Folic 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.

#1995 Runt-related Transcription Factor 1 (RUNX1) is a mediator of acute kidney injury

Abstract Background and Aims A better understanding of the pathogenesis of acute kidney injury (AKI) may lead to new therapeutic approaches. Kidney transcriptomics analyses of murine folic acid-induced AKI (FA-AKI) identified Runx1 as the most upregulated RUNX family gene. RUNX1 is a key regulator of hematopoiesis, associated to leukemias and other non-immunological cancers and modulates myoblast proliferation during muscle regeneration and cardiac remodelling after myocardial infarction. In kidney diseases, RUNX1 is overexpressed in polycystic kidney disease and kidney cancer and favours fibrosis. Method We examined the expression of RUNX1 in folic acid-AKI (FA-AKI), in bacterial lipopolysaccharide (LPS)-induced cytokine storm-AKI (CS-AKI) and in human AKI. For this, female 12- to 14-week-old C57BL/6J wild type (WT) mice, received a single intraperitoneal (i.p) injection of acid folic (250 mg/kg), LPS (5 mg/kg) or vehicle. In cultured murine tubular MCT cells, we explored the expression and role of RUNX1 in response to the cytokine TWEAK or LPS. The chemical inhibitor of RUNX1 Ro5-3335 and a specific small interfering RNA (siRNA) against RUNX1 were used in cultured cells to explore the role of RUNX1 in TWEAK and LPS-induced inflammation. Ro5-3335 was also used in murine AKI (FA-AKI and CS-AKI) to test its potential as a therapeutic target. In all experimental models, plasma samples were collected to assess renal function. Kidneys were collected for RNA (RT-PCR), protein (Western blot, ELISA, immunohistochemistry) and histopathologic studies. Results Kidney transcriptomics identified Runx1 as the most upregulated Runx gene in FA-AKI at 24 h and the most overactive transcription factor in upstream regulator analysis. RUNX1 overexpression in FA-AKI was validated at mRNA and protein levels and localized mainly to tubular cell nuclei. CS-AKI also upregulated kidney RUNX1 in nuclei. In a transcriptomics array of murine tubular cells stimulated with TWEAK for 6 h, Runx1 was upregulated compared with non-stimulated cells, while Runx2 and Runx3 were not. RT-PCR confirmed an early upregulation of Runx1 (3 h) followed by a progressive return to baseline by 24 h. TWEAK also increased total and nuclear RUNX1 protein levels from 6 h onwards. Since kidney RUNX1 is also increased in CS-AKI, we tested whether LPS regulated RUNX1 in cultured tubular cells. LPS transiently increased Runx1 mRNA expression and it also increased whole cell and nuclear RUNX1 protein levels. Mechanistically, RUNX1 bound to the Il-6 gene promoter in response to TWEAK and LPS, and RUNX1 targeting with the chemical inhibitor Ro5-3335 or a specific siRNA prevented the TWEAK- and LPS-induced upregulation of IL-6. NFκB1 p50 pathway is involved in the RUNX1/IL-6 axis since the specific p50 inhibitor SN50 decreased the nuclear accumulation of RUNX1 and IL-6 expression in TWEAK- or LPS-stimulated MCT cells. In vivo, preventive Ro5-3335 improved kidney function and reduced inflammation in FA-AKI and CS-AKI. Moreover, in FA-AKI, Ro5-3335 also prevented the expression of profibrotic genes. However, Ro5-3335 administration after the insult only improved kidney function in CS-AKI. Kidney transcriptomics identified inflammatory genes and transcription factors such as Yap1 and p53 as key targets of Ro5-3335 in CS-AKI. Conclusion RUNX1 contributes to AKI by driving the expression of genes involved in inflammation and represents a novel therapeutic target in AKI.

#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.

#1385 Dipeptidyl-peptidase-4 inhibitor teneligliptin attenuates folic acid-induced tubular cell death via inhibiting ferroptosis

Abstract Background and Aims Dipeptidyl peptidase-4 (DPP-4) inhibitors have been shown to attenuate several types of acute kidney injury (AKI) in animal models [1–3]. However, the mechanisms underlying their renoprotective effects remain to be clarified. Previous studies had identified ferroptosis as the primary cell death pathway for folic acid-induced AKI in mice [4]. Referring to that report, we previously reported that teneligliptin, a DPP-4 inhibitor, ameliorates folic acid-induced AKI. In the present study, we investigated whether teneligliptin attenuates folic acid-induced tubular cell death through its anti-ferroptotic properties. Method In in vitro experiments, we used HK-2 cells, human proximal tubular epithelial cells. HK-2 cells were co-cultured with folic acid and z-VAD-FMK (zVAD), an apoptosis inhibitor; Necrostatin-1s (Nec-1s), a necroptosis inhibitor; Ferrostatin-1 (Fer-1), a ferroptosis inhibitor; or teneligliptin. Co-incubation of erastin, a ferroptosis inducer, and teneligliptin was also performed. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted on HK-2 cells to assess cell viability. Results Fer-1, but not z-VAD or Nec-1s, inhibited folic acid-induced cell death (Fig. 1A-C), indicating that ferroptosis contributes to folic acid-induced tubular cell death, rather than apoptosis or necroptosis. Teneligliptin also inhibited folic acid- and erastin-induced tubular cell death (Fig. 1D,E), suggesting that the DPP-4 inhibitor inhibits folic acid-induced tubular cell death via its anti-ferroptotic properties. Conclusion DPP-4 inhibitor prevents folic acid-induced tubular cell death by inhibiting ferroptosis in vitro, which may contribute to the attenuation of AKI in vivo.

