SIRT1 alleviates Cd nephrotoxicity through NF-κB/p65 deacetylation-mediated pyroptosis in rat renal tubular epithelial cells.

Cadmium (Cd) is a widely distributed environmental pollutant, primarily causing nephrotoxicity through renal proximal tubular cell impairment. Pyroptosis is an inflammation-related nucleotide-binding oligomerization segment-like receptor family 3 (NLRP3)-dependent pathway for programmed cell death. We previously reported that inappropriate inflammation caused by Cd is a major contributor to kidney injury. Therefore, research on Cd-induced inflammatory response and pyroptosis may clarify the mechanisms underlying Cd-induced nephrotoxicity. In this study, we observed that Cd-induced nephrotoxicity is associated with NLRP3 inflammasome activation, leading to an increase in proinflammatory cytokine expression and secretion, as well as pyroptosis-related gene upregulation, both in primary rat proximal tubular (rPT) cells and kidney tissue from Cd-treated rats. In vitro, these effects were significantly abrogated through siRNA-based Nlrp3 silencing; thus, Cd may trigger pyroptosis through an NLRP3 inflammasome-dependent pathway. Moreover, Cd exposure considerably elevated reactive oxygen species (ROS) content. N-acetyl-l-cysteine, an ROS scavenger, mitigated Cd-induced NLRP3 inflammasome activation and subsequent pyroptosis. Mechanistically, Cd hindered the expression and deacetylase activity of SIRT1, eventually leading to a decline in SIRT1-p65 interactions, followed by an elevation in acetylated p65 levels. The administration of resveratrol (a SIRT1 agonist) or overexpression of Sirt1 counteracted Cd-induced RELA/p65/NLRP3 pathway activation considerably, leading to pyroptosis. This is the first study to reveal significant contributions of SIRT1-triggered p65 deacetylation to pyroptosis and its protective effects against Cd-induced chronic kidney injury. Our results may aid in developing potential therapeutic strategies for preventing Cd-induced pyroptosis through SIRT1-mediated p65 deacetylation.

Cadmium Induces Kidney Iron Deficiency and Chronic Kidney Injury by Interfering with the Iron Metabolism in Rats.

Cadmium (Cd) is a common environmental pollutant and occupational toxicant that seriously affects various mammalian organs, especially the kidney. Iron ion is an essential trace element in the body, and the disorder of iron metabolism is involved in the development of multiple pathological processes. An iron overload can induce a new type of cell death, defined as ferroptosis. However, whether iron metabolism is abnormal in Cd-induced nephrotoxicity and the role of ferroptosis in Cd-induced nephrotoxicity need to be further elucidated. Sprague Dawley male rats were randomly assigned into three groups: a control group, a 50 mg/L CdCl2-treated group, and a 75 mg/L CdCl2-treated group by drinking water for 1 month and 6 months, respectively. The results showed that Cd could induce renal histopathological abnormalities and dysfunction, disrupt the mitochondria's ultrastructure, and increase the ROS and MDA content. Next, Cd exposure caused GSH/GPX4 axis blockade, increased FTH1 and COX2 expression, decreased ACSL4 expression, and significantly decreased the iron content in proximal tubular cells or kidney tissues. Further study showed that the expression of iron absorption-related genes SLC11A2, CUBN, LRP2, SLC39A14, and SLC39A8 decreased in proximal tubular cells or kidneys after Cd exposure, while TFRC and iron export-related gene SLC40A1 did not change significantly. Moreover, Cd exposure increased SLC11A2 gene expression and decreased SLC40A1 gene expression in the duodenum. Finally, NAC or Fer-1 partially alleviated Cd-induced proximal tubular cell damage, while DFO and Erastin further aggravated Cd-induced cell damage. In conclusion, our results indicated that Cd could cause iron deficiency and chronic kidney injury by interfering with the iron metabolism rather than typical ferroptosis. Our findings suggest that an abnormal iron metabolism may contribute to Cd-induced nephrotoxicity, providing a novel approach to preventing kidney disease in clinical practice.

