Kidney Injury Molecule-1 Expression Research Articles

Radish red protects against early diabetic kidney disease through inhibiting inflammation, pyroptosis and insulin resistance via IRAK1 signaling suppression

Diabetic kidney disease (DKD) is a major diabetic complication and a leading cause of end-stage renal disease (ESRD), but the therapeutic strategies for DKD are still limited. Red radishes (Raphanus sativus L.) are a rich source of natural anthocyanins that have antioxidant, anti-apoptotic and anti-inflammatory activities. In this study, we attempted to explore the effects of natural pigment anthocyanin called radish red (RR) extracted from red radishes on DKD progression and the underlying molecular mechanisms. RR dose-dependently reduced the tubular cell injury, indicated by the decreased expression of kidney injury molecule 1 (KIM1). Furthermore, releases of pro-inflammatory cytokines including interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) stimulated by HF were strongly relieved by RR. Inflammatory response and pyroptosis were also identified to be induced by HF stimulation but were mitigated by RR in HK2 cells through repressing nuclear factor-κB (NF-κB) activation and NLR family, pyrin domain-containing 3 (NLRP3) inflammasome. IR and lipogenesis due to HF exposure were also significantly ameliorated by RR in HK2 cells. Mechanistically, RNA-Seq analysis showed that RR strongly depressed interleukin-1 receptor-associated kinase-1 (IRAK1)-mediated NLRP3 inflammasome, pyroptosis, IR and lipid deposition. Importantly, IRAK1 overexpression almost diminished the beneficial effects of RR, while being rescued upon NLRP3 knockdown in HF-treated HK2 cells, revealing that RR performed its nephroprotective functions in diabetic kidney via inactivating the IRAK1-NLRP3 signaling pathway. Similarly, animal studies confirmed that RR supplementation efficiently ameliorated HFF-caused metabolic disturbance, IR, renal dysfunctions in mice and improved structural kidney damage. Consistently, RR consumption dramatically reduced lipid accumulation, inflammatory response and pyroptosis in kidney tissues of HFF-challenged mice mainly through repressing IRAK1-NLRP3 axis. Our results demonstrated that dietary supplementation with RR may serve as an IRAK1-NLRP3 inhibitor for preventing and treating diabetic nephropathy.

Magnetic vagus nerve stimulation ameliorates contrast-induced acute kidney injury by circulating plasma exosomal miR-365-3p

BackgroundContrast-induced acute kidney injury (CI-AKI) is manifested by a rapid decline in renal function occurring within 48–72 h in patients exposed to iodinated contrast media (CM). Although intravenous hydration is currently the effective method confirmed to prevent CI-AKI, it has several drawbacks. Some investigations have demonstrated the nephroprotective effects of vagus nerve stimulation (VNS) against kidney ischemia-reperfusion injury, but no direct research has investigated the use of VNS for treating CI-AKI. Additionally, most current VNS treatment applies invasive electrical stimulator implantation, which is largely limited by the complications. Our recent publications introduce the magnetic vagus nerve stimulation (mVNS) system pioneered and successfully used for the treatment of myocardial infarction. However, it remains uncertain whether mVNS can mitigate CI-AKI and its specific underlying mechanisms. Therefore, we herein evaluate the potential therapeutic effects of mVNS on CM-induced nephropathy in rats and explore the underlying mechanisms.ResultsmVNS treatment was found to significantly improve the damaged renal function, including the reduction of elevated serum creatinine (Scr), blood urea nitrogen (BUN), and urinary N-acetyl-β-D-glucosaminidase (NAG) with increased urine output. Pathologically, mVNS treatment alleviated the renal tissue structure injury, and suppressed kidney injury molecule-1 (KIM-1) expression and apoptosis in renal tubular epithelial cells. Mechanistically, increased circulating plasma exosomal miR-365-3p after mVNS treatment enhanced the autophagy and reduced CM-induced apoptosis in renal tubular epithelial cells by targeting Ras homolog enriched in brain (Rheb).ConclusionsIn summary, we demonstrated that mVNS can improve CI-AKI through enhanced autophagy and apoptosis inhibition, which depended on plasma exosomal miR-365-3p. Our findings highlight the therapeutic potential of mVNS for CI-AKI in clinical practice. However, further research is needed to determine the optimal stimulation parameters to achieve the best therapeutic effects.Graphical abstract

Hollow mesoporous Prussian blue nanoenzymes as Rapamycin carrier for targeted treatment of ischemia/reperfusion-induced acute kidney injury through pro-mitophagy and oxidative stress alleviation