Baicalein alleviates cisplatin-induced acute kidney injury by inhibiting ALOX12-dependent ferroptosis

BackgroundIn acute kidney injury (AKI), ferroptosis is the main mechanism of cell death in the renal tubular epithelium. Baicalein, a traditional Chinese medicine monomer, plays a protective role in various kidney diseases; however, the effect of baicalein on ferroptosis in AKI still needs further exploration. PurposeIn this study, we explored the role of baicalein and its specific mechanism in mediating ferroptosis in cisplatin-induced AKI. MethodsWe used a cisplatin-induced AKI model to study the effects of baicalein on renal tissue and tubular epithelial cell injury. The effects of baicalein on tubular epithelial cell ferroptosis were detected in cisplatin-induced AKI and further verified by folic acid-induced AKI. The Swiss Target Prediction online database was used to predict the possible mechanism by which baicalein regulates ferroptosis, and the specific target proteins were further verified. Molecular docking and SPR were used to further determine the binding potential of baicalein to the target protein. Finally, RNA interference (RNAi) technology and enzymatic inhibition were used to determine whether baicalein regulates ferroptosis through target proteins. ResultsBaicalein alleviated cisplatin- and folic acid-induced renal dysfunction and pathological damage and improved cisplatin-induced HK2 cell injury. Mechanistically, baicalein reduced the expression of 12-lipoxygenase (ALOX12), which inhibits phospholipid peroxidation and ferroptosis in AKI. Molecular docking and SPR demonstrated direct binding between baicalein and ALOX12. Finally, we found that silencing ALOX12 had a regulatory effect similar to that of baicalein. Comparable results were also obtained with the ALOX12 inhibitor ML355. ConclusionThis was the first study to confirm that baicalein regulates ferroptosis both in vitro and in vivo in cisplatin-induced AKI and to verify the regulatory effect of baicalein in folic acid-induced AKI. Our results reveal the critical role of ALOX12 in kidney damage and ferroptosis caused by cisplatin and emphasize the regulatory effect of baicalein on renal tubular epithelial cell ferroptosis mediated by ALOX12. Baicalein is an effective drug for treating AKI, and ALOX12 is a potential drug target.

Butyrate promotes kidney resilience through a coordinated kidney protective response in tubular cells

Acute kidney injury (AKI) is common in hospitalized patients and increases short-term and long-term mortality. Treatment options for AKI are limited. Gut microbiota products such as the short-chain fatty acid butyrate have anti-inflammatory actions that may protect tissues, including the kidney, from injury. However, the molecular mechanisms of tissue protection by butyrate are poorly understood. Treatment with oral butyrate for two weeks prior to folic acid-induced AKI and during AKI improved kidney function and decreased tubular injury and kidney inflammation while stopping butyrate before AKI was not protective. Continuous butyrate preserved the expression of kidney protective factors such as Klotho, PGC-1α and Nlrp6 which were otherwise downregulated. In cultured tubular cells, butyrate blunted the maladaptive tubular cell response to a proinflammatory milieu, preserving the expression of kidney protective factors. Kidney protection afforded by this continuous butyrate schedule was confirmed in a second model of nephrotoxic AKI, cisplatin nephrotoxicity, where the expression of kidney protective factors was also preserved. To assess the contribution of preservation of kidney protective factors to kidney resilience, recombinant Klotho was administered to mice with cisplatin-AKI and shown to preserve the expression of PGC-1α and Nlrp6, decrease kidney inflammation and protect from AKI. In conclusion, butyrate promotes kidney resilience to AKI and decreases inflammation by preventing the downregulation of kidney protective genes such as Klotho. This information may be relevant to optimize antibiotic management during hospitalization.

Glycyrol Alleviates Acute Kidney Injury by Inhibiting Ferroptsis.

