Acid-induced Acute Kidney Injury Research Articles

Galectin-8 counteracts folic acid-induced acute kidney injury and prevents its transition to fibrosis

Acute kidney injury (AKI), characterized by a sudden decline in kidney function involving tubular damage and epithelial cell death, can lead to progressive tissue fibrosis and chronic kidney disease due to interstitial fibroblast activation and tissue repair failures that lack direct treatments. After an AKI episode, surviving renal tubular cells undergo cycles of dedifferentiation, proliferation and redifferentiation while fibroblast activity increases and then declines to avoid an exaggerated extracellular matrix deposition. Appropriate tissue recovery versus pathogenic fibrotic progression depends on fine-tuning all these processes. Identifying endogenous factors able to affect any of them may offer new therapeutic opportunities to improve AKI outcomes. Galectin-8 (Gal-8) is an endogenous carbohydrate-binding protein that is secreted through an unconventional mechanism, binds to glycosylated proteins at the cell surface and modifies various cellular activities, including cell proliferation and survival against stress conditions. Here, using a mouse model of AKI induced by folic acid, we show that pre-treatment with Gal-8 protects against cell death, promotes epithelial cell redifferentiation and improves renal function. In addition, Gal-8 decreases fibroblast activation, resulting in less expression of fibrotic genes. Gal-8 added after AKI induction is also effective in maintaining renal function against damage, improving epithelial cell survival. The ability to protect kidneys from injury during both pre- and post-treatments, coupled with its anti-fibrotic effect, highlights Gal-8 as an endogenous factor to be considered in therapeutic strategies aimed at improving renal function and mitigating chronic pathogenic progression.

#1955 Ferroptotic proteins ACSL4 and ALOX15 as therapeutic targets in AKI

Abstract Background and Aims Ferroptosis is a regulated form of necrosis which is dependent on cellular iron and is characterized by the accumulation of lipid peroxides and failure of cellular antioxidant defences. We have previously described in vivo that ferroptosis is the primary cause of folic acid-induced acute kidney injury (FA-AKI) and that necroinflammation secondary to ferroptosis may further worsen kidney injury, since ferroptosis inhibition improved kidney function and decreased tubular cell death and oxidative stress. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is involved in the incorporation of polyunsaturated fatty acid (PUFA) into membranes, while 15-Lipoxygenase (Alox15) catalyzes the regio- and enantioselective peroxidation of membrane-esterified PUFAs, forming the ultimate peroxidized species that induce ferroptosis. In the present work, we aim to explore the potential of both proteins as therapeutic targets in AKI, as well as the molecular characterization of kidney ferroptosis in cultured tubular cells. Method Animal model: Female 12- to 14-week-old C57BL/6J wild type mice received a single intraperitoneal (i.p) injection of folic acid or vehicle and were sacrificed 48 hours later. Troglitazone was intravenously administered for in vivo ACSL4 inhibition, and Compound 1 was i.p administered for in vivo ALOX15 inhibition. In vitro characterization of ferroptosis: HK2 human tubular kidney cells were incubated with arachidonic acid (AA) prior to sublethal RSL3 administration for sensitization to ferroptosis. We used Ferrostatin-1 (Fer-1) and Liproxstatin-1 (Lpx-1) as inhibitors of ferroptosis. The thiazolidinedione family members Rosiglitazone, Pioglitazone and Troglitazone were used as pharmacological inhibitors of ACSL4. We also performed transcriptional silencing of ACSL4 using a specific siRNA. Compound 1 and PD1646 were administered for in vitro Alox15 inhibition. Cellular lipid peroxidation was analyzed by flow cytometry with Bodipy 581/591 staining. To characterize the lipid signature of ferroptotic cells, supernatants were collected and 15-HETEs levels were measured by ELISA. Oxido-lipidomic analysis of ferroptotic cells was performed by LC-MS. Real Time PCR and Western Blot of whole kidneys and cultured cells were performed for the detection and quantification of ACSL4 and ALOX15. Cell death was assessed by measurement of cell viability (MTT assay) and cytotoxicity (LDH assay). Tissue cell death was assessed by TUNEL. Results Kidney transcriptomics identified Acsl4 as the most upregulated member of Acsl family during FA-AKI. This was validated at mRNA and protein levels. Supplementation with AA, the preferred ACSL4 substrate, sensitized HK2 tubular cells to ferroptosis under sublethal RSL3 conditions, and this was prevented with specific ferroptosis inhibitors Fer-1 and Lpx-1. Pharmacological inhibition of ACSL4 with Troglitazone and a specific siRNA protected from cell death and lipid peroxidation induced by co-stimulation of AA and RSL3 in HK2 cells. Likewise, ALOX15 proteins levels were also increased in FA-AKI at 48 hours. PD1646 and Compound 1, both used to inhibit ALOX15, protected from ferroptotic cell death and lipid peroxidation in HK2 cells. Lipidomic analysis on HK2 tubular cells stimulated with AA+RSL3 uncovered an increased content of key peroxidized lipid species involved in ferroptosis execution, which was alleviated by targeting ACSL4 or ALOX15. Additionally, renal function of FA-AKI in mice was improved by ACSL4 pharmacological inhibition with Troglitazone and with ALOX15 inhibition with Compound 1. Conclusion Our preliminary results suggest therapeutic potential of ACSL4 and ALOX15 as ferroptotic targes during AKI. High PUFA content sensitizes human tubular HK2 cells to cell death and lipid peroxidation in the presence of sublethal ferroptotic triggers, and this was prevented by targeting ACSL4 or ALOX15 both in vitro and in vivo in FA-AKI. Closely related lipid peroxide species were decreased by ACSL4 or ALOX15 inhibitors.

