Apoptosis In Acute Kidney Injury Research Articles

Unlocking renal Restoration: Mesaconine from Aconitum plants restore mitochondrial function to halt cell apoptosis in acute kidney injury

Acute kidney injury (AKI) is characterized by a sudden decline in renal function. Traditional Chinese medicine has employed Fuzi for kidney diseases; however, concerns about neurotoxicity and cardiotoxicity have constrained its clinical use. This study explored mesaconine, derived from processed Fuzi, as a promising low-toxicity alternative for AKI treatment. In this study, we assessed the protective effects of mesaconine in gentamicin (GM)-induced NRK-52E cells and AKI rat models in vitro and in vivo, respectively. Mesaconine promotes the proliferation of damaged NRK-52E cells and down-regulates intracellular transforming growth factor β1 (TGF-β1) and kidney injury molecule 1 (KIM-1) to promote renal cell repair. Concurrently, mesaconine restored mitochondrial morphology and permeability transition pores, reversed the decrease in mitochondrial membrane potential, mitigated mitochondrial dysfunction, decreased ATP production, inhibited inflammatory factor release, and reduced early apoptosis rates. In vivo, GM-induced AKI rat models exhibited elevated AKI biomarkers, in which mesaconine was effectively reduced, indicating improved renal function. Mesaconine enhanced superoxide dismutase activity, reduced malondialdehyde content, alleviated inflammatory infiltrate, mitigated tubular and glomerular lesions, and downregulated NF-κB (nuclear factor-κb) p65 expression, leading to decreased tumor necrosis factor-α (TNF-α) and IL-1β (interleukin-1β) levels in GM-induced AKI animals. Furthermore, mesaconine inhibited the expression of renal pro-apoptotic proteins (Bax, cytochrome c, cleaved-caspase 9, and cleaved-caspase 3) and induced the release of the anti-apoptotic protein bcl-2, further suppressing apoptosis. This study highlighted the therapeutic potential of mesaconine in GM-induced AKI. Its multifaceted mechanisms, including the restoration of mitochondrial dysfunction, anti-inflammatory and antioxidant effects, and apoptosis mitigation, make mesaconine a promising candidate for further exploration in AKI management.

Regulator of calcineurin 1 deletion attenuates mitochondrial dysfunction and apoptosis in acute kidney injury through JNK/Mff signaling pathway

Ischemia-reperfusion (I/R) induced acute kidney injury (AKI), characterized by excessive mitochondrial damage and cell apoptosis, remains a clinical challenge. Recent studies suggest that regulator of calcineurin 1 (RCAN1) regulates mitochondrial function in different cell types, but the underlying mechanisms require further investigation. Herein, we aim to explore whether RCAN1 involves in mitochondrial dysfunction in AKI and the exact mechanism. In present study, AKI was induced by I/R and cisplatin in RCAN1flox/flox mice and mice with renal tubular epithelial cells (TECs)-specific deletion of RCAN1. The role of RCAN1 in hypoxia-reoxygenation (HR) and cisplatin-induced injury in human renal proximal tubule epithelial cell line HK-2 was also examined by overexpression and knockdown of RCAN1. Mitochondrial function was assessed by transmission electron microscopy, JC-1 staining, MitoSOX staining, ATP production, mitochondrial fission and mitophagy. Apoptosis was detected by TUNEL assay, Annexin V-FITC staining and Western blotting analysis of apoptosis-related proteins. It was found that protein expression of RCAN1 was markedly upregulated in I/R- or cisplatin-induced AKI mouse models, as well as in HR models in HK-2 cells. RCAN1 deficiency significantly reduced kidney damage, mitochondrial dysfunction, and cell apoptosis, whereas RCAN1 overexpression led to the opposite phenotypes. Our in-depth mechanistic exploration demonstrated that RCAN1 increases the phosphorylation of mitochondrial fission factor (Mff) by binding to downstream c-Jun N-terminal kinase (JNK), then promotes dynamin related protein 1 (Drp1) migration to mitochondria, ultimately leads to excessive mitochondrial fission of renal TECs. In conclusion, our study suggests that RCAN1 could induce mitochondrial dysfunction and apoptosis by activating the downstream JNK/Mff signaling pathway. RCAN1 may be a potential therapeutic target for conferring protection against I/R- or cisplatin-AKI.

Kidney Organoids as a Novel Platform to Evaluate Lipopolysaccharide-Induced Oxidative Stress and Apoptosis in Acute Kidney Injury.