The kidney’s abundant mitochondrial content and substantial oxygen requirements make it particularly vulnerable to a series of detrimental events following ischemia/reperfusion (I/R). These events encompass oxidative stress, mitochondrial impairment, depletion of adenosine triphosphate (ATP), and inflammation, cumulatively leading to acute kidney injury (AKI). Currently, no approved therapy beyond supportive care is available for AKI. While numerous studies have focused on neutralizing pre-existing reactive oxygen species (ROS) to mitigate AKI, insufficient attention has been paid to addressing the root causes of ROS. Here, we prepared Rapamycin (Rapa)-loaded hollow mesoporous Prussian blue nanoparticles (HMPB-Rapa) and grafted injured renal-targeted hyaluronic acid (HA) onto HMPB-Rapa (HA-HMPB-Rapa), which effectively scavenge pre-existing ROS and the source (massive damaged mitochondria) through the dual function of HMPB carrier and Rapa, thus preventing the further burst of ROS. In the I/R-induced AKI rat model, a single-dose of HA-HMPB-Rapa 6 h after I/R injury effectively inhibited oxidative stress and inflammation, protected renal cells from apoptotic damage, as well as ultimately restored renal function, and attenuated pathological changes. Mechanistically, HA-HMPB-Rapa cleared damaged mitochondria by activating the pro-mitophagy signaling pathway (PINK-1/Parkin-P62-LC3), cutting off the conduction in the intrinsic apoptotic pathway mediated by the Bax-Cyt-c-Cleaved Casp-3 signaling axis, and reduced the expression of renal tubular marker kidney injury molecule-1. HA-HMPB-Rapa represents an exciting new avenue for the treatment of diseases related to I/R-induced injury.

A novel pharmacological strategy using nanoparticles with glutathione and virgin coconut oil to treat gentamicin-induced acute renal failure in rats.

In acute renal failure (ARF), the glomerular filtration rate is reduced, and nitrogenous waste products accumulate persistently, which can last anywhere from a few hours to several days. There is hope for a reversal of the rapid loss of renal function caused by this condition. This study, with gentamicin-induced acute ARF as a prospective setting, sets out to examine the reno-protective benefits of virgin coconut oil (VCO) and GSH. Furthermore, the study evaluated the effect of medication nanoparticle compositions on several kidney function markers. The induction of ARF is achieved with the intraperitoneal injection of gentamicin. To assess renal function, rats underwent 24h of dehydration and hunger before their deaths. The study examined various aspects, including kidney function tests, markers of oxidative stress, histology of kidney tissue, inflammatory cytokines, immunohistochemistry expression of nuclear factor-kappa B (NF-κB), and specific biomarkers for kidney tissue damage, such as kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL). The results of our study indicated that the combination of VCO and GSH, using both regular and nanoparticle formulations, had a better protective impact on the kidneys compared to using either drug alone. The recovery of renal tissue and serum markers, which are symptomatic of organ damage, indicates improvement. This was also demonstrated by the reduction in tubular expression of TNF-α, IL-1β, KIM-1, and NGAL. The immunohistochemical studies showed that the combination therapy, especially with the nanoforms, greatly improved the damaged cellular changes in the kidneys, as shown by higher levels of NF-κB. The study shows that VCO and GSH, when administered individually or combined, significantly improve ARF in a gentamicin-induced rat model, highlighting potential therapeutic implications. Notably, the combined nanoparticulate formulations exhibit substantial effectiveness.

Medicinal evaluation and molecular docking study of osajin as an anti-inflammatory, antioxidant, and antiapoptotic agent against sepsis-associated acute kidney injury in rats

Despite efforts to find effective drugs for sepsis-associated acute kidney injury (SA-AKI), mortality rates in patients with SA-AKI have not decreased. Our study evaluated the protective effects of isoflavone osajin (OSJ) on SA-AKI in rats by targeting inflammation, oxidative stress, and apoptosis, which represent the cornerstones in the pathophysiological mechanism of SA-AKI. Polymicrobial sepsis was induced in rats via the cecal ligation and puncture (CLP) technique. Markers of oxidative stress were evaluated in kidney tissues using biochemical methods. The expression of interleukin-33 (IL-33), 8-hydroxydeoxyguanosine (8-OHdG), caspase-3, and kidney injury molecule-1 (KIM-1) was evaluated as indicators of inflammation, DNA damage, apoptosis, and SA-AKI respectively in the kidney tissues using immunohistochemical and immunofluorescent detection methods. The CLP technique significantly (p < 0.001) increased lipid peroxidation (LPO) levels and significantly (p < 0.001) decreased the activities of superoxide dismutase and catalase in kidney tissues. In the renal tissues, strong expression of IL-33, 8-OHdG, caspase-3, and KIM-1 was observed with severe degeneration and necrosis in the tubular epithelium and intense interstitial nephritis. In contrast, the administration of OSJ significantly (p < 0.001) reduced the level of LPO, markedly improved biomarkers of antioxidant status, decreased the levels of serum creatinine and urea, lowered the expression of IL-33, 8-OHdG, caspase-3, and KIM-1 and alleviated changes in renal histopathology. A promising binding score was found via a molecular docking investigation of the OSJ-binding mode with mouse IL-33 (PDB Code: 5VI4). Therefore, OSJ protects against SA-AKI by suppressing the IL-33/LPO/8-OHdG/caspase-3 pathway and improving the antioxidant system.