Cadmium exposure exacerbates kidney damage by inhibiting autophagy in diabetic rats.

The incidence of diabetes mellitus (DM) is gradually increasing, making it a widespread global health concern. Cadmium (Cd) is a common toxic heavy metal in the environment, and cadmium exposure may be associated with diabetic nephropathy (DN). However, the mechanism of Cd-induced DN remains unclear. In this study, we aimed to determine the effect of cadmium on diabetic kidney injury and the underlying mechanism in diabetic rats and a renal tubular epithelial cell line (NRK-52E cells). Our results could provide novel insights on the nephrotoxic mechanism of cadmium. HE, PAS, and Masson staining were used to observe pathological renal injury. COL-I, COL-IV, CTSB, and CTSD protein levels were detected by immunohistochemistry and western blotting. Immunofluorescence was used to detect the fluorescence intensity of p62 and LC3 proteins in kidney tissue. TEM was used to observe the ultrastructure of mitochondria and number of autophagosomes. After cadmium exposure, DM rats showed a dramatic decrease in body weight compared to the unexposed DM group. Relative kidney weight showed a contrasting trend after cadmium exposure. Urinary microalbumin/creatinine significantly increased in normal and DM rats after cadmium exposure. However, the trend was clearer in the DM groups than in the control groups. Endogenous creatinine clearance exhibited a contrasting trend. After cadmium exposure in DM rats, MDA content significantly increased and GSH, CAT, SOD, and GSH-PX activation reduced compared to normal controls. Pathological damage was more pronounced, and the expression of autophagy related proteins and apoptosis and fibrosis proteins was significantly higher in vivo and vitro in the cadmium-exposed groups than in unexposed controls. Further, lysosomal protein levels were lower, and ROS content and autophagosome count significantly higher in the cadmium exposed groups compared to the unexposed controls. Therefore, Cadmium exposure aggravates diabetic kidney injury via autophagy inhibition.

N-acetylcysteine delayed cadmium-induced chronic kidney injury by activating the sirtuin 1-P53 signaling pathway.

With the development of modern industrial civilization, cadmium (Cd), a known nephrotoxic metal, has become a growing public safety issue due to its ability to induce various types of kidney disease. Maladaptive proximal tubule repair is a significant cause of Cd-induced chronic kidney disease (CKD), which is characterized by premature senescence and pro-fibrosis. Previously, we demonstrated that cadmium causes DNA damage and cycle arrest in renal tubular epithelial cells, which may be relevant to premature senescence regulated by sirtuin 1 (SIRT1). In this study, in vivo and in vitro studies were conducted to elucidate the role of SIRT1-mediated premature renal senescence in Cd-induced CKD. As oxidative stress is a significant cause of aging, we evaluated whether N-acetylcysteine (NAC) would inhibit Cd-induced premature aging and dysfunction in rat renal tubular epithelial cells. Cadmium induced premature renal senescence and fibrosis, and NAC inhibited premature renal senescence and fibrosis through the SIRT1-P53 pathway and delayed CKD progression. Overall, the results suggested that the SIRT1-P53 pathway mediates oxidative stress, premature renal senescence, and renal fibrosis during cadmium exposure, which may be a potential therapeutic target for Cd-induced CKD.

Nuclear factor-kappaB mediates the survival of rat kidney cells after cadmium exposure via promoting autophagy and inhibiting apoptosis.

