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Cadmium Disrupted ER Ca²⁺ Homeostasis by Inhibiting SERCA2 Expression and Activity to Induce Apoptosis in Renal Proximal Tubular Cells.

ABSTRACT: Cadmium (Cd²⁺) exposure induces chronic kidney disease and renal cancers, which originate from injury and cancerization of renal tubular cells. Previous studies have shown that Cd²⁺ induced cytotoxicity by disrupting the intracellular Ca²⁺ homeostasis that is physically regulated by the endoplasmic reticulum (ER) Ca²⁺ store. However, the molecular mechanism of ER Ca²⁺ homeostasis in Cd²⁺-induced nephrotoxicity remains unclear. In this study, our results firstly revealed that the activation of calcium-sensing receptor (CaSR) by NPS R-467 could protect against Cd²⁺ exposure-induced cytotoxicity of mouse renal tubular cells (mRTEC) by restoring ER Ca²⁺ homeostasis through the ER Ca²⁺ reuptake channel sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA). Cd²⁺-induced ER stress and cell apoptosis were effectively abrogated by SERCA agonist CDN1163 and SERCA2 overexpression. In addition, in vivo, and in vitro results proved that Cd²⁺ reduced the expressions of SERCA2 and its activity regulator phosphorylation phospholamban (p-PLB) in renal tubular cells. Cd²⁺-induced SERCA2 degradation was suppressed by the treatment of proteasome inhibitor MG132, which suggested that Cd²⁺ reduced SERCA2 protein stability by promoting the proteasomal protein degradation pathway. These results suggested that SERCA2 played pivotal roles in Cd²⁺-induced ER Ca²⁺ imbalance and stress to contribute to apoptosis of renal tubular cells, and the proteasomal pathway was involved in regulating SERCA2 stability. Our results proposed a new therapeutic approach targeting SERCA2 and associated proteasome that might protect against Cd²⁺-induced cytotoxicity and renal injury.

SUBMITTER: Li K

PROVIDER: S-EPMC10053525 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Cadmium Disrupted ER Ca2+ Homeostasis by Inhibiting SERCA2 Expression and Activity to Induce Apoptosis in Renal Proximal Tubular Cells.

Li Kongdong K Guo Chuanzhi C Ruan Jiacheng J Ning Bo B Wong Chris Kong-Chu CK Shi Haifeng H Gu Jie J

International journal of molecular sciences 20230322 6

Cadmium (Cd2+) exposure induces chronic kidney disease and renal cancers, which originate from injury and cancerization of renal tubular cells. Previous studies have shown that Cd2+ induced cytotoxicity by disrupting the intracellular Ca2+ homeostasis that is physically regulated by the endoplasmic reticulum (ER) Ca2+ store. However, the molecular mechanism of ER Ca2+ homeostasis in Cd2+-induced nephrotoxicity remains unclear. In ...[more]

PMID: 36983052

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Project description:Calcium crystal internalization into proximal tubular (PT) cells results in acute kidney injury, nephrocalcinosis, chronic kidney disease (CKD), and kidney-stone formation. Ca2+ supersaturation in PT luminal fluid induces calcium crystal formation, leading to aberrant crystal internalization into PT cells. While such crystal internalization produces reactive oxygen species (ROS), cell membrane damage, and apoptosis; the upstream signaling events involving dysregulation of intracellular Ca2+ homeostasis and ER stress, remain largely unknown. We have recently described a transepithelial Ca2+ transport pathway regulated by receptor-operated Ca2+ entry (ROCE) in PT cells. Therefore, we examined the pathophysiological consequence of internalization of stone-forming calcium crystals such as calcium phosphate (CaP), calcium oxalate (CaOx), and CaP + CaOx (mixed) crystals on the regulation of intracellular Ca2+ signaling by measuring dynamic changes in Ca2+ transients in HK2, human PT cells, using pharmacological and siRNA inhibitors. The subsequent effect on ER stress was measured by changes in ER morphology, ER stress-related gene expression, endogenous ROS production, apoptosis, and necrosis. Interestingly, our data show that crystal internalization induced G-protein-coupled receptor-mediated sustained rise in intracellular Ca2+ concentration ([Ca2+]i) via store-operated Ca2+ entry (SOCE); suggesting that the mode of Ca2+ entry switches from ROCE to SOCE following crystal internalization. We found that SOCE components-stromal interacting molecules 1 and 2 (STIM1, STIM2) and ORAI3 (SOCE) channel were upregulated in these crystal-internalized cells, which induced ER stress, ROS production, and cell death. Finally, silencing those SOCE genes protected crystal-internalized cells from prolonged [Ca2+]i rise and ER stress. Our data provide insight into the molecular mechanism of crystal-induced Ca2+ dysregulation, ER stress, and PT cell death and thus could have a translational role in treating crystal nephropathies including kidney stones. Taken together, modulation of Ca2+ signaling can be used as a tool to reverse the pathological consequence of crystal-induced conditions including cardiovascular calcification.