P2X7 receptor deficiency attenuates cisplatin-induced kidney injury via inhibiting NLRP3 inflammasome activation

Nephrotoxicity is a major constraint of cisplatin application in many solid tumors. Since the lack of preventive strategies, the necessity exists to identify critical molecular targets involved in cisplatin nephrotoxicity. The Purinergic ligand-gcotedion channel 7 receptor (P2X7R) is a ligand-gated ion channel that is predominantly implicated in inflammation and cell death. Our aim is to investigate the role P2X7R in cisplatin-induced acute and chronic kidney injury, as well as the underlying mechanism. In this study, we found that cisplatin can cause an increase in the expression of P2X7R in mouse kidney tissue, and P2X7R knockout can alleviate acute renal function damage caused by cisplatin, as well as the expression of kidney injury molecule 1 (KIM-1) and interleukin-18 (IL-18). Cisplatin can cause an increase in the expression of nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome in mouse kidney tissue. Compared with wild-type mice, P2X7R −/- mice showed decreased expression of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), cleaved Caspase-1, and cleaved IL-1β in kidney tissue after cisplatin administration, and the apoptosis of renal tubular epithelial cells were also decreased. In addition, we also found that NLRP3 knockout can improve cisplatin induced degeneration, detachment, and necrosis of renal tubular epithelial cells. Furthermore, P2X7R −/- mice also showed reduced renal fibrosis and better long-term renal prognosis. In conclusion, our study identified that P2X7R knockout can improve cisplatin induced acute renal injury and chronic renal fibrosis by inhibiting the activation of NLRP3 inflammasome.

Myeloid-specific ferritin light chain deletion does not exacerbate sepsis-associated AKI.

Sepsis-associated acute kidney injury (SA-AKI) is a key contributor to the life-threatening sequelae attributed to sepsis. Mechanistically, SA-AKI is a consequence of unabated myeloid cell activation and oxidative stress that induces tubular injury. Iron mediates inflammatory pathways directly and through regulating the expression of myeloid-derived ferritin, an iron storage protein comprising ferritin light (FtL) and ferritin heavy chain (FtH) subunits. Previous work revealed that myeloid FtH deletion leads to a compensatory increase in intracellular and circulating FtL and is associated with amelioration of SA-AKI. We designed this study to test the hypothesis that loss of myeloid FtL and subsequently, circulating FtL will exacerbate the sepsis-induced inflammatory response and worsen SA-AKI. We generated a novel myeloid-specific FtL knockout mouse (FtLLysM-/-) and induced sepsis via cecal ligation and puncture or lipopolysaccharide endotoxemia. As expected, serum ferritin levels were significantly lower in the knockout mice, suggesting that myeloid cells dominantly contribute to circulating ferritin. Interestingly, although sepsis induction led to a marked production of pro- and anti-inflammatory cytokines, there was no statistical difference between the genotypes. There was a similar loss of kidney function, as evidenced by a rise in serum creatinine and cystatin C and renal injury identified by expression of kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin. Finally, RNA sequencing revealed upregulation of pathways for cell cycle arrest and autophagy postsepsis, but no significant differences were observed between genotypes, including in key genes associated with ferroptosis, an iron-mediated form of cell death. The loss of FtL did not impact sepsis-mediated activation of NF-κB or HIF-1a signaling, key inflammatory pathways associated with dysregulated host response. Taken together, while FtL overexpression was shown to be protective against sepsis, the loss of FtL did not influence sepsis pathogenesis.NEW & NOTEWORTHY Hyperferritinemia in sepsis is often associated with a proinflammatory phenotype and poor prognosis. We previously showed the myeloid deletion of FtH results in a compensatory increase in FtL and is associated with reduced circulating cytokines and decreased rates of SA-AKI in animal sepsis models. Here, we show that myeloid deletion of FtL does not impact the severity of SA-AKI following CLP or LPS, suggesting that FtH plays the predominant role in propagating myeloid-induced proinflammatory pathways.

Sildenafil and furosemide nanoparticles as a novel pharmacological treatment for acute renal failure in rats

Hospitalized patients often develop acute renal failure (ARF), which causes severe morbidity and death. This research investigates the potential renoprotective benefits of sildenafil and furosemide in glycerol-induced ARF, and measures kidney function metrics in response to nanoparticle versions of these medications. Inducing ARF is commonly done by injecting 50% glycerol intramuscularly. Rats underwent a 24-h period of dehydration and starvation before slaughter for renal function testing. We investigated urine analysis, markers of oxidative stress, histology of kidney tissue, immunohistochemistry analysis of caspase-3 and interleukin-1 beta (IL-1 β), kidney injury molecule-1 (KIM-1), and neutrophil gelatinase–associated lipocalin (NGAL), which are specific indicators of kidney tissue damage. The results of our study showed that the combination of sildenafil and furosemide, using both traditional and nanoparticle formulations, had a greater protective effect on the kidneys compared to using either drug alone. The recovery of renal tissue indicators, serum markers, and urine markers, which are indicative of organ damage, provides evidence of improvement. This was also indicated by the reduction in KIM-1 and NGAL tubular expression. The immunohistochemistry tests showed that the combination therapy, especially with the nanoforms, greatly improved the damaged cellular changes in the kidneys, as shown by higher levels of caspase-3 and IL-1β. According to the findings, a glycerol-induced rat model demonstrates that sildenafil and furosemide, either alone or in combination, in conventional or nanoparticulate forms, improve ARF dysfunction. The synergistic nanoparticulate compositions show remarkable effectiveness. This observation highlights the possible therapeutic implications for ARF treatment.