Critical roles of tubular mitochondrial ATP synthase dysfunction in maleic acid-induced acute kidney injury.

Maleic acid (MA) induces renal tubular cell dysfunction directed to acute kidney injury (AKI). AKI is an increasing global health burden due to its association with mortality and morbidity. However, targeted therapy for AKI is lacking. Previously, we determined mitochondrial-associated proteins are MA-induced AKI affinity proteins. We hypothesized that mitochondrial dysfunction in tubular epithelial cells plays a critical role in AKI. In vivo and in vitro systems have been used to test this hypothesis. For the in vivo model, C57BL/6 mice were intraperitoneally injected with 400mg/kg body weight MA. For the in vitro model, HK-2 human proximal tubular epithelial cells were treated with 2mM or 5mM MA for 24h. AKI can be induced by administration of MA. In the mice injected with MA, the levels of blood urea nitrogen (BUN) and creatinine in the sera were significantly increased (p < 0.005). From the pathological analysis, MA-induced AKI aggravated renal tubular injuries, increased kidney injury molecule-1 (KIM-1) expression and caused renal tubular cell apoptosis. At the cellular level, mitochondrial dysfunction was found with increasing mitochondrial reactive oxygen species (ROS) (p < 0.001), uncoupled mitochondrial respiration with decreasing electron transfer system activity (p < 0.001), and decreasing ATP production (p < 0.05). Under transmission electron microscope (TEM) examination, the cristae formation of mitochondria was defective in MA-induced AKI. To unveil the potential target in mitochondria, gene expression analysis revealed a significantly lower level of ATPase6 (p < 0.001). Renal mitochondrial protein levels of ATP subunits 5A1 and 5C1 (p < 0.05) were significantly decreased, as confirmed by protein analysis. Our study demonstrated that dysfunction of mitochondria resulting from altered expression of ATP synthase in renal tubular cells is associated with MA-induced AKI. This finding provides a potential novel target to develop new strategies for better prevention and treatment of MA-induced AKI.

DIA proteomics analysis reveals the mechanism of folic acid-induced acute kidney injury and the effects of icariin

The complexities of acute kidney injury (AKI), a multifaceted pathological occurrence, are not fully understood. At present, there is a lack of effective pharmaceutical treatments in clinical practice. Studies have shown that icariin has beneficial effects in models of acute kidney injury (AKI) caused by cisplatin and lipopolysaccharide (LPS). The aim is to explore the mechanisms that cause folic acid (FA)-induced AKI and examine the protective effects of icariin against this condition. To establish a mouse model of AKI, FA was administered via intraperitoneal injection. Icariin was used as the drug intervention. The model and the impact of drug intervention were assessed using measurements of renal function parameters, staining with hematoxylin and eosin, and Q-PCR. The analysis of protein expression changes in the control, model, and icariin treatment groups was conducted using proteomics. KEGG signaling pathway analysis indicates that differential expressed proteins are enriched in the component and coagulation cascades signaling pathway. Through protein-protein interaction network analysis, it was found that compared to the normal group, the expression of Fibrinogen and other proteins was significantly upregulated at the center of the protein interaction network in the model group. After drug treatment, the expression of these proteins was significantly downregulated. The validation experiment supports the above results. In conclusion, this study clarified the molecular mechanism of FA induced acute renal injury from the proteomics level, and provided target selection for AKI; At the same time, the mechanism of icariin in the treatment of AKI was analyzed from the proteomics level.

