Proximal tubular FHL2, a novel downstream target of hypoxia inducible factor 1, is a protector against ischemic acute kidney injury.

Four-and-a-half LIM domains protein 2 (FHL2) is an adaptor protein that may interact with hypoxia inducible factor 1α (HIF-1α) or ß-catenin, two pivotal protective signaling in acute kidney injury (AKI). However, little is known about the regulation and function of FHL2 during AKI. We found that FHL2 was induced in renal tubular cells in patients with acute tubular necrosis and mice model of ischemia-reperfusion injury (IRI). In cultured renal proximal tubular cells (PTCs), hypoxia induced FHL2 expression and promoted the binding of HIF-1 to FHL2 promoter. Compared with control littermates, mice with PTC-specific deletion of FHL2 gene displayed worse renal function, more severe morphologic lesion, more tubular cell death and less cell proliferation, accompanying by downregulation of AQP1 and Na, K-ATPase after IRI. Consistently, loss of FHL2 in PTCs restricted activation of HIF-1 and ß-catenin signaling simultaneously, leading to attenuation of glycolysis, upregulation of apoptosis-related proteins and downregulation of proliferation-related proteins during IRI. In vitro, knockdown of FHL2 suppressed hypoxia-induced activation of HIF-1α and ß-catenin signaling pathways. Overexpression of FHL2 induced physical interactions between FHL2 and HIF-1α, ß-catenin, GSK-3ß or p300, and the combination of these interactions favored the stabilization and nuclear translocation of HIF-1α and ß-catenin, enhancing their mediated gene transcription. Collectively, these findings identify FHL2 as a direct downstream target gene of HIF-1 signaling and demonstrate that FHL2 could play a critical role in protecting against ischemic AKI by promoting the activation of HIF-1 and ß-catenin signaling through the interactions with its multiple protein partners.

miR-125a-5p/miR-125b-5p contributes to pathological activation of angiotensin II-AT1R in mouse distal convoluted tubule cells by the suppression of Atrap.

The renin-angiotensin system plays a crucial role in the regulation of blood pressure. Activation of the angiotensin II (Ang II)-Ang II type 1 receptor (AT1R) signaling pathway contributes to the pathogenesis of hypertension and subsequent organ damage. AT1R-associated protein (ATRAP) has been identified as an endogenous inhibitory protein of the AT1R pathological activation. We have shown that mouse Atrap (Atrap) represses various Ang II-AT1R-mediated pathologies, including hypertension in mice. The expression of human ATRAP (ATRAP)/Atrap can be altered in various pathological states in humans and mice, such as Ang II stimulation and serum starvation. However, the regulatory mechanisms of ATRAP/Atrap are not yet fully elucidated. miRNAs are 21 to 23 nucleotides of small RNAs that post-transcriptionally repress gene expression. Single miRNA can act on hundreds of target mRNAs, and numerous miRNAs have been identified as the Ang II-AT1R signaling-associated disease phenotype modulator, but nothing is known about the regulation of ATRAP/Atrap. In the present study, we identified miR-125a-5p/miR-125b-5p as the evolutionarily conserved miRNAs that potentially act on ATRAP/Atrap mRNA. Further analysis revealed that miR-125a-5p/miR-125b-5p can directly repress both ATRAP and Atrap. In addition, the inhibition of miR-125a-5p/miR-125b-5p resulted in the suppression of the Ang II-AT1R signaling in mouse distal convoluted tubule cells. Taken together, miR-125a-5p/miR-125b-5p activates Ang II-AT1R signaling by the suppression of ATRAP/Atrap. Our results provide new insights into the potential approaches for achieving the organ-protective effects by the repression of the miR-125 family associated with the enhancement of ATRAP/Atrap expression.

Podocyte-derived soluble RARRES1 drives kidney disease progression through direct podocyte and proximal tubular injury.