Megalin-related mechanism of hemolysis-induced acute kidney injury and the therapeutic strategy.

Hemolysis-induced acute kidney injury (AKI) is attributed to heme-mediated proximal tubule epithelial cell (PTEC) injury and tubular cast formation due to intratubular protein condensation. Megalin is a multiligand endocytic receptor for proteins, peptides, and drugs in PTECs and mediates the uptake of free hemoglobin and the heme-scavenging protein α1-microglobulin. However, understanding of how megalin is involved in the development of hemolysis-induced AKI remains elusive. Here, we investigated the megalin-related pathogenesis of hemolysis-induced AKI and a therapeutic strategy using cilastatin, a megalin blocker. A phenylhydrazine-induced hemolysis model developed in kidney-specific mosaic megalin knockout (MegKO) mice confirmed megalin-dependent PTEC injury revealed by the co-expression of kidney injury molecule-1 (KIM-1). In the hemolysis model in kidney-specific conditional MegKO mice, the uptake of hemoglobin and α1-microglobulin as well as KIM-1 expression in PTECs was suppressed, but tubular cast formation was augmented, likely due to the nonselective inhibition of protein reabsorption in PTECs. Quartz crystal microbalance analysis revealed that cilastatin suppressed the binding of megalin with hemoglobin and α1-microglobulin. Cilastatin also inhibited the specific uptake of fluorescent hemoglobin by megalin-expressing rat yolk sac tumor-derived L2 cells. In a mouse model of hemolysis-induced AKI, repeated cilastatin administration suppressed PTEC injury by inhibiting the uptake of hemoglobin and α1-microglobulin and also prevented cast formation. Hemopexin, another heme-scavenging protein, was also found to be a novel ligand of megalin, and its binding to megalin and uptake by PTECs in the hemolysis model were suppressed by cilastatin. Mass spectrometry-based semiquantitative analysis of urinary proteins in cilastatin-treated C57BL/6J mice indicated that cilastatin suppressed the reabsorption of a limited number of megalin ligands in PTECs, including α1-microglobulin and hemopexin. Collectively, cilastatin-mediated selective megalin blockade is an effective therapeutic strategy to prevent both heme-mediated PTEC injury and cast formation in hemolysis-induced AKI. © 2024 The Pathological Society of Great Britain and Ireland.

Interaction of Sex Hormones and Dopamine D2 Receptor Polymorphisms in Human Renal Proximal Tubule Cells

The renal inflammatory response can be modulated by the dopamine D2 receptor (D2R) subtype. The DRD2 gene is highly polymorphic in humans, and single nucleotide polymorphisms (SNPs), such as rs6276 and rs6277 ( DRD2 SNPs), decrease its expression and function. We hypothesized that the response of human renal proximal tubular cells (hRPTCs) to hormones in the culture medium differs by sex and DRD2 SNPs, leading to differences in the inflammatory response. We determined the effects of dihydrotestosterone (DHT) and estradiol (ES) on hRPTCs genotyped for the presence or absence of DRD2 SNPs. We studied four cell lines from males (M) and females (F) with wild-type (WT) DRD2 (M- DRD2 WT and F- DRD2 WT) and DRD2 SNPs (M- DRD2 SNPs and F- DRD2 SNPs). The cells were cultured in a medium containing charcoal-stripped fetal bovine serum (FBS) for 24 hours, without (control group, CG) and with dihydrotestosterone (DHT, 5 nM) or estradiol (ES, 20 nM). We quantified the renal protein expression (protein of interest/GAPDH) of proinflammatory and profibrotic factors and markers of injury and proliferation. DHT decreased the expression of transforming growth factor (TGF-β) by ≍50% in both M- DRD2 WT (0.4±0.1 vs. 1.0±0.2) and M- DRD2 SNPs (1.0±0.1 vs. 1.6±0.1) (n=5-6, p&lt;0.05). However, there was no significant effect of DHT or ES on the expression of TGF-β in F- DRD2 WT and F- DRD2 SNPs. DHT did not affect the fibronectin 1 (FN1) expression in M- DRD2 WT and M- DRD2 SNPs. In F- DRD2 WT, DHT did not affect FN1 expression but decreased in F- DRD2 SNPs (0.9±0.1 vs. 1.7±0.1, n=6, p&lt;0.05). Similar to DHT, ES did not affect FN1 expression in M- DRD2 WT. However, ES decreased FN1 by 50% in M- DRD2 SNPs (0.5±0.1 vs. 1.1±0.1, n=6, p&lt;0.05). Similar to the studies in males, ES did not affect FN1 expression in F- DRD2 WT but also decreased FN1 (1.0±0.1 vs. 1.7±0.1, n=6, p&lt;0.05) in F-DRD2 SNPs. Neither DHT nor ES affected the expression of Kidney Injury Molecule-1 (KIM-1) in M- DRD2 WT. However, in M- DRD2 SNPs, both DHT and ES decreased KIM-1 (1.4±0.2 vs. 1.8±0.2 and 1.1±0.1 vs. 1.8±0.2, respectively; n=5, p&lt;0.05). Unlike in M- DRD2-WT, in F- DRD2 WT, ES increased KIM-1 expression (1.0±0.2 vs. 0.52±0.05, n=5, p&lt;0.05), while DHT had no significant effect. Unlike in M- DRD2 SNPs, where both DHT and ES decreased KIM-1, in F- DRD2 SNPs, neither DHT nor ES affected KIM-1. DHT did not affect the expression of Ki67 in M- DRD2 WT. However, in M- DRD2 SNPs, DHT decreased Ki67 expression (0.60±0.08 vs. 1.6±0.2, n=6, p&lt;0.05). Similar to M- DRD2-WT, DHT did not affect Ki67 expression in F- DRD2-WT. ES also did not affect Ki67 expression in M- DRD2 WT but decreased Ki67 expression in M-DRD2 SNPs (0.91± 0.18 vs. 1.6±0.2, n=6, p&lt;0.05). Similar to the male data, the treatment with ES did not affect the Ki67 expression in F- DRD2 WT but decreased Ki67 expression in F- DRD2 SNPs (1.5±0.2 vs. 0.9±0.1, n=6, p&lt;0.05). Our results demonstrate similarities (FN1 and Ki67) and differences (TGF-β and KIM-1) in profibrotic and proliferation markers expressed in hRPTCs between males and females. These differences are more pronounced in cell lines expressing DRD2 SNPs than those expressing the DRD2 WT. While both DHT and ES decreased KIM-1 expression in males with DRD2 polymorphisms, they had no effect in females with DRD2 polymorphisms. These results indicate a complex interaction of the effect of hormones and DRD2 polymorphisms in regulating the inflammatory response in hRPTCs. Their roles in regulating renal physiology and blood pressure remain to be defined. R01 DK039308 P01 HL068686 R01 HL023081 R37 HL023081 R01 DK119652/ NIH HH/ US. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Renal epithelial-derived miR-17~92 inhibits STAT3 signaling, serving a sex-specific and renoprotective role in the development of AKI

