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in part by ER Ca2+ store release signaling pathway. We further analyzed our data obtained from Figure 2A-C and confirmed that NPS-2143 and SKF-96365 have an equal footing as far as the inhibitory role in concern, whereas 2-APB effect was significantly lower (Figure 2D; 2-APB and NPS-2143: P = 0.024, 2-APB and SKF-96365: P = 0.026). Next, we performed whole-cell patch clamp experiments on the HK-2 cells for directly measuring the Ca2+ channel activity. Our data show that L-ornithine induced current was blocked by NPS-2143, SKF-96365 and 2-APB. Notably, NPS-2143 (Figure 2E; (P < 0.001) and SKF-96365 (Figure 2F) have inhibited the majority of the current (P < 0.001), but 2-APB has inhibited nearly all the L-ornithine activated

Journal Pre-proof current back to baseline (Figure 2G; P < 0.001). As it confirms the activation of a TRPC channel, however, the mode of action is not clear, since 2-APB have been often described as a nonspecific inhibitor of ER-Ca2+ release and/or Ca2+ entry blocker (Leon-Aparicio, Chavez-Reyes, & Guerrero-Hernandez, 2017). Therefore, the complete activation of current by 2-APB should be tested further with newly developed pharmacological inhibitor to understand the mode of Ca 2+ entry by L-ornithine. Together, these results show that activation of CaSR by L-ornithine mostly

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target TRPC channels to mediate the Ca2+ entry response in HK-2 cells.

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3.3 CaSR activation by L-ornithine triggers G-protein coupled ROCE pathway in HK-2 cells Because we observed some effect of 2-APB, which blocks that IP3 -mediated pathway, it is

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possible that L-ornithine-induced activation of TRPC channel can stimulate IP3 -induced Ca2+

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release, a component of SOCE. In fact, the CaSR activation of a TRPC channel can generate

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both ER Ca2+-store release due to IP3 generation, activating a SOCE component and/or could be due to a direct activation via DAG, via receptor-operated Ca2+ entry (ROCE; Brown & Macleod,

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2001). Therefore, we applied pyrazole compounds Pyr6, a known SOCE inhibitor, and Pyr10, a

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known ROCE inhibitor, for differentiating the modes of Ca2+ entry due to L-ornithine activation of CaSR (Schleifer et al., 2012) using Ca2+ imaging experiments on HK-2 cells. While the Lornithine activated Ca2+ entries for both treatments were decreased, the Ca2+ entry inhibited by Pyr10 (P < 0.001) have shown a significantly substantial decrease from the control when compared to that of Pyr6 (Figure 3A; P = 0.047). To confirm our findings from Ca2+ imaging experiments, we measured Ca2+ channel activity by performing whole-cell patch clamp experiments on HK-2 cells using Pyr6 and Pyr10. Our results show that L-ornithine application generated an outwardly rectifying current which was substantially blocked by Pyr10 (inhibited

Journal Pre-proof the L-ornithine current almost to baseline; P < 0.001), while Pyr6 has diminished ~48% of the current (Figure 3B-C; P = 0.010). These data thus suggest that in human PT cells primary mode of Ca2+ entry by L-ornithine activated CaSR mediated pathway, is via ROCE, similar to what we found in murine PT cells in response to L-phenylalanine mediated activation of CaSR (Ibeh et al., 2019). These data thus confirm our aggregate findings of L-amino acid mediated Ca2+ signaling in human PT cells and thus suggest for a positive effect of L-ornithine on PT cells due

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to more involvement of ROCE.

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3.4 L-ornithine has a protective effect against ROS generation in HK-2 cells

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Oxidative stress through ROS production and related redox signaling has been shown to play a major role in several pathophysiological cellular processes and is a hallmark of both acute and

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chronic kidney diseases (Nita & Grzybowski, 2016). Exogenous ROS intermediate, H2 O2 , has

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been shown to be a potent inducer of further ROS formation and oxidative stress (Ogawa et al., 2004). Interestingly, our recent demonstration show, that when switched the mechanism form

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SOCE to ROCE, the physiological function of PT cells demonstrated a reduction of ROS-