Ameliorative Effect of Galium Verum (Rubiaceae Family) Methanolic Extract on Folic Acid-induced Acute kidney Injury in Male Rats.

Background: Antioxidant, sedative, anticancer, and antibacterial properties are among the numerous pharmacological characteristics of Galium verum. Aim: The primary goal of this research was to investigate the therapeutic effects of G. verum extract against folic acid-induced acute kidney injury (AKI). Materials and methods: 18 male rats were assigned into three groups: Control, AKI, and G. verum. AKI was induced by a dose of folic acid (250 mg/kg, i.p.) while G. verum (250 mg/kg) was administrated for 7 consecutive days. Results: G. verum methanol extract contains flavonoids, anthraquinones, tannins, iridoids, triterpenes, steroids, phenols, and saponins, while it free of cardiac glycosides and alkaloids. G. verum extract significantly decreased levels of creatinine, urea, uric acid, sodium, chloride, potassium, microalbumin, malondialdehyde and nitric oxide while it increased creatinine clearance, glutathione reduced, and catalase levels. Meanwhile, histological examination of the kidney’s tissues revealed apparently normal structure. Conclusions: Underlying renal therapeutic mechanisms of G. verum action include a diuretic, antioxidant, and anti-inflammatory properties of G. verum extract resulting in improving kidney structure and enhance recovery of renal function.

CU06-1004 alleviates oxidative stress and inflammation on folic acid-induced acute kidney injury in mice

PurposeAcute kidney injury (AKI) is characterized by reduced renal function, oxidative stress, inflammation, and renal fibrosis. CU06-1004, an endothelial cell dysfunction blocker, exhibits anti-inflammatory effects by reducing vascular permeability in pathological conditions. However, the potential effects of CU06-1004 on AKI have not been investigated. We investigated the renoprotective effect of CU06-1004 against oxidative stress, inflammation, and fibrotic changes in a folic acid-induced AKI model. MethodsAKI was induced by intraperitoneal injection of high dose (250 mg/kg) folic acid in mice. CU06-1004 was orally administered a low (10 mg/kg) or high dose (20 mg/kg). ResultsCU06-1004 ameliorated folic acid-induced AKI by decreasing serum blood urea nitrogen and creatinine levels, mitigating histological abnormalities, and decreasing tubular injury markers such as kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin in folic acid-induced AKI mice. Additionally, CU06-1004 alleviated folic acid-induced oxidative stress by reducing 4-hydroxynonenal and malondialdehyde levels. Furthermore, it attenuated macrophage infiltration and suppressed the expression of the proinflammatory factors, including tumor necrosis factor-α, intercellular adhesion molecule-1, and vascular cell adhesion protein-1. Moreover, CU06-1004 mitigated folic acid-induced tubulointerstitial fibrosis by decreasing α-smooth muscle actin and transforming growth factor-β expression. ConclusionThese findings suggest CU06-1004 as a potential therapeutic agent for folic acid-induced AKI.

Accumulation of Heavy Metals in Sepia officinalis Extract Aggravate Acute Kidney Injury Induced by a High Folic Acid Dosage in Wistar Rats

Background: Seafood is an important source of food for the majority of people. Marine species have a wide spectrum of pharmacological actions, including antibacterial, antiviral, antiparasitic, anti-inflammatory, and anti-diabetic properties. Objective: The purpose of this study was to examine the effects of Sepia officinalis extract (SoE) on folic acid-induced acute kidney injury in Wistar rats. Methods: A single dosage of folic acid (250 mg/kg) was injected intraperitoneally to cause kidney injury induced (AKI). The study contained three groups of six rats each: control, folic acid, and folic acid + SoE groups. The SoE group received SoE (45 mg/kg, orally) daily for one week, while the control and folic acid groups were administered distilled water. Results: The crude extract of Sepia officianlis contains heavy metals such as Fe, Cr, Cd, Pb, and Zn, according to our findings. The LD50 value of SoE was 450 mg/kg. SoE treatment increases creatinine, urea, uric acid, sodium, potassium, chloride, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyltransferase, malondialdehyde, and nitric oxide levels while decreasing total proteins, albumin, glutathione reduced, glutathione-S-transferase, and catalase. Several histological alterations were found in the liver and kidney of the SoE rats. Conclusion: The heavy metal content of S. officinalis extract has a synergistic effect with folic acid to induce hepatorenal injury. Natural extracts of marine species should be used with caution as a component of medications or natural remedies.