Prerenal acute kidney injury (AKI) is a sudden loss of kidney excretory function from reduced blood flow to the kidney. Sex differences in the degree of injury have been recognized, the causes of which are ill-defined. While a role for miRs (microRNAs) in AKI has been recognized, there are no reports of sex differences in miR regulation after injury. Here we investigated sex differences in miR regulation in proximal tubular epithelial cells during AKI with IRI (ischemia reperfusion injury) in vivo and miR regulation of signal transducer and activator of transcription 3 (STAT3) signaling in vitro. We hypothesize that sex-differentially expressed miR-17~92 serves as a negative regulator of STAT3 signaling, repressing proinflammatory signaling in tubular epithelium during AKI. We recapitulated the phenotype that males have a higher level of injury compared to females with proportional intensity and duration of IRI in a murine AKI model. C57BL/6J mice underwent 25 minutes of unilateral IRI with a contralateral nephrectomy and sacrificed 18 hours post-reperfusion. Injured male tubules showed a significant increase in Kidney injury molecule-1 (Kim-1) expression (n³3, fold change=75.28 ± 35.92) whereas injured females showed no change, confirming that injured males experience a higher level of tubular injury than females with a matched degree of ischemia. Control female tubules showed significantly higher baseline expression of miR-17~92 compared to control males (n ³ 5, difference between means ± SEM: miR-17a, 2.337 ± 0.6322; miR-18a, 5.841 ± 2.008; miR-19a, 5.041 ± 1.232; miR- 19b, 3.826 ± 0.9791; miR-20a, 3.826 ± 1.053; miR-92a, 5.627 ± 1.588). MiR-17~92 expression in injured male tubules significantly increased compared to male controls. Even after this increase, miR-17~92 expression in male tubules was lower compared to miR-17~92 in injured females. Mir-17~92 is predicted to target STAT3 and components of the interleukin-6 (IL6)/STAT3 pathway. qPCR showed a significant increase in Stat3 expression after IRI in both sexes, but only a significant increase in IL6 and heme oxygenase-1 in injured males. To investigate miR regulation of STAT3 in vitro, we used a human proximal tubule cell line (HK2) model. Recombinant IL6 induced STAT3 protein expression and phosphorylation, and increased miR-17~92 expression. By altering miR-17~92 expression in HK2 cells we demonstrated that expression of STAT3 in response to IL6 is regulated, in part, by these miRs. These data provide the first example of sex-differential miR regulation in response to IRI. The elevated baseline expression of miR-17~92 in the female mice may provide a novel mechanism to account for sex-differences in IRI-AKI and may form part of the mechanism to protect female kidney epithelia from IRI and AKI. This work is supported by the NIH (DK102843 to MBB), (NIH R01DK102843-08S1 to AS), NIH (DK137419 and DK125015 to JH) and NIH (5R01DK131991 and 5R01DK064005 to RT). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Nephroprotective Effect of Hibiscus Sabdariffa Leaf Flavonoid Extracts via KIM-1 and TGF-1β Signaling Pathways in Streptozotocin-Induced Rats.