Cadmium (Cd) is a heavy metal pollutant in the environment, and the kidney is one of the target organs after Cd exposure. Previous studies have shown that apoptosis and autophagy disorders are the main mechanisms of Cd-induced nephrotoxicity in rats. As a transcription factor that balances cell survival and death, nuclear factor-kappaB (NF-κB) protein plays dual regulatory effects on apoptosis and autophagy in multiple renal diseases. However, the regulatory mechanisms of NF-κB in Cd-induced kidney injury remain unclear. Therefore, the normal rat kidney cell line (NRK-52E cells) was applied to investigate the above questions in this study. Here, we found that Cd promotes the nuclear translocation and activation of NF-κB in a concentration-dependent manner, and activated NF-κB mediates NRK-52E cells survival after Cd exposure. Next, our study elaborated the mechanisms of NF-κB in antagonizing Cd-induced renal cytotoxicity. Inhibition of NF-κB by inhibitor BAY 11-7082 (BAY) and NF-κB p65 siRNA (siNF-κB p65) exacerbate Cd-induced apoptosis and autophagy inhibition, and then aggravate Cd-induced NRK-52E cells injury. Activation of NF-κB by activator phorbol-12-myristate-13-acetate (PMA) alleviates Cd-induced apoptosis and autophagy inhibition, and then attenuates Cd-induced NRK-52E cells injury. In conclusion, Cd exposure promotes the activation of NF-κB, and activated NF-κB mediates the survival of NRK-52E cells after Cd exposure via promoting autophagy and inhibiting apoptosis.

Puerarin alleviates cadmium-induced rat neurocyte injury by alleviating Nrf2-mediated oxidative stress and inhibiting mitochondrial unfolded protein response.

Cadmium (Cd) is a highly neurotoxic environmental pollutant. Puerarin (Pur) is a natural antioxidant isolated from Kudzu root that exhibits a powerful neuroprotective effect. Herein, we illustrated the mechanism underlying the protective effect of Pur on Cd-induced rat neurocyte injury in an in vivo rat model as well as in vitro using PC12 cells and primary rat cerebral cortical neurons. First, the results showed that Pur alleviated Cd-induced cerebral cortical pathological damage and decreased the viability of neurocytes. Furthermore, Cd activated the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, which plays a negative role in Cd-induced rat neurocyte injury. In addition, Pur alleviated Cd-induced oxidative stress by enhancing antioxidant defense, reducing reactive oxygen species (ROS) accumulation and lipid peroxidation, and inhibiting activation of the Nrf2 signaling pathway in rat neurocytes. Moreover, Pur inhibited the Cd-induced mitochondrial unfolded protein response (UPRmt) in rat neurocytes. Overall, Pur alleviated Cd-induced rat neurocyte injury by alleviating Nrf2-mediated oxidative stress and inhibiting UPRmt.

Honokiol Antagonizes Cadmium-Induced Nephrotoxicity in Quail by Alleviating Autophagy Dysfunction, Apoptosis and Mitochondrial UPR Inhibition with Its Antioxidant Properties.

Japanese quail is a highly economically valuable bird due to its commercial production for meat and eggs. Although studies have reported Cadmium (Cd) is a ubiquitous heavy metal that can cause injury to various organs, the molecular mechanisms of Cd on quail kidney injury remain largely unknown. It has been reported that Honokiol (HKL), a highly functional antioxidant, can protect cells against oxidative stress effectively. This study was conducted to investigate the effects of Cd on quail kidneys injury and the protective effect of HKL on Cd-induced nephrotoxicity. A total of 40 Japanese quails were randomly divided into four groups: the control group, Cd treatment group, Co-treatment group and HKL treatment group. The results showed that Cd resulted in significant changes in growth performance, kidney histopathology and kidney biochemical status, antioxidant enzymes and oxidative stress parameters, and ultrastructure of renal tubular epithelial cells, compared with controls. Cd increased the expression of autophagy-related and apoptosis-related genes, but decreased expression of lysosomal function-related and UPRmt-related genes. The co-treatment group ameliorated Cd-induced nephrotoxicity by alleviating oxidative stress, inhibiting apoptosis, repairing autophagy dysfunction and UPRmt disorder. In conclusion, dietary supplementation of HKL showed beneficial effects on Japanese quail kidney injury caused by Cd.

Melatonin improves mitochondrial function by preventing mitochondrial fission in cadmium-induced rat proximal tubular cell injury via SIRT1-PGC-1α pathway activation.