A novel method for automated crystal visualization and quantification in murine folic acid-induced acute kidney injury.

Folic acid (FA)-induced acute kidney injury (FA-AKI) is an increasingly prevalent rodent disease model involving the injection of a high dose of FA that culminates in renal FA crystal deposition and injury. However, the literature characterizing the FA-AKI model is sparse and dated in part due to the absence of a well-described methodology for the visualization and quantification of renal FA crystals. Using widely available materials and tools, we developed a straightforward and crystal-preserving histological protocol that can be coupled with automated imaging for renal FA crystal visualization and generated an automated macro for downstream crystal content quantification. The applicability of the method was demonstrated by characterizing the model in male and female C57BL6/JRj mice after 3 and 30 h of FA treatment. Kidneys from both sexes and timepoints showed a bimodal distribution of FA crystal deposition in the cortical and medullary regions while, compared with males, females exhibited higher renal FA crystal content at the 30-h timepoint accompanied by greater kidney weight and higher plasma urea. Despite comparable plasma phosphate concentrations, FA-AKI resulted in a substantially more elevated plasma intact fibroblast growth factor 23 (FGF23) in females, reflected by a similar pattern in osseous Fgf23 mRNA expression. Therefore, the presented method constitutes a valuable tool for the quantification of renal FA crystals, which can aid the mechanistic characterization of the FA-AKI model and serves as a means to control for confounding changes in FA crystallization when using the model for investigating early and prophylactic AKI therapeutic interventions.NEW & NOTEWORTHY Here, we describe a novel method for the visualization and quantification of renal folic acid (FA) crystals in the rodent FA-induced acute kidney injury (FA-AKI) model. The protocol involves a straightforward histological approach followed by fully automated imaging and quantification steps. Applicability was confirmed by showing that the FA-AKI model is sex-dependent. The method can serve as a tool to aid in characterizing FA-AKI and to control for studies investigating prophylactic therapeutic avenues using FA-AKI.

Krüppel-like Factor 15 Suppresses Ferroptosis by Activating an NRF2/GPX4 Signal to Protect against Folic Acid-Induced Acute Kidney Injury.

Acute kidney injury (AKI) is a common and serious disease with high morbidity and mortality, and its pathophysiological mechanisms are not fully understood. Increasing evidence suggests an important role of ferroptosis in AKI. Krüppel-like factor 15 (KLF15) is a transcription factor involved in several metabolic diseases, but its role in AKI and ferroptosis remains unclear. In this study, we explored the potential role of KLF15 using a folic acid-induced AKI model. Our study showed that KLF15 expression was reduced in kidney tissues of AKI mice, and KLF15 knockout exacerbated folic acid-induced ferroptosis and kidney injury. In vitro studies revealed that the ferroptosis inducer erastin significantly suppressed KLF15 expression in human tubular epithelial cells. Notably, the overexpression of KLF15 attenuated ferroptosis, as evidenced by a decrease in the lipid peroxidation marker of malondialdehyde and the upregulation of glutathione peroxidase 4 (GPX4), while KLF15 knockdown with shRNA exerted the opposite effect. Mechanistically, KLF15 stabilized the protein of nuclear factor erythroid 2-related factor 2 (NRF2) and subsequently increased the GPX4 level. Collectively, KLF15 plays an important role in the modulation of ferroptosis in AKI and may be a potential therapeutic target for treating AKI.

Renal Tubular CD24 Upregulation Aggravates Folic Acid Induced Acute Kidney Injury: A Possible Role for T Regulatory Cells Inhibition in Mice.