Sepsis-associated acute kidney injury (SA-AKI) is a life-threatening syndrome. Lipopolysaccharide (LPS) is a widely used inducer for modeling SA-AKI both in vivo and in vitro. However, due to the innate complexity of the kidney architecture, the mechanisms underlying the pathogenesis of SA-AKI, as well as those involved in LPS-induced kidney injury remain to be clarified. Kidney organoids derived from human pluripotent stem cells (hPSCs) act as a model of multiple types of kidney cells in vitro and eliminate potential confounders in vivo. In the current study, we established LPS-induced kidney injury models both in vivo and in human kidney organoids. Kidney function, pathological changes, and markers of oxidative stress were evaluated with/without the presence of methylprednisolone (MP) treatment both in vivo and in vitro. The extent of LPS-induced oxidative stress and apoptosis in kidney organoids was further investigated in vitro. LPS-induced acute kidney injury in mice, together with pathological changes and increased oxidative stress, as well as enhanced apoptosis in kidney cells were evaluated. These phenomena were ameliorated by MP treatment. Experiments in kidney organoids showed that the LPS-induced apoptotic effects occurred mainly in podocytes and proximal tubular cells. Our experiments demonstrated the efficacy of using kidney organoids as a solid platform to study LPS-induced kidney injury. LPS induced oxidative stress as well as apoptosis in kidney cells independently of changes in perfusion or immune cell infiltration. MP treatment partially alleviated LPS-induced injury by reducing kidney cell oxidative stress and apoptosis.

Curcumin prevents renal cell apoptosis in acute kidney injury in a rat model of dry-heat environment heatstroke via inhibition of the mitochondrial apoptotic pathway.

Heatstroke is a life-threatening illness that is characterised by a core body temperature >40°C and central nervous system dysfunction. Acute kidney injury (AKI) is a common complication of heatstroke, and the mitochondrial apoptotic pathway has been demonstrated to be one of the leading causes of tissue damage and cell death in AKI. Curcumin is a phenol that is extracted from turmeric and demonstrates anti-apoptotic properties. To test if curcumin can protect the kidney from injury caused by heat stress, the effect of curcumin administration on renal injury and apoptosis of renal tissue was examined in a rat model of dry-heat environment. A total of 50 Sprague-Dawley rats were randomly divided into five groups (n=10): Standard temperature control, dry-heat control and curcumin treatment groups (50, 100 and 200 mg/kg groups). After exposure to a dry-heat environment for 150 min, the rats were anesthetized and euthanized. Blood, urine and renal tissue were collected to quantify the expression of specific mitochondrial apoptosis-related molecules. Curcumin pre-treatment decreased blood urea nitrogen and serum creatinine, urinary kidney injury molecule-1, and neutrophil gelatinase-associated lipocalin levels compared with the dry-heat control group. Curcumin was also revealed to downregulate c-Jun N-terminal kinases (JNK), cytochrome c, caspase-3 and caspase-9 expression upon treatment with 100 and 200 mg/kg curcumin, which may result in inhibition of the mitochondrial apoptotic pathway in renal cells. The current study revealed that Curcumin may to have potential for preventing heatstroke-induced AKI.

Apoptosis in Acute Kidney Injury

Abstract Apoptosis is an inborn process that has been preserved during evolution; it allows the cells to systematically inactivate, destroy and dispose of their own components thus leading to their death. This program can be activated by both intra and extracellular mechanisms. The intracellular components involve a genetically defined development program while the extracellular aspects regard endogenous proteins, cytokines and hormones as well as xenobiotics, radiations, oxidative stress and hypoxia. The ability of a cell to enter apoptosis as a response to a „death” signal depends on its proliferative status, the position in the cell cycle and also on the controlled expression of those genes that have the capacity of promoting and inhibiting cell death. The fine regulation of these parameters needs to be maintained in order to ensure the physiological environment required for the induction of apoptosis. In this review, we first describe evidence for the role of apoptotic pathways in ischemic acute renal failure, and then consider the potential mechanisms that may participate in this model of acute renal tubular injury. Potential therapeutic interventions to prevent tubular apoptosis in renal disease include angiotensin system inhibition, whereby the angiotensin II AT2 receptor blockade seems more promising in apoptosis inhibition than the inhibition of other receptor subtypes. A better understanding of the mechanisms of apoptosis could lead to safer and more specific therapeutic interventions for acute kidney injury.