Acute kidney injury (AKI) is a common clinical problem with high morbidity and mortality. The discovery of ferroptosis has provided novel insights into the mechanisms underlying AKI and paves the way for developing ferroptosis-based approaches to treat AKI. Glycyrol (GC) is a representative coumarin compound isolated from licorice that demonstrates various pharmacological activities. However, its potential for a protective effect against kidney injury remains unknown. We hypothesized that GC might be able to protect against AKI via suppression of ferroptosis. This hypothesis was tested in a cell-culture model of RSL3-induced nephrocyte ferroptosis and a mouse model of folic acid-induced AKI. The results showed that GC exerted a significant protective effect against nephrocyte ferroptosis in vitro and was effective against folic acid-induced AKI in vivo, where it was mechanistically associated with suppressing HO-1-mediated heme degradation. Collectively, the findings of the present study support the hypothesis that GC holds considerable potential to be developed as a novel agent for treating ferroptosis-related 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.

Phosphate Restriction Prevents Metabolic Acidosis and Curbs Rise in FGF23 and Mortality in Murine Folic Acid-Induced AKI.

Patients with AKI suffer a staggering mortality rate of approximately 30%. Fibroblast growth factor 23 (FGF23) and phosphate (P i ) rise rapidly after the onset of AKI and have both been independently associated with ensuing morbidity and mortality. This study demonstrates that dietary P i restriction markedly diminished the early rise in plasma FGF23 and prevented the rise in plasma P i , parathyroid hormone, and calcitriol in mice with folic acid-induced AKI (FA-AKI). Furthermore, the study provides evidence for P i -sensitive osseous Fgf23 mRNA expression and reveals that P i restriction mitigated calciprotein particles (CPPs) formation, inflammation, acidosis, cardiac electrical disturbances, and mortality in mice with FA-AKI. These findings suggest that P i restriction may have a prophylactic potential in patients at risk for AKI. In AKI, plasma FGF23 and P i rise rapidly and are independently associated with disease severity and outcome. The effects of normal (NP) and low (LP) dietary P i were investigated in mice with FA-AKI after 3, 24, and 48 hours and 14 days. After 24 hours of AKI, the LP diet curbed the rise in plasma FGF23 and prevented that of parathyroid hormone and calcitriol as well as of osseous but not splenic or thymic Fgf23 mRNA expression. The absence of Pth prevented the rise in calcitriol and reduced the elevation of FGF23 in FA-AKI with the NP diet. Furthermore, the LP diet attenuated the rise in renal and plasma IL-6 and mitigated the decline in renal α -Klotho. After 48 hours, the LP diet further dampened renal IL-6 expression and resulted in lower urinary neutrophil gelatinase-associated lipocalin. In addition, the LP diet prevented the increased formation of CPPs. Fourteen days after AKI induction, the LP diet group maintained less elevated plasma FGF23 levels and had greater survival than the NP diet group. This was associated with prevention of metabolic acidosis, hypocalcemia, hyperkalemia, and cardiac electrical disturbances. This study reveals P i -sensitive FGF23 expression in the bone but not in the thymus or spleen in FA-AKI and demonstrates that P i restriction mitigates CPP formation, inflammation, acidosis, and mortality in this model. These results suggest that dietary P i restriction could have prophylactic potential in patients at risk for AKI.

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.

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.

NAC Pre-Administration Prevents Cardiac Mitochondrial Bioenergetics, Dynamics, Biogenesis, and Redox Alteration in Folic Acid-AKI-Induced Cardio-Renal Syndrome Type 3.

The incidence of kidney disease is increasing worldwide. Acute kidney injury (AKI) can strongly favor cardio-renal syndrome (CRS) type 3 development. However, the mechanism involved in CRS development is not entirely understood. In this sense, mitochondrial impairment in both organs has become a central axis in CRS physiopathology. This study aimed to elucidate the molecular mechanisms associated with cardiac mitochondrial impairment and its role in CRS development in the folic acid-induced AKI (FA-AKI) model. Our results showed that 48 h after FA-AKI, the administration of N-acetyl-cysteine (NAC), a mitochondrial glutathione regulator, prevented the early increase in inflammatory and cell death markers and oxidative stress in the heart. This was associated with the ability of NAC to protect heart mitochondrial bioenergetics, principally oxidative phosphorylation (OXPHOS) and membrane potential, through complex I activity and the preservation of glutathione balance, thus preventing mitochondrial dynamics shifting to fission and the decreases in mitochondrial biogenesis and mass. Our data show, for the first time, that mitochondrial bioenergetics impairment plays a critical role in the mechanism that leads to heart damage. Furthermore, NAC heart mitochondrial preservation during an AKI event can be a valuable strategy to prevent CRS type 3 development.