Acute kidney injury (AKI) is characterized by cell death and inflammation. CD24 is a protein induced during tissue damage and is not expressed in mature renal tissue. We explored the role of CD24 in the pathogenesis of folic acid-induced AKI (FA-AKI) in mice. A single Intraperitoneal (IP) injection of folic acid induced AKI in WT and CD24-/- mice. Renal function tests, histological analysis, immunohistochemistry, Western blot analysis, and ELISA were performed to assess the severity of renal damage and the intensity of the inflammatory response. FA-AKI induced CD24 in the distal tubular epithelial cells. Compared to WT mice, FA-AKI CD24-/- mice exhibited an attenuated reduction in renal function and histological injury, lower serum IL-10 and interferon γ, and decreased expression of renal TNFα. In contrast, renal and systemic IL-33 upregulation were augmented. CD24-/- FA-AKI animals exhibited increased splenic margination and renal infiltration of regulatory T cells (Tregs). At day 7, FA-AKI CD24-/- mice exhibited increased expression of tubular pro-apoptotic and decreased anti-apoptotic proteins compared to WT animals. Anti-CD24 antibody administration to FA-AKI mice attenuated the decrease in renal function as well as the histological injury. Renal biopsies from patients with ATN stained strongly for CD24 in the distal tubules. In conclusion, during AKI, upregulation of CD24 promotes renal inflammation through inhibition of Treg infiltration and diversion of cell death towards necrosis rather than apoptosis. Neutralization of CD24 may prove a target for future therapies in AKI.

Identification of a group of bisbenzylisoquinoline (BBIQ) compounds as ferroptosis inhibitors

Ferroptosis induced by detrimental accumulation of lipid peroxides has been recently linked to a variety of pathological conditions ranging from acute tissue injuries to chronic degenerative diseases and suppression of ferroptosis by small chemical inhibitors is beneficial to the prevention and treatment of these diseases. However, in vivo applicable small chemical ferroptosis inhibitors are limited currently. In this study, we screened an alkaloid natural compound library for compounds that can inhibit RSL3-induced ferroptosis in HT1080 cells and identified a group of bisbenzylisoquinoline (BBIQ) compounds as novel ferroptosis-specific inhibitors. These BBIQ compounds are structurally different from known ferroptosis inhibitors and they do not appear to regulate iron homeostasis or lipid ROS generation pathways, while they are able to scavenge 1,1-diphenyl-2-picryl-hydrazyl (DPPH) in cell-free reactions and prevent accumulation of lipid peroxides in living cells. These BBIQ compounds demonstrate good in vivo activities as they effectively protect mice from folic acid-induced renal tubular ferroptosis and acute kidney injury. Several BBIQ compounds are approved drugs in Japan and China for traditional uses and cepharanthine is currently in clinical trials against SARS-CoV-2, our discovery of BBIQs as in vivo applicable ferroptosis inhibitors will expand their usage to prevent ferroptotic tissue damages under various pathological conditions.

Protective effects of mefunidone on ischemia-reperfusion injury/Folic acid-induced acute kidney injury.

Renal ischemia-reperfusion injury (IRI) is one of the most common causes of acute kidney injury (AKI). It poses a significant threat to public health, and effective therapeutic drugs are lacking. Mefunidone (MFD) is a new pyridinone drug that exerts a significant protective effect on diabetic nephropathy and the unilateral ureteral obstruction (UUO) model in our previous study. However, the effects of mefunidone on ischemia-reperfusion injury-induced acute kidney injury remain unknown. In this study, we investigated the protective effect of mefunidone against ischemia-reperfusion injury-induced acute kidney injury and explored the underlying mechanism. These results revealed that mefunidone exerted a protective effect against ischemia-reperfusion injury-induced acute kidney injury. In an ischemia-reperfusion injury-induced acute kidney injury model, treatment with mefunidone significantly protected the kidney by relieving kidney tubular injury, suppressing oxidative stress, and inhibiting kidney tubular epithelial cell apoptosis. Furthermore, we found that mefunidone reduced mitochondrial damage, regulated mitochondrial-related Bax/bcl2/cleaved-caspase3 apoptotic protein expression, and protected mitochondrial electron transport chain complexes III and V levels both in vivo and in vitro, along with a protective effect on mitochondrial membrane potential in vitro. Given that folic acid (FA)-induced acute kidney injury is a classic model, we used this model to further validate the efficacy of mefunidone in acute kidney injury and obtained the same conclusion. Based on the above results, we conclude that mefunidone has potential protective and therapeutic effects in both ischemia-reperfusion injury- and folic acid-induced acute kidney injury.