Retinoic acid receptor responder protein-1 (RARRES1) is a podocyte-enriched transmembrane protein whose increased expression correlates with human glomerular disease progression. RARRES1 promotes podocytopenia and glomerulosclerosis via p53-mediated podocyte apoptosis. Importantly, the cytopathic actions of RARRES1 are entirely dependent on its proteolytic cleavage into a soluble protein (sRARRES1) and subsequent podocyte uptake by endocytosis, as a cleavage mutant RARRES1 exerted no effects in vitro or in vivo. As RARRES1 expression is upregulated in human glomerular diseases, here we investigated the functional consequence of podocyte-specific overexpression of RARRES1 in mice in the experimental focal segmental glomerulosclerosis and diabetic kidney disease. We also examined the effects of long-term RARRES1 overexpression on slowly developing aging-induced kidney injury. As anticipated, the induction of podocyte overexpression of RARRES1 (Pod-RARRES1WT) significantly worsened glomerular injuries and worsened kidney function in all three models, while overexpression of RARRES1 cleavage mutant (Pod-RARRES1MT) did not. Remarkably, direct uptake of sRARRES1 was also seen in proximal tubules of injured Pod-RARRES1WT mice and associated with exacerbated tubular injuries, vacuolation, and lipid accumulation. Single-cell RNA sequence analysis of mouse kidneys demonstrated RARRES1 led to a marked deregulation of lipid metabolism in proximal tubule subsets. We further identified matrix metalloproteinase 23 (MMP23) as a highly podocyte-specific metalloproteinase and responsible for RARRES1 cleavage in disease settings, as adeno-associated virus 9-mediated knockdown of MMP23 abrogated sRARRES1 uptake in tubular cells in vivo. Thus, our study delineates a previously unrecognized mechanism by which a podocyte-derived protein directly facilitates podocyte and tubular injury in glomerular diseases and suggests that podocyte-specific functions of RARRES1 and MMP23 may be targeted to ameliorate glomerular disease progression in vivo.

High CD133 expression in proximal tubular cells in diabetic kidney disease: good or bad?

BACKGROUND: Proximal tubular cells (PTCs) play a critical role in the progression of diabetic kidney disease (DKD). As one of important progenitor markers, CD133 was reported to indicate the regeneration of dedifferentiated PTCs in acute kidney disease. However, its role in chronic DKD is unclear. Therefore, we aimed to investigate the expression patterns and elucidate its functional significance of CD133 in DKD. METHODS: Data mining was employed to illustrate the expression and molecular function of CD133 in PTCs in human DKD. Subsequently, rat models representing various stages of DKD progression were established. The expression of CD133 was confirmed in DKD rats, as well as in human PTCs (HK-2 cells) and rat PTCs (NRK-52E cells) exposed to high glucose. The immunofluorescence and flow cytometry techniques were utilized to determine the expression patterns of CD133, utilizing proliferative and injury indicators. After overexpression or knockdown of CD133 in HK-2 cells, the cell proliferation and apoptosis were detected by EdU assay, real-time cell analysis and flow analysis. Additionally, the evaluation of epithelial, progenitor cell, and apoptotic indices was performed through western blot and quantitative RT-PCR analyses. RESULTS: The expression of CD133 was notably elevated in both human and rat PTCs in DKD, and this expression increased as DKD progressed. CD133 was found to be co-expressed with CD24, KIM-1, SOX9, and PCNA, suggesting that CD133+ cells were damaged and associated with proliferation. In terms of functionality, the knockdown of CD133 resulted in a significant reduction in proliferation and an increase in apoptosis in HK-2 cells compared to the high glucose stimulus group. Conversely, the overexpression of CD133 significantly mitigated high glucose-induced cell apoptosis, but had no impact on cellular proliferation. Furthermore, the Nephroseq database provided additional evidence to support the correlation between CD133 expression and the progression of DKD. Analysis of single-cell RNA-sequencing data revealed that CD133+ PTCs potentially play a role in the advancement of DKD through multiple mechanisms, including heat damage, cell microtubule stabilization, cell growth inhibition and tumor necrosis factor-mediated signaling pathway. CONCLUSION: Our study demonstrates that the upregulation of CD133 is linked to cellular proliferation and protects PTC from apoptosis in DKD and high glucose induced PTC injury. We propose that heightened CD133 expression may facilitate cellular self-protective responses during the initial stages of high glucose exposure. However, its sustained increase is associated with the pathological progression of DKD. In conclusion, CD133 exhibits dual roles in the advancement of DKD, necessitating further investigation.

O-linked ß-N-acetylglucosamine (O-GlcNAc) modification: Emerging pathogenesis and a therapeutic target of diabetic nephropathy.