Melatonin is an indoleamine produced in the pineal gland and has many physiological roles. There is increasing evidence that melatonin ameliorates cadmium (Cd)-induced nephrotoxicity. The potential protective impact of melatonin against Cd-induced nephrotoxicity and the mechanisms behind this protection are unknown. The relevance of mitochondrial dynamics in Cd-induced nephrotoxicity and the putative mechanism of melatonin-mediated protection were examined in this study. We show that melatonin prevents Cd-induced nephrotoxicity by inhibiting dynamin-related protein 1 (Drp1)- and mitochondrial fission protein 1 (Fis1)-mediated mitochondrial fission. Melatonin treatment attenuated cytotoxicity, suppressed oxidative stress, restored mitochondrial membrane potential, and increased mitochondrial mass in response to Cd exposure. Consistent with this finding, melatonin treatment increased Cd-inhibited sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) expression and inhibited Drp1- and Fis1-mediated mitochondrial fission. Like melatonin, SIRT1 overexpression via resveratrol attenuated Drp1- and Fis1-mediated mitochondrial fission and other Cd-induced mitochondrial oxidative injuries effectively. Melatonin has significant pharmacological potential for protecting against Cd-induced nephrotoxicity by preventing excessive mitochondrial fission.

Effect of cadmium on Rho GTPases signal transduction during osteoclast differentiation.

Osteoclasts are the key target cells for cadmium (Cd)-induced bone metabolism diseases, while Rho GTPases play an important role in osteoclast differentiation and bone resorption. To identify new therapeutic targets of Cd-induced bone diseases; we evaluated signal transduction through Rho GTPases during osteoclast differentiation under the influence of Cd. In osteoclastic precursor cells, 10 nM Cd induced pseudopodia stretching, promoted cell migration, upregulated the levels of Cdc42, and RhoQ mRNAs and downstream Rho-associated coiled-coil kinase 1 (ROCK1) and ROCK2 proteins, and downregulated the actin-related protein 2/3 (ARP2/3) levels. Cd at 2 and 5 µM shortened the pseudopodia, inhibited cell migration, and decreased ROCK1, ROCK2, and ARP2/3 protein levels; Cd at 5 µM also reduced the mRNA expression levels of Rac1, Rac2, and RhoU mRNAs and decreased the level of phosphorylated (p)-cofilin. In osteoclasts, 10 nM Cd induced the formation of sealing zones, slightly upregulated Cdc42 mRNA levels and ROCK2 and ARP2/3 protein levels and significantly reduced p-cofilin levels. Cd at 2 µM and 5 µM Cd blocked the fusion of precursor cells; and 5 µM Cd downregulated the expression levels of RhoB, Rac1, Rac3, and RhoU mRNAs, and ROCK1, p-cofilin and ARP2/3 protein levels, significantly. In vivo, Cd (at 5 or 25 mg/L) increased the levels of key proteins RhoA, Rac1/2/3, Cdc42, and RhoU and their mRNAs in bone marrow cells. In summary, the results suggested that Cd affected the differentiation process of osteoclast and altered the expression of several Rho GTPases, which might be crucial targets of Cd during the differentiation of osteoclasts.

Paeonol protects renal tubular cells against cadmium-induced cytotoxicity via alleviating oxidative stress, inhibiting inflammatory responses and restoring autophagy.