Potential Therapeutic Effects of Morin Hydrate in Folic Acid-Induced Acute Kidney Injury

Abstract Introduction/Objective Acute kidney injury (AKI) causes significant morbidity and mortality and, if left unmanaged, can progress to chronic kidney disease. Despite the advances in our understanding of the pathogenesis of AKI, there are no available therapeutics to combat it effectively. Folic acid (FA)-induced AKI is a well established rat model characterized by acute renal injury due to the disturbance of the antioxidant system with subsequent renal fibrosis. Morin hydrate, a well-known bioactive flavonoid, has been proven to alleviate oxidative stress and exhibits anti- inflammatory, anti-apoptotic and neuroprotective activity. Methods/Case Report We investigated the potential protective and therapeutic effects of morin hydrate on FA-induced AKI. Thirty-five rats were divided randomly into 5 groups (6 rats/ group) as follows: control group received a vehicle for 10 days, FA group received a vehicle for 10 days with an intraperitoneal (IP) injection of a single dose (SD) of FA (250 mg/kg) on the 7thday, FA-Withdrawal group injected with a SD of FA (250 mg/kg IP) on the 1st day with a vehicle for 10 days, morin-pretreated group received morin hydrate (40 mg/kg orally) for 7 days, followed by a SD of FA (250 mg/kg, i.p), and morin-treated group injected with a SD of FA (250 mg/kg IP), then treated with morin hydrate (40 mg/kg orally) for 7 consecutive days. All animals were sacrificed after 10 days and then, blood samples and kidneys were collected for biochemical and histopathological examination. Results (if a Case Study enter NA) FA group showed extensive structural damage in renal tubules and glomeruli with no significant improvement in withdrawal and morin-pretreated groups. Morin treatment significantly attenuated histopathological changes and reduced FA-induced increase in serum creatinine, albumin and urea levels. This therapeutic role of morin was associated with suppression of inflammation, oxidative stress, and apoptosis. Conclusion These findings suggest that morin hydrate constitutes a potential natural therapeutic product for treating AKI.

Melatonin Attenuates Mitochondrial Damage in Aristolochic Acid-Induced Acute Kidney Injury.

Aristolochic acid (AA), extracted from Aristolochiaceae plants, plays an essential role in traditional herbal medicines and is used for different diseases. However, AA has been found to be nephrotoxic and is known to cause aristolochic acid nephropathy (AAN). AA-induced acute kidney injury (AKI) is a syndrome in AAN with a high morbidity that manifests mitochondrial damage as a key part of its pathological progression. Melatonin primarily serves as a mitochondria-targeted antioxidant. However, its mitochondrial protective role in AA-induced AKI is barely reported. In this study, mice were administrated 2.5 mg/kg AA to induce AKI. Melatonin reduced the increase in Upro and Scr and attenuated the necrosis and atrophy of renal proximal tubules in mice exposed to AA. Melatonin suppressed ROS generation, MDA levels and iNOS expression and increased SOD activities in vivo and in vitro. Intriguingly, the in vivo study revealed that melatonin decreased mitochondrial fragmentation in renal proximal tubular cells and increased ATP levels in kidney tissues in response to AA. In vitro, melatonin restored the mitochondrial membrane potential (MMP) in NRK-52E and HK-2 cells and led to an elevation in ATP levels. Confocal immunofluorescence data showed that puncta containing Mito-tracker and GFP-LC3A/B were reduced, thereby impeding the mitophagy of tubular epithelial cells. Furthermore, melatonin decreased LC3A/B-II expression and increased p62 expression. The apoptosis of tubular epithelial cells induced by AA was decreased. Therefore, our findings revealed that melatonin could prevent AA-induced AKI by attenuating mitochondrial damage, which may provide a potential therapeutic method for renal AA toxicity.

Ruscogenin Prevents Folic Acid-Induced Acute Kidney Damage by Inhibiting Rev-erbα/β-Mediated Ferroptosis.