AIMS: O-Linked ß-N-acetylglucosamine (O-GlcNAc) modification, a unique post-translational modification of proteins, is elevated in diabetic nephropathy. This review aims to summarize the current knowledge on the mechanisms by which O-GlcNAcylation of proteins contributes to the pathogenesis and progression of diabetic nephropathy, as well as the therapeutic potential of targeting O-GlcNAc modification for its treatment. METHODS: Current evidence in the literature was reviewed and synthesized in a narrative review. RESULTS: Hyperglycemia increases glucose flux into the hexosamine biosynthesis pathway, which activates glucosamino-fructose aminotransferase expression and activity, leading to the production of O-GlcNAcylation substrate UDP-GlcNAc and an increase in protein O-GlcNAcylation in kidney cells. Protein O-GlcNAcylation regulates the function of kidney cells including mesangial cells, podocytes, and proximal tubular cells, and promotes renal interstitial fibrosis, resulting in kidney damage. Current treatments for diabetic nephropathy, such as sodium-glucose cotransporter 2 (SGLT-2) inhibitors and renin-angiotensin-aldosterone system (RAAS) inhibitors, delay disease progression, and suppress protein O-GlcNAcylation. CONCLUSIONS: Increased protein O-GlcNAcylation mediates renal cell damage and promotes renal interstitial fibrosis, leading to diabetic nephropathy. Although the full significance of inhibition of O-GlcNAcylation is not yet understood, it may represent a novel target for treating diabetic nephropathy.

STING1 deficiency ameliorates immune-mediated crescentic glomerulonephritis in mice.

Rapidly progressive/crescentic glomerulonephritis (RPGN/CGN) involves the formation of glomerular crescents by maladaptive differentiation of parietal epithelial cells that leads to rapid loss of renal function. The molecular mechanisms of crescent formation are poorly understood. Therefore, new insights into molecular mechanisms could identify alternative therapeutic targets for RPGN/CGN. Analysis of kidney biopsies from patients with RPGN revealed increased interstitial, glomerular, and tubular expression of STING1, an accessory protein of the c-GAS-dependent DNA-sensing pathway, which was also observed in murine nephrotoxic nephritis induced by an anti-GBM antibody. STING1 was expressed by key cell types involved in RPGN and crescent formation such as glomerular parietal epithelial cells, and tubular cells as well as by inflammation accessory cells. In functional in vivo studies, Sting1-/- mice with nephrotoxic nephritis had lower kidney cytokine expression, milder kidney infiltration by innate and adaptive immune cells, and decreased disease severity. Pharmacological STING1 inhibition mirrored these findings. Direct STING1 agonism in parietal and tubular cells activated the NF-κB-dependent cytokine response and the interferon-induced genes (ISGs) program. These responses were also triggered in a STING1-dependent manner by the pro-inflammatory cytokine TWEAK. These results identify STING1 activation as a pathological mechanism in RPGN/CGN and TWEAK as an activator of STING1. Pharmacological strategies targeting STING1, or upstream regulators may therefore be potential alternatives to treat RPGN. © 2023 The Pathological Society of Great Britain and Ireland.

Human scattered tubular cells represent a heterogeneous population of glycolytic dedifferentiated proximal tubule cells.

Scattered tubular cells (STCs) are a phenotypically distinct cell population in the proximal tubule that increase in number after acute kidney injury. We aimed to characterize the human STC population. Three-dimensional human tissue analysis revealed that STCs are preferentially located within inner bends of the tubule and are barely present in young kidney tissue (<2 years), and their number increases with age. Increased STC numbers were associated with acute tubular injury (kidney injury molecule 1) and interstitial fibrosis (alpha smooth muscle actin). Isolated CD13+ CD24- CD133- proximal tubule epithelial cells (PTECs) and CD13+ CD24+ and CD13+ CD133+ STCs were analyzed using RNA sequencing. Transcriptome analysis revealed an upregulation of nuclear factor κB, tumor necrosis factor alpha, and inflammatory pathways in STCs, whereas metabolism, especially the tricarboxylic acid cycle and oxidative phosphorylation, was downregulated, without showing signs of cellular senescence. Using immunostaining and a publicly available single-cell sequencing database of human kidneys, we demonstrate that STCs represent a heterogeneous population in a transient state. In conclusion, STCs are dedifferentiated PTECs showing a metabolic shift toward glycolysis, which could facilitate cellular survival after kidney injury. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Scopoletin alleviates high glucose-induced toxicity in human renal proximal tubular cells via inhibition of oxidative damage, epithelial-mesenchymal transition, and fibrogenesis.