Salusin-β mediates tubular cell apoptosis in acute kidney injury: Involvement of the PKC/ROS signaling pathway

Salusin-β is abundantly expressed in many organs and tissues including heart, blood vessels, brain and kidneys. Recent studies have identified salusin-β as a bioactive peptide that contributes to various diseases, such as atherosclerosis, hypertension, diabetes and metabolic syndrome. However, the role of salusin-β in the pathogenesis of acute kidney injury (AKI) is largely unclear. In the present study, we investigated the roles of salusin-β in cisplatin or lipopolysaccharide (LPS)-induced renal injury. Herein, we found that salusin-β expression was upregulated in both renal tubular cells and kidney tissues induced by both cisplatin and LPS. In vitro, silencing of salusin-β diminished, whereas overexpression of salusin-β exaggerated the increased PKC phosphorylation, oxidative stress, histone γH2AX expression, p53 activation and apoptosis in either cisplatin or LPS-challenged renal tubular cells. More importantly, salusin-β overexpression-induced tubular cell apoptosis were abolished by using the PKC inhibitor Go 6976, reactive oxygen species (ROS) scavenger NAC, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin (Apo) or p53 inhibitor Pifithrin-α. In animals, blockade of salusin-β alleviated PKC phosphorylation, ROS accumulation, DNA damage, and p53 activation as well as renal dysfunction in mice after administration of cisplatin or LPS. Taken together, these results suggest that overexpressed salusin-β is deleterious in AKI by activation of the PKC/ROS signaling pathway, thereby priming renal tubular cells for apoptosis and death.

Overexpression of augmenter of liver regeneration (ALR) mitigates the effect of H2O2-induced endoplasmic reticulum stress in renal tubule epithelial cells.

Ischemia/reperfusion is a major cause of acute kidney injury and can induce apoptosis in renal epithelial tubule (HK-2) cells. Accumulating evidence indicates that endoplasmic reticulum (ER) stress is a major contributor to apoptosis in acute kidney injury. We previously reported that augmenter of liver regeneration (ALR) functions as an anti-apoptotic factor in H2O2-treated HK-2 cells although the precise mechanism underlying this action remains unclear. In the present study, we investigate the role of ALR in H2O2-induced ER stress-mediated apoptosis. We overexpressed ALR and established a H2O2-induced ER stress model in HK-2 cells. Overexpression of ALR reduced the level of reactive oxygen species and the rate of apoptosis in H2O2-treated HK-2 cells. Using confocal microscopy and western blot, we observed that ALR colocalized with the ER and mitochondria compartment. Moreover, ALR suppressed ER stress by maintaining the morphology of the ER and reducing the levels of the ER-related proteins, glucose-regulated protein 78 (GRP78), phospho-protein kinase-like ER kinase (p-PERK), phospho-eukaryotic initiation factor 2α (p-eIF2α) and C/EBP-homologous protein (CHOP) significantly (p < 0.05). Mechanistically, ALR promoted Bcl-2 expression and suppressed Bax and cleaved-caspase-3 expression significantly during ER-stress induced apoptosis (p < 0.05). Furthermore, ALR attenuated calcium release from the ER, and transfer to mitochondria, under ER stress. To conclude, ALR alleviates H2O2-induced ER stress-mediated apoptosis in HK-2 cells by suppressing ER stress response and by maintaining calcium homeostasis. Consequently, ALR may protect renal tubule epithelial cells from ischemia/reperfusion induced acute kidney injury.

Downregulation of TIMP2 attenuates sepsis-induced AKI through the NF-κb pathway

Acute kidney injury (AKI) is a frequent complication of sepsis and contributes to increased morbidity and mortality. Urinary tissue inhibitor of metalloproteinases-2 (TIMP2) has been recently recognized as an early biomarker to predict AKI in critically ill patients. However, the biological functions of TIMP2 remain largely unknown. In this study, we investigated the role of TIMP2 in mediating inflammation and tubular cell apoptosis in AKI. In kidney tissue taken from mice exposed to cecal ligation and puncture (CLP) and in human kidney 2 (HK-2) cells exposed to lipopolysaccharide (LPS) in culture, TIMP2 expression was significantly upregulated. The expression of TIMP2 in the kidney tissue correlated with the severity of AKI in vivo. In cultured HK-2 cells, LPS challenge markedly induced cytokine release, and recombinant cytokines promoted TIMP2 expression and apoptosis. However, TIMP2 silencing ameliorated LPS-induced cytokine release, apoptosis, and cell injury. We further found that the effects of downregulation of TIMP2 on a suppression of release of inflammatory cytokines were mediated by p-P65. Stable, kidney-specific TIMP2 knockdown mice were transduced by injecting the TIMP2 knockdown lentiviral vector into kidney parenchyma. TIMP2 silencing ameliorated CLP-induced proinflammatory cytokines, kidney dysfunction as measured by serum creatinine level, and histopathological changes. Downregulation of TIMP2 showed renoprotective effects on endotoxin-induced AKI, which was associated with the anti-inflammatory activity through inhibition of the nuclear factor (NF)-κB pathway. Collectively, our results indicate that TIMP2 plays an important role in mediating sepsis-induced AKI through regulation of NF-κB. These findings reveal the pathogenic role of TIMP2 in AKI and suggest a novel target for the treatment of AKI.