To investigate the pharmacodynamic effects of ruscogenin on acute kidney injury and the Rev-erbα/β regulation of ferroptosis intervention mechanism. The C57BL-6 mice were induced acute kidney injury with folic acid. Plasma, urine, and kidney samples were collected after intraperitoneal injection of ruscogenin (0.01, 0.1, and 1 mg/kg). We measured mouse kidney function indicators, including creatinine (CRE), blood urea nitrogen (BUN), N-acetyl-β-D-glucosidase (NAG), albumin, albumin and creatinine rate (ACR), renal index, and renal injury molecule-1 expression. Meanwhile, we detected the levels of ferroptosis indicators malondialdehyde (MDA), carbonylated proteins, iron ions, glutathione peroxidase 4 (GPX-4), and glutathione (GSH). The expression of solute carrier family 7 member 11 (Slc7a11), heme oxygenase-1 (HO-1), and Rev-erbα/β were detected by the Western blot and quantitative reverse transcription polymerase chain reaction (qRT-PCR), respectively. Ruscogenin (1 mg/kg) significantly reduced the index of folic acid-induced acute kidney injury and alleviated acute kidney injury. In kidney tissues, ruscogenin inhibited folic acid-induced Rev-erbα/β expression, restored HO-1 and SLC7A11 expression to normal levels, and alleviated ferroptosis. Ruscogenin ameliorates acute kidney injury via suppressing ferroptosis in kidney tissues through modulation of the Rev-erbα/β-SLC7A11/HO-1 pathway.

Therapeutic Effect of Trigonella foenum-graecum l Seeds Extract on Folic Acid-Induced Acute Kidney Injury

Therapeutic Effect of Trigonella foenum-graecum l Seeds Extract on Folic Acid-Induced Acute Kidney Injury

MO276: Renal Tubular CD24 Upregulation Aggravates Folic Acid-Induced Acute Kidney Injury Through Inhibition of Regulatory T Cells in Mice

Abstract BACKGROUND AND AIMS Acute kidney injury (AKI) is characterized by cell death and inflammation. CD24 is a sialoglycoprotein which is induced during tissue damage and is not expressed in matured renal tissue. We explored the role of CD24 in the pathogenesis of folic acid-induced AKI (FA-AKI) in mice. METHOD A single i.p. injection of folic acid induced AKI in WT and CD24-/- mice. Renal function tests, histological analysis, immunohistochemistry, Western blot analysis and ELISA were performed to assess the severity of renal damage and the intensity of the inflammatory response. RESULTS FA-AKI induced the expression of CD24 in the distal tubular epithelial cells. Compared to WT mice, FA-AKI CD24-/- mice exhibited an attenuated reduction in renal function and histological injury, lower serum IL-10 and interferon γ, and decreased expression of renal TNFα. Renal and systemic IL-33 were upregulated in both groups. However, it was more pronounced in FA-AKI CD24-/- animals. FA-AKI CD24-animals exhibited increased splenic margination and renal infiltration of regulatory T cells (Tregs). At day 7, FA-AKI CD24-/- mice demonstrated increased expression of tubular pro-apoptotic (activated caspase 3) and decreased anti-apoptotic (Bcl-xL) proteins compared with WT animals. Administration of anti CD24 antibody to FA-AKI mice attenuated the decrease in renal function and ameliorated the histological injury in comparison with untreated animals. Renal biopsies from patients with ATN stained strongly for CD24 in the distal tubules. CONCLUSION During AKI, upregulation of CD24 promotes renal inflammation through inhibition of Tregs infiltration and diversion of cell death towards necrosis rather than apoptosis. Neutralization of CD24 may prove as a target for future therapies in AKI.

MO284: Dipeptidyl-Peptidase-4 Inhibitor, Teneligliptin, Ameliorates Folic Acid-Induced Acute Kidney Injury in Mice

Abstract BACKGROUND AND AIMS Acute kidney injury (AKI) is histologically characterized by tubular cell death and inflammation. It has demonstrated that cell death in renal tubule cells on AKI could be introduced by regulated necrosis or apoptosis, but the molecular mechanisms are reported to be different between AKI models [1]. Recently, Dipeptidyl -peptidase-4(DPP4) inhibitors have been reported to ameliorate cisplatin-induced AKI and ischemia-reperfusion-induced AKI [2, 3]. In this study, we examined the effect of teneligliptin, a DPP4 inhibitor, on folic acid (FA)-induced AKI(FA-AKI) model in mice, which is reported to be mainly induced by ferroptosis, one of the regulated necrotic pathways [4]. METHOD C57BL/6J mice (12–14 weeks old) received a single intraperitoneal injection of FA of 250 mg/kg in 0.3 mol/L sodium bicarbonate, and were euthanized 48 h later. Teneligliptin or vehicle were orally administered just before and 24 h after FA infusion, respectively. Blood samples and renal tissue samples were collected at the time of euthanasia for evaluation. RESULTS The increase of serum creatinine (mean 0.297 mg/dL in teneligliptin-treated mice versus 0.833 mg/dL in vehicle-treated mice; P &amp;lt; 0.01) and blood urea nitrogen (mean 72.22mg/dL in teneligliptin-treated mice versus 177.7mg/dL in teneligliptin-treated mice; P = 0.016) were suppressed in teneligliptin-treated mice, compared to vehicle-treated mice (Fig. 1A and B). Histologically, teneligliptin treatment reduced tubular injury score, tubular cell death evaluated with the terminal uridine nick-end labeling (TUNEL) and kidney injury molecule-1 (kim-1) expression in the kidney (Fig. 2A-C). In addition, number of infiltrating Ly6G + neutrophil and number of infiltrating F4/80 + macrophages were decreased in the kidney of teneligliptin-treated mice, compared with that of vehicle-treated mice (Fig. 2D and E). CONCLUSION We found that teneligliptin ameliorated FA-AKI in mice by suppressing necroinflammation in the kidney. Further studies are needed to clarify the underlying mechanisms of the renoprotection.