Diabetes-induced kidney damage represents a substantial health hazard, emphasizing the imperative to explore potential therapeutic interventions. This study investigates the nephroprotective activity of flavonoid-rich extracts from Hibiscus sabdariffa leaves in streptozotocin-induced diabetic rats. The flavonoid-rich extracts of H. sabdariffa leaves was obtained using a standard procedure. The animals were induced with streptozotocin and thereafter treated with both low (LDHSFL) and high doses (HDHSFL) of flavonoid-rich extracts from H. sabdariffa leaves and metformin (MET), and other groups are diabetic control (DC) and normal control (NC). The study assesses diverse renal parameters, encompassing kidney redox stress biomarkers, serum electrolyte levels, kidney inflammatory biomarkers, serum concentrations of creatinine, urea, and uric acid, kidney phosphatase activities, renal histopathology, and relative gene expressions of kidney injury molecule-1 (KIM-1) and transforming growth factor beta-1 (TGF-1β), comparing these measurements with normal and diabetic control groups (NC and DC). The findings indicate that the use of extracts from H. sabdariffa leaves markedly (p < 0.05) enhanced renal well-being by mitigating nephropathy, as demonstrated through the adjustment of various biochemical and gene expression biomarkers, indicating a pronounced antioxidative and anti-inflammatory effect, improved kidney morphology, and mitigation of renal dysfunction. These findings suggest that H. sabdariffa leaf flavonoid extracts exhibit nephroprotective properties, presenting a potential natural therapeutic approach for the treatment of diabetic nephropathy.

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.

Myricetin induces M2 macrophage polarization to alleviate renal tubulointerstitial fibrosis in diabetic nephropathy via PI3K/Akt pathway.

Development of end-stage renal disease is predominantly attributed to diabetic nephropathy (DN). Previous studies have indicated that myricetin possesses the potential to mitigate the pathological alterations observed in renal tissue. Nevertheless, the precise molecular mechanism through which myricetin influences the progression of DN remains uncertain. To investigate the effects of myricetin on DN and explore its potential therapeutic mechanism. Db/db mice were administered myricetin intragastrically on a daily basis at doses of 50 mg/kg or 100 mg/kg for a duration of 12 wk. Subsequently, blood and urine indexes were assessed, along with examination of renal tissue pathology. Kidney morphology and fibrosis were evaluated using various staining techniques including hematoxylin and eosin, periodic acid-Schiff, Masson's trichrome, and Sirius-red. Additionally, high-glucose culturing was conducted on the RAW 264.7 cell line, treated with 25 mM myricetin or co-administered with the PI3K/Akt inhibitor LY294002 for a period of 24 h. In both in vivo and in vitro settings, quantification of inflammation factor levels was conducted using western blotting, real-time qPCR and ELISA. In db/db mice, administration of myricetin led to a mitigating effect on DN-induced renal dysfunction and fibrosis. Notably, we observed a significant reduction in expressions of the kidney injury markers kidney injury molecule-1 and neutrophil gelatinase associated lipocalin, along with a decrease in expressions of inflammatory cytokine-related factors. Furthermore, myricetin treatment effectively inhibited the up-regulation of tumor necrosis factor-alpha, interleukin-6, and interluekin-1β induced by high glucose in RAW 264.7 cells. Additionally, myricetin modulated the M1-type polarization of the RAW 264.7 cells. Molecular docking and bioinformatic analyses revealed Akt as the target of myricetin. The protective effect of myricetin was nullified upon blocking the polarization of RAW 264.7 via inhibition of PI3K/Akt activation using LY294002. This study demonstrated that myricetin effectively mitigates kidney injury in DN mice through the regulation of macrophage polarization via the PI3K/Akt signaling pathway.

Oleoylethanolamide attenuates acute-to-chronic kidney injury: in vivo and in vitro evidence of PPAR-α involvement

Chronic kidney disease (CKD) development after acute kidney injury (AKI) involves multiple mechanisms, including inflammation, epithelial-mesenchymal transition (EMT), and extracellular matrix deposition, leading to progressive tubulointerstitial fibrosis. Recently, a central role for peroxisome-proliferator activated receptor (PPAR)-α has been addressed in preserving kidney function during AKI. Among endogenous lipid mediators, oleoylethanolamide (OEA), a PPAR-α agonist, has been studied for its metabolic and anti-inflammatory effects. Here, we have investigated OEA effects on folic acid (FA)-induced kidney injury in mice and the underlying mechanisms. OEA improved kidney function, normalized urine output, and reduced serum BUN, creatinine, and albuminuria. Moreover, OEA attenuated tubular epithelial injury, as shown by histological analysis, and decreased expression of neutrophil gelatinase-associated lipocalin and kidney injury molecule-1. Gene expression analysis of kidney tissue indicated that OEA limited immune cell infiltration and inflammation. Moreover, OEA significantly inhibited Wnt7b and Catnb1 gene transcription and α-smooth muscle actin expression, indicating suppression of EMT. Accordingly, OEA exhibited an anti-fibrotic effect, as shown by Masson staining and the reduced levels of transforming growth factor (TGF)-β1, fibronectin, and collagen IV. Mechanistically, the nephroprotective effect of OEA was related to PPAR-α activation since OEA failed to exert its beneficial activity in FA-insulted PPAR-α-/- mice. PPAR-α involvement was also confirmed in HK2 cells where GW6471, a PPAR-α antagonist, blunted OEA activity on the TGF-β1 signalling pathway and associated pro-inflammatory and fibrotic patterns. Our findings revealed that OEA counteracts kidney injury by controlling inflammation and fibrosis, making it an effective therapeutic tool for limiting AKI to CKD progression.