Project description:We examined the roles of Notch1 signaling and its cross-talk with other signaling pathways, including p53 and phosphatidylinositol-3-kinase (PI3K)/Akt, in cadmium-induced cellular damage in HK-2 human renal proximal tubular epithelial cells. Following exposure to cadmium chloride (CdCl2), the level of Notch intracellular domain (NICD), the cleaved form of the Notch1 receptor, was increased and accumulated in the nuclear fraction. Knockdown of Notch1 with siRNA or treatment with the γ-secretase inhibitor, DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester), prevented CdCl2-induced morphological change of HK-2 cells and reduction of cell viability. Knockdown of Jagged1 or Jagged2, the ligands of the Notch1 receptor, partially suppressed cadmium cytotoxicity. Inhibition of p53 activity with pifithrin-α or inhibition of PI3K with LY294002 suppressed CdCl2-induced cellular damage and elevation of Notch1-NICD. In addition, treatment with the epidermal growth factor receptor (EGFR) inhibitor, AG1478, and the insulin-like growth factor-1 receptor inhibitor, PPP, suppressed both Notch1-NICD accumulation and Akt phosphorylation in HK-2 cells exposed to CdCl2. However, knockdown of Notch1 did not affect CdCl2-induced p53 accumulation and phosphorylation but suppressed phosphorylation of EGFR, Akt, and p70 S6 kinase. Depletion of Notch1 suppressed CdCl2-induced reduction of E-cadherin expression and elevation of Snail expression. Furthermore, treatment with SB216763, an inhibitor of glycogen synthase kinase-3, suppressed the potency of LY294002 treatment to reduce Snail expression in HK-2 cells exposed to CdCl2. Knockdown of Snail with siRNA partially prevented HK-2 cells from CdCl2-induced reduction of E-cadherin expression and cellular damage. These results suggest that cadmium exposure induces the activation of Notch1 signaling in renal proximal tubular cells with cooperative activation by the p53 and PI3K/Akt signaling pathways; the resultant expression of Snail, a repressor of E-cadherin expression, might lead to cellular damage by decreasing cell-cell adhesion.

Project description:RationaleWe recently discovered pivotal contributions of stress kinase JNK2 (c-Jun N-terminal kinase isoform 2) in increased risk of atrial fibrillation through enhanced diastolic sarcoplasmic reticulum (SR) calcium (Ca2+) leak via RyR2 (ryanodine receptor isoform 2). However, the role of JNK2 in the function of the SERCA2 (SR Ca2+-ATPase), essential in maintaining SR Ca2+ content cycling during each heartbeat, is completely unknown.ObjectiveTo test the hypothesis that JNK2 increases SERCA2 activity SR Ca2+ content and exacerbates an arrhythmic SR Ca2+ content leak-load relationship.Methods and resultsWe used confocal Ca2+ imaging in myocytes and HEK-RyR2 (ryanodine receptor isoform 2-expressing human embryonic kidney 293 cells) cells, biochemistry, dual Ca2+/voltage optical mapping in intact hearts from alcohol-exposed or aged mice (where JNK2 is activated). We found that JNK2, but not JNK1 (c-Jun N-terminal kinase isoform 1), increased SERCA2 uptake and consequently elevated SR Ca2+ content load. JNK2 also associates with and phosphorylates SERCA2 proteins. JNK2 causally enhances SERCA2-ATPase activity via increased maximal rate, without altering Ca2+ affinity. Unlike the CaMKII (Ca2+/calmodulin-dependent kinase II)-dependent JNK2 action in SR Ca2+ leak, JNK2-driven SERCA2 function was CaMKII independent (not prevented by CaMKII inhibition). With CaMKII blocked, the JNK2-driven SR Ca2+ loading alone did not significantly raise leak. However, with JNK2-CaMKII-driven SR Ca2+ leak present, the JNK2-enhanced SR Ca2+ uptake limited leak-induced reduction in SR Ca2+, normalizing Ca2+ transient amplitude, but at a higher arrhythmogenic SR Ca2+ leak. JNK2-specific inhibition completely normalized SR Ca2+ handling, attenuated arrhythmic Ca2+ activities, and alleviated atrial fibrillation susceptibility in aged and alcohol-exposed myocytes and intact hearts.ConclusionsWe have identified a novel JNK2-induced activation of SERCA2. The dual action of JNK2 in CaMKII-dependent arrhythmic SR Ca2+ leak and a CaMKII-independent uptake exacerbates atrial arrhythmogenicity, while helping to maintain normal levels of Ca2+ transients and heart function. JNK2 modulation may be a novel therapeutic target for atrial fibrillation prevention and treatment.

Dataset Information

Cadmium Disrupted ER Ca²⁺ Homeostasis by Inhibiting SERCA2 Expression and Activity to Induce Apoptosis in Renal Proximal Tubular Cells.

Publications

Cadmium Disrupted ER Ca<sup>2+</sup> Homeostasis by Inhibiting SERCA2 Expression and Activity to Induce Apoptosis in Renal Proximal Tubular Cells.

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