BACKGROUND: Recent years of evidence suggest the crucial role of renal tubular cells in developing diabetic kidney disease. Scopoletin (SCOP) is a plant-based coumarin with numerous biological activities. This study aimed to determine the effect of SCOP on renal tubular cells in developing diabetic kidney disease and to elucidate mechanisms. METHODS AND RESULTS: In this study, SCOP was evaluated in vitro using renal proximal tubular (HK-2) cells under hyperglycemic conditions to understand its mechanism of action. In HK-2 cells, SCOP alleviated the high glucose-generated reactive oxygen species (ROS), restored the levels of reduced glutathione, and decreased lipid peroxidation. High glucose-induced alteration in the mitochondrial membrane potential was markedly restored in the SCOP-treated cells. Moreover, SCOP significantly reduced the high glucose-induced apoptotic cell population in the Annexin V-FITC flow cytometry study. Furthermore, high glucose markedly elevated the mRNA expression of fibrotic and extracellular matrix (ECM) components, namely, transforming growth factor (TGF)-ß, alfa-smooth muscle actin (α-SMA), collagen I, and collagen III, in HK-2 cells compared to the untreated cells. SCOP treatment reduced these mRNA expressions compared to the high glucose-treated cells. Collagen I and TGF-ß protein levels were also significantly reduced in the SCOP-treated cells. Further findings in HK-2 cells revealed that SCOP interfered with the epithelial-mesenchymal transition (EMT) in the high glucose-treated HK-2 cells by normalizing E-cadherin and downregulating the vimentin and α-SMA proteins. CONCLUSIONS: In conclusion, SCOP modulates the high glucose-generated renal tubular cell oxidative damage and accumulation of ECM components and may be a promising molecule against diabetic nephropathy.

Bioenergetics disruption, oxidative stress, and inflammation as underlying mechanisms of tramadol-induced nephrotoxicity.

Tramadol (TR), a commonly prescribed pain reliever for moderate to severe pain, has been associated with kidney damage. This study investigates TR-induced nephrotoxicity mechanisms, focusing on its effects on renal proximal tubular cells (PTCs). The study findings demonstrate that TR disrupts PTC bioenergetic processes, leading to oxidative stress and inflammation. Significant toxicity to PTCs was observed with estimated effective concentration 50 values of 9.8 and 11.5 µM based on 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays, respectively. TR also interferes with the function of PTC transporters, including organic cation uptake transporter 1, organic cation transporter 2, P-glycoprotein, and multidrug resistance protein 2. Furthermore, bioenergetics assays showed that TR reduced the activities of mitochondrial complexes I and III, adenosine triphosphate production, mitochondrial membrane potential, and oxygen consumption rate while increasing lactate release. TR also increased the production of reactive oxygen species, lipid peroxidation thiobarbituric acid reactive substances end products, and the expression of the NRf2 gene while decreasing reduced glutathione (GSH-R) stores and catalase and superoxide dismutase antioxidant activities. Additionally, TR increased the production of inflammatory cytokines (TNF-α and IL-6) and their coding genes expression. Interestingly, the study found that antioxidants like GSH-R, inhibitors of IL-6 and TNF-α, and mitochondrial activating Co-Q10 could protect cells against TR-induced cytotoxicity. The study suggests that TR causes nephrotoxicity by disrupting bioenergetic processes, causing oxidative stress and inflammation, but antioxidants and agents targeting mitochondria may have protective and curative potential.

Mitochondrial calcium uniporter promotes kidney aging in mice through inducing mitochondrial calcium-mediated renal tubular cell senescence.