Abstract 16879: Endothelial Prolyl Hydroxylase 3 Regulates Post-Ischemic Kidney Injury Through HIF Dependent Mechanisms

Introduction: Acute kidney injury (AKI) is highly prevalent clinical condition after cardiac surgeries and is associated with high morbidity and mortality in hospitalized patients. Ischemia reperfusion injury (IRI) is one of the major causes of AKI. Previously, we have reported a critical role for the endothelial oxygen sensor prolyl hydroxylase (PHD2) in ischemic AKI. Recently, we found that prolyl hydroxylase 3 (PHD3) shows robust increase in kidney endothelium after renal IRI. However, its role in ischemic AKI is unknown. Hypothesis: We hypothesized that endothelial PHD3 regulates post-ischemic kidney injury. Methods: To delete endothelial PHD3, VECadherin (Cdh5)-CreER transgenic mice were crossed to mice carrying conditional PHD3 allele, generating PHD3 iEC mice. To test the HIF-dependent role of PHD3, we generated Cdh5CreER; Phd3 flox/flox Arnt flox/flox mice ( PHD3ARNT iEC ). Inactivating ARNT (aryl hydrocarbon receptor nuclear translocator), also known as Hif-1β, blocks HIF signaling. Unilateral IRI was induced by renal artery clamping, and tamoxifen treatment was started at day 1 post IRI, followed by analysis at day 14. Results: Compared to Cre - controls, inactivation of Phd3 in endothelial cells (ECs) following IRI exacerbated kidney damage and inflammation, as indicated by increased expression of kidney injury molecule 1 ( Kim1 ) and profibrotic genes lysyl oxidase-like 2 ( Loxl2 ), transforming growth factor-beta 1 ( Tgfb1 ) , smooth muscle actin ( Acta2 ) and collagen deposition assessed by Picro Sirius red staining (n=6-7; p&lt; 0.05). Post-ischemic inactivation of ARNT along with PHD3 ameliorated the effects of only PHD3 inactivation and normalized levels of Kim1 , Loxl2 , Tgfb1 and collagen deposition ( n=7; p&lt; 0.05) . In vitro suppression of PHD3 in human primary EC using PHD3-siRNA showed significant upregulation of inflammatory molecules such as ICAM1 (Intercellular Adhesion Molecule 1), VCAM1 (Vascular Cell Adhesion Molecule 1), C-X-C motif chemokine ligand 9 and 10 ( CXCL9 and CXCL10 ), which were again blunted by ARNT- siRNA treatment (n= 3; p&lt; 0.05). Conclusions: In summary, our data support a critical role for endothelial PHD3 in regulating post-ischemic kidney inflammation and fibrosis through HIF-dependent mechanisms.

Prostaglandin F2α analogue, bimatoprost ameliorates colistin-induced nephrotoxicity

Colistin (polymyxin E) is an antibiotic that is effective against multidrug-resistant gram-negative bacteria. However, the high incidence of nephrotoxicity caused by colistin limits its clinical use. To identify compounds that might ameliorate colistin-induced nephrotoxicity, we obtained 1707 compounds from the Korea Chemical Bank and used a high-content screening (HCS) imaging-based assay. In this way, we found that bimatoprost (one of prostaglandin F2α analogue) ameliorated colistin-induced nephrotoxicity. To further assess the effects of bimatoprost on colistin-induced nephrotoxicity, we used in vitro and in vivo models. In cultured human proximal tubular cells (HK-2), colistin induced dose-dependent cytotoxicity. The number of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells, indicative of apoptosis, was higher in colistin-treated cells, but this effect of colistin was ameliorated by cotreatment with bimatoprost. The generation of reactive oxygen species, assessed using 2,7-dichlorodihydrofluorescein diacetate, was less marked in cells treated with both colistin and bimatoprost than in those treated with colistin alone. Female C57BL/6 mice (n = 10 per group) that were intraperitoneally injected with colistin (10 mg/kg/12 hr) for 14 days showed high blood urea nitrogen and serum creatinine concentrations that were reduced by the coadministration of bimatoprost (0.5 mg/kg/12 hr). In addition, kidney injury molecule-1 (KIM1) and Neutrophil gelatinase-associated lipocalin (NGAL) expression also reduced by bimatoprost administration. Further investigation in tubuloid and kidney organoids also showed that bimatoprost attenuated the nephrotoxicity by colistin, showing dose-dependent reducing effect of KIM1 expression. In this study, we have identified bimatoprost, prostaglandin F2α analogue as a drug that ameliorates colistin-induced nephrotoxicity.