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induced ER stress and apoptosis pathway (Gombedza, Shin, Kanaras & Bandyopadhyay, 2019). Since L-ornithine exert its effect mainly though ROCE pathway, we explored if L-ornithine can offer such protecting effect. Therefore, we applied L-ornithine to examine its protective effect from peroxide-induced ROS production using HypeRed, a genetically-encoded ROS indicator, sensitive to peroxide production. In this experiment HK-2 cells were treated with none (control), 300 µM of L-ornithine, H2 O2 , H2 O 2 + L-ornithine 100 µM, and H2 O2 + 300 µM L-ornithine, and then measured the expression of induction of HypeRed using fluorescence microscopy. As expected, the H2 O2 induced HK-2 cells displayed a significant amount of ROS compared to the

Journal Pre-proof control group (Figure 4A-B; P < 0.001). However, the cells incubated with H2 O2 that were treated with L-ornithine reduced the ROS amount, indicating that L-ornithine can have a protective function against ROS (P = 0.001). We confirmed the results of our experiment by performing Ca2+ imaging experiment. We first induced a Ca2+ entry response by using H2 O2 as our control, which elicited a profound Ca2+ entry response (Figure 4C and D). After incubating the HK-2 cells with L-ornithine, we performed the same experiment and found that the peak Ca 2+

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entry has greatly diminished (Figure 4D: P = 0.010). To confirm the ROCE mediated protection

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of L-ornithine from ROS induced Ca2+ entry, we also incubated the cells with Pyr10. Our data

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shows that the ROS induced Ca2+ entry has increased, verifying that the ROCE has been

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inhibited (Figure 4D; P = 0.030). Together these data thus suggest the functional confirmation of

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the protection of L-ornithine against ROS formation in HK-2 cells via ROCE pathway.

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3.5 L-ornithine can protect against cell death due to pathophysiological condition Renal epithelial cells, specially the PT cells, are much more vulnerable to damage by

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endogenous calcium crystals formed in renal tubular fluid (Schepers, van Ballegooijen, Bangma,

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& Verkoelen, 2005; Khan et al., 1999). We have recently demonstrated that calcium phosphate (CaP), calcium oxalate (CaOx) and mixed (CaP+CaOx) can lead to apoptosis (Gombedza et al., 2019). Moreover, such crystal induced ER stress and apoptosis can be mediated by SOCE, which can be reversed when ROCE comes into play (Gombedza, Shin, Kanaras & Bandyopadhyay, 2019). Since L-ornithine can provide protection in the experimental conditions of ROS induction via ROCE, we wanted to find out what extent L-ornithine can provide protection in more translational pathophysiological conditions, i.e. in crystal-induced renal injury. Thus, we treated these human PT cells with or without L-ornithine and then exposed cells to crystals to observe

Journal Pre-proof the effect on cell damage. We stained the cells afterwards with DAPI to visualize cellular damage and found that crystal internalization resulted in significant nuclear fragmentation (damage; P < 0.001); importantly, L-ornithine preincubated cells were protected against crystalinduced cell damage (Figure 5A-B; P < 0.001). Since we found that L-ornithine normalized H2 O 2 -induced Ca2+ elevation via ROCE, we next assessed the effect of ROS (H2 O2 ) alone, plus L-ornithine and its blockade due to ROCE on apoptosis by annexin-V staining and necrosis by

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propidium iodide (PI) staining (dead cells uptake the stains as an indicator necrosis). To examine

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the effect, we preincubated the cells with L-ornithine and then exposed to with or without H2 O2 ,

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adding Pyr10 as ROCE pathway blocker. Remarkably, our data showed that L-ornithine

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protected from H2 O2 -induced necrosis (P < 0.001) and apoptosis (P < 0.001) and that the ROCE

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0.016 and P = 0.025, respectively).

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blockade diminished the effect of L-ornithine on H2 O2 -induced cell death (Figure 5C-D; P =

3.6 L-ornithine can provide protection against oxidative stress-induced cell death

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In accordance, we next evaluated the protective effect of L-ornithine on cell survival by

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examining the mitochondrial oxidative stress components. Cell viability percentage of those induced with H2 O2 has diminished by nearly half compared to the control (Figure 6A; P < 0.001). However, cells induced with H2 O 2 after incubation of L-ornithine has maintained viability, further conferring L-ornithine as a protecting agent against H2 O2 induced PT cell death (Figure 6A; P = 0.154). As a confirmatory evidence, addition of Pyr10 to H2 O2 + L-ornithine diminished the cell viability, suggesting that the involvement of ROCE in the protection of PT cells (Figure 6A; P = 0.038). Since L-ornithine protection against cell death is via ROCE we evaluated the BAX/BCL-2 ratio to determine L-ornithine effect on apoptotic potential due to