Renal tubular epithelial cell senescence plays a critical role in promoting and accelerating kidney aging and age-related renal fibrosis. Senescent cells not only lose their self-repair ability, but also can transform into senescence-associated secretory phenotype (SASP) to trigger inflammation and fibrogenesis. Recent studies show that mitochondrial dysfunction is critical for renal tubular cell senescence and kidney aging, and calcium overload and abnormal calcium-dependent kinase activities are involved in mitochondrial dysfunction-associated senescence. In this study we investigated the role of mitochondrial calcium overload and mitochondrial calcium uniporter (MCU) in kidney aging. By comparing the kidney of 2- and 24-month-old mice, we found calcium overload in renal tubular cells of aged kidney, accompanied by significantly elevated expression of MCU. In human proximal renal tubular cell line HK-2, pretreatment with MCU agonist spermine (10 µM) significantly increased mitochondrial calcium accumulation, and induced the production of reactive oxygen species (ROS), leading to renal tubular cell senescence and age-related kidney fibrosis. On the contrary, pretreatment with MCU antagonist RU360 (10 µM) or calcium chelator BAPTA-AM (10 µM) diminished D-gal-induced ROS generation, restored mitochondrial homeostasis, retarded cell senescence, and protected against kidney aging in HK-2 cells. In a D-gal-induced accelerated aging mice model, administration of BAPTA (100 µg/kg. i.p.) every other day for 8 weeks significantly alleviated renal tubuarl cell senescence and fibrosis. We conclude that MCU plays a key role in promoting renal tubular cell senescence and kidney aging. Targeting inhibition on MCU provides a new insight into the therapeutic strategy against kidney aging.

Tanshinone IIA ameliorates cisplatin-induced toxicology and cisplatin resistance via regulating SLC7A11 expression.

Cisplatin, a potent chemotherapy agent, is highly effective against various cancers but is hindered by resistance and toxicities. This study aims to investigate the roles of SLC7A11, a cystine/glutamate transporter, in cisplatin resistance, and explored Tanshinone IIA as a therapeutic option. Cisplatin reduced SLC7A11 in renal cells, worsening toxicity. Cisplatin-resistant gastric cancer cells show increased SLC7A11, driving resistance, while SLC7A11 knockdown curbed resistance. Tanshinone IIA showed promise in alleviating cisplatin toxicity by enhancing SLC7A11 expression and reducing associated adverse effects, while it effectively reversed cisplatin resistance in gastric cancer cells by suppressing SLC7A11. Additionally, Tanshinone IIA counteracted cisplatin resistance by inhibiting PIAS4-mediated SUMOylation of SLC7A11. Simultaneously, overexpressing miR-375, which has been shown to target SLC7A11, exacerbated cisplatin toxicity via SLC7A11 downregulation, which Tanshinone IIA attenuates. In summary, our study unveils complex SLC7A11 regulation in cisplatin resistance and toxicity. Tanshinone IIA emerges as a promising modulator of SLC7A11 through individual pathways, offering novel insights into overcoming cisplatin resistance and reducing toxicities in cancer therapy.

Lemon-Derived Extracellular Vesicle-like Nanoparticles Block the Progression of Kidney Stones by Antagonizing Endoplasmic Reticulum Stress in Renal Tubular Cells.

Kidney stones, represented by the calcium oxalate (CaOx) type, are highly prevalent and recrudescent. Cumulative evidence shows regular consumption of lemonade intervenes with stone development. However, the detailed mechanism remains obscure. Here, extracellular vesicle-like nanoparticles (LEVNs) isolated from lemonade are demonstrated to traffick from the gut to the kidney, primarily enriched in tubule cells. Oral administration of LEVNs significantly alleviates the progression of kidney stones in rats. Mechanistically, in addition to altering the crystallization of CaOx toward a less stable subtype, LEVNs suppress the CaOx-induced endoplasmic reticulum stress response of tubule cells, as indicated by homeostasis of specific signaling molecules and restoration of subcellular function, thus indirectly inhibiting stone formation. To exercise this regulation, endocytosed LEVNs traffick along the microtubules throughout the cytoplasm and are eventually recruited into lysosomes. In conclusion, this study reveals a LEVNs-mediated mechanism against renal calculi and provides positive evidence for consumption of lemonade preventing stone formation.

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

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

Prostaglandin Transporter and Dipeptidyl Peptidase-4 as New Pharmacological Targets in the Prevention of Acute Kidney Injury in Diabetes: An In Vitro Study.