Cadmium (Cd) is a widespread environmental pollutant with highly toxic to kidney. Paeonol (Pae) is a natural flavonoid isolated from Moutan Cortex that exhibits antioxidant and anti-inflammatory properties. Herein, we investigated the protective effects of Pae against Cd-induced nephrotoxicity and elucidated its underlying mechanisms. First, we screened the optimum treatment conditions for Pae and confirmed its ability to protect NRK-52E cells against Cd-induced cytotoxicity. The results showed that Pae alleviated Cd-induced oxidative stress by scavenging excessive reactive oxygen species. Furthermore, Pae suppressed Cd-induced inflammatory responses by inhibiting the activation of nuclear factor-κB and the transcriptions of pro-inflammatory cytokines including cytokines including interleukin (IL)-1ß, IL-6, monocyte chemotactic protein (MCP)-1 and tumour necrosis factor (TNF)-α. Pae alleviated Cd-induced autophagy inhibition, which was partly attributable to alleviation of oxidative stress. Meanwhile, Pae improved the structural integrity and degradation function of lysosomes, indicating that Pae can also target lysosomes to restore Cd-inhibited autophagic flux. Collectively, Pae alleviated Cd-induced renal cytotoxicity by alleviating oxidative stress, inhibiting inflammatory responses and restoring autophagy, implicating that Pae may serve as new candidate drug to treat Cd-induced nephrotoxicity.

Cadmium exposure induces rat proximal tubular cells injury via p62-dependent Nrf2 nucleus translocation mediated activation of AMPK/AKT/mTOR pathway.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a nuclear transcription factor of great concern which is widely involved in physiological and pathological processes of the organism, but the role and regulatory mechanism of Nrf2 in kidney exposed to cadmium (Cd) remain largely unknown. Here we demonstrated that Cd exposure induced injury in primary rat proximal tubular (rPT) cells and NRK-52E cell line, which was accompanied by autophagic flux blockade and subsequent accumulation of p62. Cd-activated nucleus translocation of Nrf2 depended on p62, which promoted antioxidant genes transcription, but it failed to against Cd-induced cell injury and ultimately succumbed to Cd toxicity. CDDO Methyl Ester (CDDO-ME) or ML385 treatment aggravated or alleviated rPT cells injury induced by Cd respectively, indicating that Nrf2 nucleus translocation played a negative role during Cd-induced rPT cells injury. Phosphorylation of 5' AMP-activated protein kinase (AMPK) decreased together with enhanced Nrf2 nucleus translocation in rPT cells exposed to Cd. Dephosphorylation of AMPK induced by Cd were facilitated or restored by CDDO-ME or ML385 treatment, which confirmed AMPK is a downstream factor of Nrf2. Simultaneously, CDDO-ME further enhanced Phosphorylation of mTOR and AKT which increased during Cd exposure. While, Cd-induced phosphorylation of mTOR and AKT were reversed by ML385 treatment. These results illustrated that Cd mediated Nrf2 nucleus translocation depends on p62 accumulation which results from autophagic flux inhibition. The enhanced nucleus translocation of Nrf2 suppresses phosphorylation of AMPK to inactivate AKT/mTOR signaling, and results in rPT cells injury finally.

Puerarin prevents cadmium-induced disorder of testicular lactic acid metabolism in rats by activating 5' AMP-activated protein kinase (AMPK)/sirtuin 1 (SIRT1) signaling pathway.

Cadmium (Cd) interferes with the function of the male reproductive system; however, the molecular mechanism is poorly understood. This study aimed to evaluate the effect of puerarin (PU) on Cd-induced testicular lactic acid metabolism disorder. Weaning male Sprague-Dawley rats were pre-fed for 7 days, weighed, and randomly divided into four groups: Control group, CdAc2 group, CdAc2 + PU group, PU group. The results showed that Cd accumulated in the testis, the testicles became congested and shrank, and the testis index decreased in the rats treated in the CdAc2 group. Cadmium exposure reduced the serum concentration of testosterone, and the concentration of lactic acid and pyruvate in the testis. Cd decreased testicular superoxide dismutase activity and total antioxidant capacity, and increased testicular malondialdehyde levels. Cd reduced the level of ATP, glycolytic gene expression, and lactate production-related proteins in the testis. Cd also decreased the expression of 5' AMP-activated protein kinase (AMPK)/sirtuin 1 (SIRT1) signaling pathway-related proteins in the testis. However, these negative effects were attenuated by PU administration. In summary, Cd reduces the production of lactic acid in the testis of rats, while PU administration restores the production of lactic acid and reduces the toxicity of Cd to the testis of rats.