Journal Pre-proof ROS production. Our data showed that H2 O2 treatment resulted in a BAX/ BCL-2 ratio greater than 1.0, which is indicative of apoptosis (Figure 6C). Significantly, L-ornithine treatment reduced H2 O 2 -induced apoptotic potential (P = 0.002) and this effect was mediated by ROCE, because Pyr10 reversed such effect (Figure 6C; P = 0.009), whereas neither L-ornithine (P = 0.419) nor Pyr10 (P = 0.411) alone had any apoptotic effect. Taken together, our data confirm that L-ornithine can reverse the ROS and oxidative damage in HK-2 cells that are exposed to

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H2 O 2 .

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3.6 p38 MAPK mediates nephroprotective effect of L-ornithine

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Mitogen-activated protein kinases (MAPK) activation in intracellular signal transduction has long been understood to be pro-apoptotic, however, studies have emerged, which demonstrated

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the protective/beneficial role of several MAPKs including p38 signaling (Clark, Sarafraz, &

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Marber, 2007; Rose, Force, & Wang, 2010; Christov & Alper, 2010). It has been shown that p38 can protect against oxidative stress-induced cell death (Hernández et al., 2011). To examine the

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role of L-ornithine signaling, we exposed HK-2 cells to L-ornithine in a concentration and time-

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dependent manner and analyzed downstream process such as phosphorylation of MAPK. In HK2 cells, we find that among the MAPK, p38 is more responsive to ROS (Figure S1). Therefore, we looked at that p38 phosphorylation, which was optimally activated by 100 μM L-ornithine after 60 mins of exposure (P = 0.034), and we maintained similar conditions for further experimentation (Figure 7A-B).

To

understand the molecular mechanism of L-ornithine

protection, we next examined p38 phosphorylation in the presence of H2 O 2 and/or Pyr10. Our data show that while H2 O2 did not alter L-ornithine induced p38 activity (P = 0.601), ROCE blockade with Pyr10 markedly reduced L-ornithine-induced p38 activation (Figure 7C-D; P =

Journal Pre-proof 0.005), which suggest that ROCE pathway regulate the ROS (H2 O2 )-induced activation of p38 phosphorylation. However, residual amount of p38 activity remains, which may be regulated by other MAPKs. Importantly, H2 O2 (P = 0.237) and Pyr10 (P = 0.761) did not have any change on p38 phosphorylation over the basal (control) activity, suggesting the specificity of our experiment. Together, our findings suggest that the protection conferred by L-ornithine on ROS-

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induced apoptosis was mediated by p38 MAPK activation through ROCE pathway.

4 DISCUSSION

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Ca2+ sensing by L-amino acids has been well established in the gut, specifically, L-phenylalanine

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and L-tryptophan, causing [Ca2+]i mobilization (Ojha, 2018). Following these, we have

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conducted functional studies in PT cells, which suggest that activation of the CaSR by L-

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phenylalanine and L-tryptophan resulted in Ca2+ entry mainly through the ROCE pathway (Ibeh et al., 2019). Our study evaluates for the first time the physiological role which L-ornithine,

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which present in highest concentration in PT luminal fluid among other amino acids when

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compared to other tubular segments (Gonzalez-Vicente & Garvin, 2017), in the activation of

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CaSR. While other studies have shown that L-ornithine could also allosterically activate a similarly structured GPCR called GPRC6, the receptor expression in human PT cells is not well understood (Oya et al., 2012, Wellendorph et al., 2005). Moreover, besides using a known CaSR inhibitor, NPS-2143, we have undertaken CaSR-gene-specific (CaSR siRNA transfected HK-2 cells) knockdown to show in our physiological experiments a concentration-dependent effect on CaSR-mediated, Ca2+-entry induced by L-ornithine, is indeed, through the CaSR activation pathway. The data presented above show for the first time that in human PT cells, L-ornithine can allosterically activate CaSR and generate a PLC-mediated Ca2+ entry, leading to a rise in [Ca2+]i. We show that such elevation of [Ca2+]i, is mainly caused by ROCE, via the direct