Aldosterone induces NRK-52E cell apoptosis in acute kidney injury via rno-miR-203 hypermethylation and Kim-1 upregulation.

Acute kidney injury (AKI) is characterized by an acute reduction in kidney function as identified by an increase in serum creatinine levels and reduction in urine output. Kidney injury molecule-1 (Kim-1) is a hallmark of kidney diseases, since it is typically non-detectable in the non-injured kidney, but upregulated and excreted in the urine during AKI. Aldosterone (Aldo) is a mediator of the renin-angiotensin-Aldo system with a pivotal role in the regulation of salt and extracellular fluid metabolism. In the present study, mice subjected to renal ischemia/reperfusion-induced AKI were investigated. The mice exhibited elevated levels of Aldo and angiotensin II, together with increased Kim-1 expression levels in renal tissue. Treatment of the mice with the Aldo receptor antagonist spironolactone decreased Kim-1 expression levels. These results suggest that Aldo may be associated with the expression of Kim-1 during AKI. However, the molecular mechanism underlying the role of Aldo in Kim-1 expression is unclear, and thus was investigated using NRK-52E cells. Aldo was found to induce the apoptosis of NRK-52E cells via the hypermethylation of rno-microRNA (miR)-203 and upregulation of Kim-1. In addition, luciferase reporter assays demonstrated that Kim-1 was a target gene of rno-miR-203 in NRK-52E cells. Furthermore, Aldo-induced NRK-52E cell apoptosis was reduced by treatment with pre-miR-203 and spironolactone to a greater extent when compared with either alone. The results may provide a promising diagnostic marker or novel therapeutic target for AKI.

Augmenter of Liver Regeneration Attenuates Tubular Cell Apoptosis in Acute Kidney Injury in Rats: The Possible Mechanisms

Augmenter of liver regeneration (ALR), the expression of which increased in rat kidneys after renal ischemia/reperfusion (I/R) injury, enhances renal tubular cell regeneration in vivo and in vitro. We aimed to investigate the effects of ALR on apoptosis of renal tubular cells after renal I/R injury in vivo and consider the possible mechanisms. Rats that were subjected to bilateral renal ischemia for 60 min followed by reperfusion were administered with either vehicle or recombinant human ALR (rhALR). Renal dysfunction and histologic injury were assessed by the measurement of serum biochemical markers and histological grading. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL). Caspase-3 activity was measured using a colorimetric protease assay. Expression of Bcl-2, Bax Fas, phosphorylated-Akt (p-Akt), and phosphorylated-p53 (p-p53) was determined by western blotting. Compared with vehicle-treated rats, renal dysfunction and histologic injury were significantly attenuated by administration of rhALR. The number of TUNEL-positive tubular cells and caspase-3 activity were decreased, Bcl-2 and p-Akt expression was up-regulated, and Bax and p-p53 expression was down-regulated by administration of rhALR. However, administration of rhALR had no effect on Fas protein expression. These results indicate that the protective effect of rhALR on renal I/R injury is associated with its anti-apoptotic action in renal tubular cells. RhALR inhibits apoptosis by increasing the ratio of Bcl-2 to Bax and by decreasing the activity of caspase-3. The activation of Akt and inactivation of p53 are involved in the rhALR anti-apoptosis process.

Glutamine attenuates tubular cell apoptosis in acute kidney injury via inhibition of the c-Jun N-terminal kinase phosphorylation of 14-3-3*