Puerarin restores the autophagic flux to alleviate cadmium­induced endoplasmic reticulum stress in NRK­52E cells.

Cadmium (Cd) is a heavy metal that can accumulate and cause damage to a variety of tissues and organs. The kidney is the primary target organ for Cd accumulation and toxic damage. Autophagy, which is a critical intracellular process, serves an important role in maintaining the homeostasis of the intracellular environment. Endoplasmic reticulum stress (ERS) is another key process that functions to promote cell survival or results in cell injury and death. Both autophagy and ERS are associated with oxidative stress; however, the mechanism by which ERS is regulated by autophagy in Cd­induced nephrotoxicity remains unclear. The present study employed a rat NRK­52E cell model, where alterations in cell morphology, density and viability, the accumulation of reactive oxygen species, an increase in malondialdehyde generation and a decrease in antioxidant enzyme activity and apoptosis were induced by Cd treatment. Cd induced the activation of nuclear factor erythroid 2­related factor 2 (NRF2), an obstruction of autophagic flux and ERS, which were attenuated by puerarin administration. Furthermore, puerarin failed to alleviate ERS following knockdown of autophagy­related protein 7 in NRK­52E cells. Overexpression of Ras­related protein Rab­7, which promotes the fusion of autophagosomes and lysosomes, efficiently reduced ERS. Taken together, these results indicate that puerarin administration restored the autophagic flux to alleviate ERS, via blocking the activation of NRF2.

ERK1/2 MAPK promotes autophagy to suppress ER stress-mediated apoptosis induced by cadmium in rat proximal tubular cells.

Cadmium (Cd) is a toxic heavy metal and its toxic mechanism is not entirely clear. The goal of the present study was to investigate the toxic mechanism of Cd on rPT cells, and to elucidate the role of ERK1/2 signaling pathway in mediating the relationship between apoptosis and autophagy. We evaluated the cell morphology, cell cycle distribution, apoptosis rates, and the expression of related proteins. We observed that increased Cd concentration disrupted cell morphology, increased apoptosis and induced autophagy. Additionally, activation of JNK1/2 and p38 MAPK promoted apoptosis, while activation of ERK1/2 inhibited apoptosis. Upon inhibition of autophagy, apoptosis rate and the expression of ER proteins related to the apoptosis were increased. Following inhibition of the ERK1/2 signaling pathway, the number of LC3 aggregates, the rate of LC3II/LC3I and the expression of Beclin-1were decreased, but the expression level of ER proteins related to apoptosis were increased. Our results indicated that Cd exposure damages cells also induces apoptosis and autophagy, meanwhile demonstrate that the ERK1/2 signaling pathway plays an important role in this process. Besides, these data suggest that autophagy can inhibit Cd-induced apoptosis and the ERK1/2 signaling pathway can suppress ER stress-mediated apoptosis by activating autophagy.

PARP-1 overexpression contributes to Cadmium-induced death in rat proximal tubular cells via parthanatos and the MAPK signalling pathway.

Parthanatos is a newly discovered form of PARP-1-dependent programmed cell death. It has been reported to play an important role in several cancer or tumour cells; however, few studies have been performed in normal cells. Cadmium is a highly toxic pollutant and is reported to induce autophagy and apoptosis in multiple cell types. Although cadmium toxicity induces cell death, the underlying mechanism is not fully understood. Therefore, in this study we aimed to investigate the mechanism of Cadmium -induced cell damage using rat proximal tubular cell line NRK-52E and primary rat proximal tubular (rPT) cells. Our results indicated that parthanatos and the MAPK signalling pathway contribute to Cadmium-induced cell death, and that oxidative stress and mitochondrial damage play key roles in this process. In addition, parthanatos with oxidative stress has a synergistic effect on apoptosis, and JNK1/2 and p38 contribute to parthanatos.