Journal Pre-proof activation of TRPC channel. Our previous studies show that melamine-stimulated CaSRmediated Ca2+ signaling resulted in a sustained Ca2+ entry, which can prolong the rise in [Ca2+]i. In contrast, Ca2+ entry response by L-ornithine shows a rapid onset with a steady decline, which might work in the alleviation of ER stress response. Such intracellular Ca2+ signaling pattern may reduce the ROS generation, which can further slowdown mitochondrial oxidative stress, which could cause DNA damage, resulting in apoptotic and necrotic cell death. In fact,

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extracellular L-amino acid, such as L-arginine, has been shown to be required for proper ER

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function and fight against oxidative damage arise within cardiomyocytes (Ramachandran &

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Peluffo, 2017). Our recent study shows that crystal-mediated ER-stress response can be reversed

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when Ca2+ entry pathway switch from SOCE to ROCE (Gombedza, Shin, Kanaras, & Bandyopadhyay, 2019). Such protection against cell damage could be also be mediated through

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1) suppression of mitochondrial pro-apoptotic (BAX-1/BCL-2) pathway, and 2) reduction in

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ROS-mediated cytotoxicity, which attenuates necrosis (Redza-Dutordoir & Averill-Bates, 2016). Our data therefore provide evidence that L-ornithine generate its protective response via a ROCE

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rather than SOCE, which is summarized a possible mechanism of action of L-ornithine in

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schematic diagram (Figure 8).

Several studies described the roles of the three major MAPKs [extracellular signal-regulated kinase

(ERK)1/2,

c-Jun

NH2-terminal

survival/apoptosis (Cowan & Storey, 2003).

kinase

(JNK),

and

p38]

in

mediating

cell

However, the regulation of these intracellular signal

kinases in response to different upstream physiological signaling such as Ca 2+ signaling, that results in pro-apoptotic and/or survival effect is not clear. Moreover, cell types and variety of experimental conditions are also the factors resulting the divergence of signaling pathway (Brozovic & Osmak, 2007). In our case, the ERK phosphorylation expression in HK-2 cells did

Journal Pre-proof not change when the cells are exposed to either L-ornithine or H2 O2 (Figure S1). We found that L-ornithine mediates its reduction in oxidative stress via induction of p38 MAPK, thus targeted by us as a key mediator to alleviate the stress signals for protecting against apoptosis. We propose that L-ornithine induced a protective [Ca2+]i signaling to reduce a caspase-mediated apoptosis pathway by phosphorylation of p38 MAPK, which protects from tubular cell injury. The exact role of p38 MAPK in the protection of programmed cell death remains to be

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determined. Although activation of MAPKs was observed following L-ornithine mediated

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reduction in oxidative stress and p38 MAPK activation are rather implicated in cellular

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resistance against the oxidative stress. We think that the individual isoforms of the p38 MAPK

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may possess distinct biological functions determination of which is out of the scope of the present manuscript. Importantly, this finding in the revisal of cell death can provide a greater role

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of p38 MAPK in many aspects of protective effect.

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Reabsorption of L-ornithine in PT is mediated by amino acid transporter proteins expressed at

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the luminal membrane (Thomson et al., 2001; Ashman et al., 2005). Notably, vertebrates are

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dependent on the formation L-ornithine, as being absent from that food (not a constituent of proteins), therefore, L-ornithine activation of CaSR may not be implicated in GI tract (Levillain et al., 2004). In this study, L-ornithine has been described as a signaling molecule, which is present in the PT fluid in vivo, and can trigger the activation of CaSR to protect the PT cells from undergoing cell damage. Renal tubular cells are especially vulnerable to free radicals due to environmental factors. Moreover, oxidative damage has been linked to many kidney pathologies including Chronic Kidney Disease (CKD; Kao, Ang, Pall, & Struthers, 2010). We propose that L-ornithine can prevent such process via the operation of Ca 2+ signaling pathway. Such

Journal Pre-proof regulation by Ca2+ entry pathway to counteracts apoptosis has been established by others (Huang et al., 2017)

Kidney

diseases

such

as

ischemic

acute

renal

failure,

renal graft

rejection,

acute

glomerulonephritis, and toxic renal diseases are mediated by ROS production (Baud, & Ardaillou, 1986). To investigate the protective effect of L-ornithine, we simulated deleterious

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effects using H2 O2 /crystal internalization and assessed ROS production using genetically linked

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HypeRed indicator (Ermakova et al., 2014). Our results also showed L-ornithine significantly reduced H2 O 2 induced ROS production and protected against crystal-induced cytotoxic effects.