Tubular cell apoptosis is linked to the development of acute kidney injury (AKI), which is a frequent complication of traumatic rhabdomyolysis. The 14-3-3 protein, a multifunctional regulatory protein, binds a variety of apoptotic proteins and is a target of c-Jun N-terminal kinase (JNK) in the cell death signaling pathway. Therefore, we examined whether JNK phosphorylates 14-3-3 and downstream mitochondrial death pathway mediates apoptosis in myoglobinuric acute kidney injury to determine whether these events are regulated by glutamine, which is known to induce heat shock protein 70 (Hsp70), or involved in the synthesis of glutathione (GSH). A prospective, randomized, controlled animal trial. University research laboratory. Male Sprague-Dawley rats. We utilized a rat model of myoglobinuric AKI. Glutamine or saline was administered intraperitoneally before and after glycerol injection. Apoptotic cell death was determined via transferase-mediated deoxyuridine triphosphate nick-end labeling staining, and Hsp70, JNK, phospho-JNK, 14-3-3, phospho-14-3-3, and many other apoptotic proteins were examined via Western blot. Relative interactions between these proteins were tested by coimmunoprecipitation analyses. Also, GSH levels were determined to further test whether glutamine affects apoptotic cell death in myoglobinuric AKI. Glutamine treatment elevated levels of Hsp70 or reduced GSH and attenuated tubular cell apoptosis in kidney tissues of rats with myoglobinuric AKI. Further, Hsp70 physically associated with JNK, thereby limiting its activation. In addition, JNK evidently interacted with 14-3-3, leading to its phosphorylation, Bad or Bax dissociation from 14-3-3, and subsequent Bax mitochondrial translocation and caspase activation in rats with acute renal failure. Glutamine treatment very modestly lowered elevated levels of serum creatinine in AKI rats. A signaling link between JNK and 14-3-3 and subsequent mitochondrial death pathway may partly act as an early signaling that promotes apoptotic cell death leading to AKI, and glutamine may at least partially prevent apoptosis via enhancing Hsp70 or GSH levels.

MMP9 and SCF Protect from Apoptosis in Acute Kidney Injury

Apoptosis of tubular epithelial cells is a hallmark of acute kidney injury (AKI), but the cellular events preceding apoptosis in this setting are incompletely understood. Because matrix metalloproteinase 9 (MMP9) degrades matrix components involved in cell survival, we studied the role of MMP9 in AKI. In the mouse model of folic acid-induced AKI, we observed a marked increase of MMP9 activity in the S3 segment of the proximal tubule (S3PT), correlating with the apoptotic phase. MMP9 deficiency increased apoptosis and the severity of renal lesions and substantially delayed recovery of renal function. MMP9-/- mice exhibited significant apoptosis in the S3PT and the intercalated cells of the collecting duct (I-CD), whereas wild-type mice exhibited none in these segments. Stem cell factor (SCF), an MMP9 substrate, was identified in the S3PT, and its receptor, c-Kit, was expressed in both the S3PT and I-CD. MMP9 released the soluble form of SCF (sSCF) from kidney cells in vivo and in vitro. In addition, SCF inhibited apoptosis of tubular cells in vitro, rescued MMP9-/- S3PT and I-CD from apoptosis in vivo, and improved renal function. An ischemia-reperfusion model of AKI produced similar results. In patients with AKI, urinary sSCF increased with acute tubular necrosis but not with prerenal azotemia. In conclusion, these data show that MMP9 protects the S3 segment of the proximal tubule and the I-CD from apoptosis in AKI, most likely by releasing sSCF.

The role of p53 pro‐apoptotic target, PERP, induces mitochondrial permeability and apoptosis in hypoxic renal cells

PERP, a direct target of p53, has lately been implicated in apoptosis, and we reported earlier a p53‐dependent upregulation of PERP in proximal tubular epithelia subsequent to ischemia/reperfusion injury (IRI); nonetheless, the role of PERP in IRI is unknown. To test our hypothesis that PERP alters mitochondrial membrane permeability (MMP) to induce apoptosis following renal IRI, we developed renal tubular epithelial cell lines (LLCPK1) stably overexpressing PERP (P‐Epi) or control vector (C‐Epi) and subjected to 4 hour (hr) hypoxia in a glucose‐free buffer followed by 1hr normoxia with normal medium replacement. By 1hr post‐injury, the mitochondrial membrane potential is significantly altered in P‐Epi cells compared to C‐Epi or uninjured cells (p&lt; 0.001; n=3) as analyzed by flow cytometry with JC‐1 probing. In addition, AIF and cytochrome c were released from mitochondria earlier by 3hrs post‐injury in P‐Epi cells compared to C‐Epi cells. Further, the number of P‐Epi cells undergoing apoptosis by 3hrs post‐injury was higher compared to control cells (p&lt; 0.05; n=3). Thus we conclude, upregluation of PERP accelerates the release pro‐apoptotic molecules by altering MMP after IRI in renal tubular epithelia. Targeting a p53 downstream molecule rather than p53 per se may offer a specific therapeutic strategy to inhibit p53‐mediated apoptosis in acute kidney injury. Support by Univ Nebraska Med Cntr and NKF Nebraska.