Nephroprotective effect of diosmin against sodium arsenite-induced renal toxicity is mediated via attenuation of oxidative stress and inflammation in mice

Arsenic compounds, which are used in different industries like pesticide manufacturing, cause severe toxic effects in almost all organs, including the kidneys. Since the primary route of exposure to arsenic is through drinking water, and millions of people worldwide are exposed to unsafe levels of arsenic that can pose a threat to their health, this research was performed to investigate the nephroprotective effects of Diosmin (Dios), a flavonoid found in citrus fruits, against nephrotoxicity induced by sodium arsenite (SA). To induce nephrotoxicity, SA (10 mg/kg, oral gavage) was administered to mice for 30 days. Dios (25, 50, and 100 mg/kg, oral gavage) was given to mice for 30 days prior to SA administration. After the study was completed, animals were euthanized and blood and kidney samples were taken for biochemical and histopathological assessments. Results showed that SA-treated mice significantly increased the blood urea nitrogen and creatinine levels in the serum. This increase was associated with significant kidney tissue damage in SA-treated mice, which was confirmed by histopathological studies. Furthermore, SA enhanced the amounts of renal thiobarbituric acid reactive substances and decreased total thiol reserves, as well as the activity of antioxidant enzymes such as catalase, superoxide dismutase, and glutathione peroxidase. Also, in the SA-exposed group, an increase in the levels of kidney inflammatory biomarkers, including nitric oxide and tumor necrosis factor-alpha was observed. The western blot analysis indicated an elevation in the protein expression of kidney injury molecule-1 and nuclear factor-kappa B in SA-treated mice. However, pretreatment with Dios ameliorated the SA-related renal damage in mice. Our findings suggest that Dios can protect the kidneys against the nephrotoxic effects of SA by its antioxidant and anti-inflammatory characteristics.

Pharmacological inhibition of the mixed lineage leukemia 1-menin interaction aggravates acute kidney injury induced by folic acid and ischemia-reperfusion in mice.

Mixed lineage leukemia 1 (MLL1) is a methyltransferase that induces histone H3 lysine 4 trimethylation (H3K4me3) and partially exerts its untoward functional effects by interacting with multiple subunits including menin and WD repeat-containing protein 5 (WDR5). In this study, we investigated the role and mechanisms of MLL1 in murine models of acute kidney injury induced by folic acid (FA) and ischemia-reperfusion. Injury to the kidney elevated the expression of MLL1, menin, WDR5, and H3K4Me3, which was accompanied by increased serum creatinine and blood urea nitrogen, renal tubular injury, and apoptosis. Pharmacological inhibition of MLL1 activity with MI503 to disrupt the interaction between MLL1 with menin further increased serum creatinine and blood urea nitrogen levels, enhanced expression of neutrophil gelatinase-associated lipocalin and kidney injury molecule-1, and induced more apoptosis in the kidney following FA and ischemia-reperfusion injury. In contrast, MI503 treatment decreased the expression of vimentin and proliferating cell nuclear antigens. Similarly, treatment with MM102 to disrupt the interaction between MLL1 and WDR5 also worsened renal dysfunction, aggravated tubular cell injury, increased apoptosis, and inhibited cellular dedifferentiation and proliferation in mice following FA injection. Moreover, MI503 inhibited FA-induced phosphorylation of epidermal growth factor receptor, signal transducer and activator of transcription 3, and extracellular signal-regulated kinase-1/2 in injured kidneys. Collectively, these data suggest that MLL1 contributes to renal protection and functional recovery and promotes renal regeneration through a mechanism associated with activation of the epidermal growth factor receptor signaling pathway.NEW & NOTEWORTHY Mixed lineage leukemia 1 (MLL1) is a methyltransferase that induces histone H3 lysine 4 trimethylation and exerts its functional roles by interacting with multiple subunits. In this study, we demonstrated that inhibition of MLL1 activity by MI503 or MM102 aggravated renal injury and apoptosis and suppressed renal tubular cell dedifferentiation and proliferation, suggesting that MLL1 activation during acute kidney injury acts as an intrinsic protective mechanism to mediate renal tubular cell survival and regeneration.

Nrf2 protects against methamphetamine-induced nephrotoxicity by mitigating oxidative stress and autophagy in mice

Methamphetamine (MA) is a widely abused drug that can cause kidney damage. However, the molecular mechanism remains unclear. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that regulates resistance to oxidative and proteotoxic stress. In this study, we investigated the role of Nrf2 in MA-induced renal injury in mice. Nrf2 was pharmacologically activated and genetically knocked-out in mice. The animal model of MA-induced nephrotoxicity was established by injecting MA (2 mg/kg) intraperitoneally twice a day for 5 days. Histopathological alterations were shown in the MA-exposed kidneys. MA significantly increased renal function biomarkers and kidney injury molecule-1 (KIM-1) levels. MA decreased superoxide dismutase activity and increased malondialdehyde levels. Autophagy-related factors (LC3 and Beclin 1) were elevated in MA-treated mice. Furthermore, Nrf2 increased in the MA-exposed kidneys. Activation of Nrf2 may attenuate histopathological changes in the kidneys of MA-treated mice. Pre-administration of Nrf2 agonist significantly decreased KIM-1 expression, oxidative stress, and autophagy in the kidneys after MA toxicity. In contrast, Nrf2 knockout mice treated with MA lost renal tubular morphology. Nrf2 deficiency increased KIM-1 expression, oxidative stress, and autophagy in the MA-exposed kidneys. Our results demonstrate that Nrf2 may protect against MA-induced nephrotoxicity by mitigating oxidative stress and autophagy.