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Previous studies have reported that L-ornithine can propagate the formation of ROS in its studies

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with pancreatic acinar cells; however, it appears that higher concentrations were used to induce

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pathophysiological conditions (Chvanov et al., 2015; Zhang et al., 2019). Importantly, we observed the protective effects of L-ornithine following oxidative stress or crystal internalization

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in HK-2. Our study provides evidence for the nephroprotective function of L-ornithine and

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crystal nephropathies.

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suggests a potential new therapeutic role for acute kidney injury, involving oxidative stress or

ACKNOWLEDGMENTS

We would like to thank our Lab members Cliff-Lawrence Ibeh, Yianni L. Kanaras, and Dr. Peijun Li for helping in some Ca2+ imaging and patch clamp experiments and data analysis. We also thank National Institute of Diabetes and Digestive and Kidney Diseases (DK102043) and National Institute of Biomedical Imaging and Bioengineering (EB021483) for their funding support. FUNDING National Institute of Diabetes and Digestive and Kidney Diseases (DK102043) and National Institute of Biomedical Imaging and Bioengineering (EB021483) funding support to B.C.B supported this study. Although there was no involvement of those funding agencies in the

Journal Pre-proof preparation of this manuscript, design, collection, analyses, and interpretation of the data, writing of the report, or decision to submit this article for publication. CONFLICT OF INTERESTS The authors declare that there is no conflict of interests. AUTHOR CONTRIBUTIONS

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Conception and design: B. C. B., S. S. and F.C. G.; Acquisition and analysis (e.g., statistical analysis) of experimental data: S. S., F. C. G.; Interpretation of data: B. C. B., S. S., F. C. G.; Writing the manuscript draft, review and revision of the manuscript: S. S., F. C. G., B. C. B. Funding acquisition: B. C. B.

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DATA ACCEESSIBILITY

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The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Figures Legends

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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 [Ca 2+]i after L-orn application. B. Whole-cell patch clamp current of HK-2 cell upon concentration dependent application of Lornithine (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.

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Figure 2. Mean Fura-2 fluorescent traces of HK-2 cells bathed in 0.5 mM [Ca2+]o solution incubated in A. 12 µM 2-APB, B. 1 uM NPS, or C. 1 uM SKF-96163, 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 ttest statistical analysis performed for D, E, F, and G. *, p<0.05; **, p<0.01.

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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 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-ornithine (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. Two-tailed t-test statistical analysis performed for A, B, and C. *, p<0.05; **, p<0.01. Figure 4. A. HK-2 cells were transfected with HypeRed (H2 O2 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: 50µM of H2 O 2 and 2mM Ca2+ were added at their indicated positions. A substantial and prolonged Ca 2+ entry was observed. HK-2 cells were incubated with 1mM L-Orn or 1mM L-Orn and 1µM Pyr10 for 1 hour and

Journal Pre-proof 50µM H2 O2 and 2mM 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. Statistical analysis for unpaired two-tailed t-test performed for B and D; *, p < 0.05; **, p < 0.01. Figure 5. L-ornithine protects HK-2 cells from mixed crystal induced apoptosis and necrosis. HK-2 cells were treated or not treated with L-ornithine (L-Orn) for an hour prior to incubating cells with or without CaP/CaOx mixed crystals. Cells were stained with A. DAPI, B. PI, or C. Annexin staining to assess cell death. White arrows indicate defragmentation of the nuclear DNA. 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.

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Figure 6. HK-2 cells were pretreated with or without L-ornithine (L-Orn; 10 µM) before inducing ROS with H2 O2 (500 µM). A. Cell viability assay was performed on these cells. B. RTPCR 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. The Ca2+ entry was not substantial or prolonged indicating a protective property of LOrn.

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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, H2 O2 , 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.

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Figure 8. Schematic diagram of L-ornithine activated ROCE pathway protecting PT cell. Lornithine (L-Orn) allosterically activates the CaSR and triggers the ROCE Ca 2+ 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. Figure S1. ERK phosphorylation induction by L-ornithine (L-Orn) or H2 O2 . Western analysis of expression of phosphorylated ERK (p-ERK) and total ERK (T-ERK) expression untreated HK-2 cells (control) incubated with A. L-orn or B. H2 O2 . B. Bar diagram represents A in mean+s.e.m. Statistical analysis performed on A. and B. with two-tailed t-test. *, p < 0.05; **, p < 0.01. Graphical abstract

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