IRF-4 deficiency reduces inflammation and kidney fibrosis after folic acid-induced acute kidney injury

The chronic phase following toxin-induced acute kidney injury (AKI) is characterized by robust inflammation and progressive kidney fibrosis. Interferon regulatory factor 4 (IRF-4) is a type of multifunctional transcription factor that has been deeply linked to inflammation and fibrotic diseases. However, the role of IRF-4 in kidney damage and renal fibrosis after toxin-induced AKI remain to be explored. In this work, we examined the effect of IRF-4 deficiency on inflammation and kidney fibrosis in an AKI-chronic kidney disease (CKD) transition model induced by folic acid (FA) injury. We showed that FA treatment resulted in severe acute tubular injury followed by inflammatory reaction and interstitial fibrosis in wild-type mice. A sharp elevation of IRF-4 levels was observed in FA-injured kidneys. IRF-4 knockout led to a substantial reduction of extracellular matrix (ECM) proteins deposition and inhibited myofibroblasts transformation in the kidneys of mice subjected to FA treatment. In addition, IRF-4 ablation impaired F4/80+ macrophages and CD3+ T lymphocytes infiltration into the FA-injured kidneys. Loss of IRF-4 reduced the production of inflammatory molecules such as CXCL16, IL-18, IL-6, and TGF-β1 in the kidneys in response to FA stress. Following FA injury, the kidneys of IRF-4 knockout mice had fewer bone marrow-derived myofibroblasts than wild-type controls. Moreover, IRF-4 disruption inhibited macrophages to myofibroblasts differentiation in the kidneys in response to FA stimuli. In vitro, IL-4 stimulated expression of α-smooth muscle actin and ECM proteins and promoted M2 macrophages to myofibroblasts transition in mouse bone marrow-derived monocytes, which was abolished in the absence of IRF-4. Thus, we identified an important role of IRF-4 in the pathogenesis of progressive CKD following FA-induced AKI.

C-Jun Amino Terminal Kinase Signaling Promotes Aristolochic Acid-Induced Acute Kidney Injury.

Aristolochic acid (AA) is a toxin that induces DNA damage in tubular epithelial cells of the kidney and is the cause of Balkan Nephropathy and Chinese Herb Nephropathy. In cultured tubular epithelial cells, AA induces a pro-fibrotic response via the c-Jun amino terminal kinase (JNK) signaling pathway. This study investigated the in vivo role of JNK signaling with a JNK inhibitor (CC-930) in mouse models of acute high dose AA-induced kidney injury (day 3) and renal fibrosis induced by chronic low dose AA exposure (day 22). CC-930 treatment inhibited JNK signaling and protected from acute AA-induced renal function impairment and severe tubular cell damage on day 3, with reduced macrophage infiltration and expression of pro-inflammatory molecules. In the chronic model, CC-930 treatment inhibited JNK signaling but did not affect AA-induced renal function impairment, tubular cell damage including the DNA damage response and induction of senescence, or renal fibrosis; despite a reduction in the macrophage pro-inflammatory response. In conclusion, JNK signaling contributes to acute high dose AA-induced tubular cell damage, presumably via an oxidative stress-dependent mechanism, but is not involved in tubular atrophy and senescence that promote chronic kidney disease caused by ongoing DNA damage in chronic low dose AA exposure.