Dietary docosahexaenoic acid plays an opposed role in ferroptotic and non-ferroptotic acute kidney injury

Ferroptosis due to polyunsaturated fatty acid (PUFA) peroxidation has been implicated in the pathogenesis of acute kidney injury (AKI), suggesting the risk of dietary intake of PUFA for people susceptible to AKI. Clinically, however, in addition to ferroptosis, other mechanisms also contribute to different types of AKI such as inflammation associated necroptosis and pyroptosis. Therefore, the role of PUFA, especially ω3 PUFA which is a common food supplement, in various AKIs deserves further evaluation. In this study, rhabdomyolysis- and folic acid-induced AKI (Rha-AKI and FA-AKI) were established in mice fed with different fatty acids Histology of kidney, blood urea nitrogen and creatinine, lipid peroxidation, and inflammatory factors were examined. Results showed that these two types of AKIs had diametrically different pathogenesis indicated by that ferrostatin-1 (Fer-1), a lipid antioxidant, can attenuate FA-AKI rather than Rha-AKI. Further, dietary DHA (provided by fish oil) reduced tubular injury and renal lesion by inhibiting peroxidation and inflammation in mice with Rha-AKI while increasing cell death, tissue damage, peroxidation and inflammation in mice with FA-AKI. In human renal tubular epithelial cell line HK-2, MTT assay and DHE staining showed that both myoglobin and ferroptosis inducers can cause cell death and oxidative stress. Ferroptosis inducer-induced cell death was promoted by DHA, while such result was not observed in myoglobin-induced cell death when adding DHA. This study illustrates that the mechanisms of AKI might be either ferroptosis dependent or -independent and the deterioration effect of dietary DHA depends on whether ferroptosis is involved.

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.

Use of novel structural features to identify urinary biomarkers during acute kidney injury that predict progression to chronic kidney disease

BackgroundA significant barrier to biomarker development in the field of acute kidney injury (AKI) is the use of kidney function to identify candidates. Progress in imaging technology makes it possible to detect early structural changes prior to a decline in kidney function. Early identification of those who will advance to chronic kidney disease (CKD) would allow for the initiation of interventions to halt progression. The goal of this study was to use a structural phenotype defined by magnetic resonance imaging and histology to advance biomarker discovery during the transition from AKI to CKD.MethodsUrine was collected and analyzed from adult C57Bl/6 male mice at four days and 12 weeks after folic acid-induced AKI. Mice were euthanized 12 weeks after AKI and structural metrics were obtained from cationic ferritin-enhanced-MRI (CFE-MRI) and histologic assessment. The fraction of proximal tubules, number of atubular glomeruli (ATG), and area of scarring were measured histologically. The correlation between the urinary biomarkers at the AKI or CKD and CFE-MRI derived features was determined, alone or in combination with the histologic features, using principal components.ResultsUsing principal components derived from structural features, twelve urinary proteins were identified at the time of AKI that predicted structural changes 12 weeks after injury. The raw and normalized urinary concentrations of IGFBP-3 and TNFRII strongly correlated to the structural findings from histology and CFE-MRI. Urinary fractalkine concentration at the time of CKD correlated with structural findings of CKD.ConclusionsWe have used structural features to identify several candidate urinary proteins that predict whole kidney pathologic features during the transition from AKI to CKD, including IGFBP-3, TNFRII, and fractalkine. In future work, these biomarkers must be corroborated in patient cohorts to determine their suitability to predict CKD after AKI.

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.

Mitochondrial Targeted Antioxidant SKQ1 Ameliorates Acute Kidney Injury by Inhibiting Ferroptosis.

Emerging evidence suggests that ferroptosis is highly correlated with the pathogenesis of acute kidney injury (AKI). Ferroptosis, an iron-dependent form of cell death, is manifested by a toxic accumulation of lipid peroxides and ultrastructural changes in mitochondria. We herein investigated the effect of Visomitin (SKQ1), a novel mitochondria-targeting antioxidant, on several AKI models in vivo and in vitro. Our results revealed that SKQ1 treatment greatly reversed renal outcomes in cisplatin, ischemia-reperfusion injury (IRI), or folic acid-induced AKI models. These effects were reflected in attenuated levels of renal injury biomarkers, histologic indices of tubular injury, and inflammatory infiltration in the SKQ1-treated groups. Transcriptomics analysis depicted ferroptosis signaling as the most pronounced pathway downregulated after SKQ1 treatment. Consequently, administration of SKQ1 significantly ameliorated lipid peroxide accumulation and inhibited ferroptosis in the kidneys of mice with AKI. In cultured human proximal tubule epithelial cells (HK2), SKQ1 treatment markedly mitigated cisplatin-induced mitochondrial reactive oxygen species (ROS) production, resulting in lower levels of lipid peroxidation and ferroptosis. In conclusion, SKQ1 treatment protected against ischemic- or nephrotoxic-induced AKI by inhibiting ferroptosis in vivo and in vitro. These results could facilitate a broader understanding of the interaction between mitochondrial antioxidants and ferroptotic defense mechanisms, providing a possible therapeutic strategy in AKI.