The probability of acute kidney injury (AKI) is higher in septic diabetic patients, which is associated with, among other factors, proximal tubular cell (PTC) injury induced by the hypoxic/hyperglycemic/inflammatory microenvironment that surrounds PTCs in these patients. Here, we exposed human PTCs (HK-2 cells) to 1% O2/25 mM glucose/inflammatory cytokines with the aim of studying the role of prostaglandin uptake transporter (PGT) and dipeptidyl peptidase-4 (DPP-4, a target of anti-hyperglycemic agents) as pharmacological targets to prevent AKI in septic diabetic patients. Our model reproduced two pathologically relevant mechanisms: (i) pro-inflammatory PTC activation, as demonstrated by the increased secretion of chemokines IL-8 and MCP-1 and the enhanced expression of DPP-4, intercellular leukocyte adhesion molecule-1 and cyclo-oxygenase-2 (COX-2), the latter resulting in a PGT-dependent increase in intracellular prostaglandin E2 (iPGE2); and (ii) epithelial monolayer injury and the consequent disturbance of paracellular permeability, which was related to cell detachment from collagen IV and the alteration of the cell cytoskeleton. Most of these changes were prevented by the antagonism of PGE2 receptors or the inhibition of COX-2, PGT or DPP-4, and further studies suggested that a COX-2/iPGE2/DPP-4 pathway mediates the pathogenic effects of the hypoxic/hyperglycemic/inflammatory conditions on PTCs. Therefore, inhibitors of PGT or DPP-4 ought to undergo testing as a novel therapeutic avenue to prevent proximal tubular damage in diabetic patients at risk of AKI.

Selenoprotein-P1 (SEPP1) Expression in Human Proximal Tubule Cells after Ischemia-Reperfusion Injury: An In Vitro Model.

Background and Objectives: Selenium deficiency represents a risk factor for the occurrence of severe diseases, such as acute kidney injury (AKI). Recently, selenoprotein-p1 (SEPP1), a selenium transporter, mainly released by the liver, has emerged as a promising plasmatic biomarker of AKI as a consequence of cardio-surgery operations. The aim of the present study was to investigate, on an in vitro model of hypoxia induced in renal tubular cells, HK-2, the effects of sodium selenite (Na2SeO3) and to evaluate the expression of SEPP1 as a marker of injury. Materials and Methods: HK-2 cells were pre-incubated with 100 nM Na2SeO3 for 24 h, and then, treated for 24 h with CoCl2 (500 µM), a chemical hypoxia inducer. The results were derived from an ROS assay, MTT, and Western blot analysis. Results: The pre-treatment determined an increase in cells' viability and a reduction in reactive oxygen species (ROS), as shown by MTT and the ROS assay. Moreover, by Western blot an increase in SEPP1 expression was observed after hypoxic injury as after adding sodium selenite. Conclusions: Our preliminary results shed light on the possible role of selenium supplementation as a means to prevent oxidative damage and to increase SEPP1 after acute kidney injury. In our in vitro model, SEPP1 emerges as a promising biomarker of kidney injury, although further studies in vivo are necessary to validate our findings.

Exploring the role of urinary extracellular vesicles in kidney physiology, aging, and disease progression.

Extracellular vesicles (EVs), membranous vesicles present in all body fluids, are considered important messengers, carrying their information over long distance and modulating the gene expression profile of recipient cells. EVs collected in urine (uEVs) are mainly originated from the apical part of urogenital tract, following the urine flow. Moreover, bacterial-derived EVs are present within urine and may reflect the composition of microbiota. Consolidated evidence has established the involvement of uEVs in renal physiology, being responsible for glomerular and tubular cross talk and among different tubular segments. uEVs may also be involved in other physiological functions such as modulation of innate immunity, coagulation, or metabolic activities. Furthermore, it has been recently remonstrated that age, sex, endurance excise, and lifestyle may influence uEV composition and release, modifying their cargo. On the other hand, uEVs appear modulators of different urogenital pathological conditions, triggering disease progression. uEVs sustain fibrosis and inflammation processes, both involved in acute and chronic kidney diseases, aging, and stone formation. The molecular signature of uEVs collected from diseased patients can be of interest for understanding kidney physiopathology and for identifying diagnostic and prognostic biomarkers.

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.

MiR-20a-5p alleviates kidney ischemia/reperfusion injury by targeting ACSL4-dependent ferroptosis.

Ischemia/reperfusion injury (IRI) is prone to occur after kidney transplantation, leading to delayed graft function (DGF). MicroRNAs play a crucial role in the pathogenesis of ischemia/reperfusion-induced acute kidney injury, and miR-20a-5p was found to be the most significantly upregulated gene in a DGF patient cohort. However, the roles of microRNAs in transplanted kidneys remain largely unknown. In this study, we found that miR-20a-5p was upregulated in the kidneys of acute kidney injury mice and in patients with DGF. We identified early growth response-1 as a critical upstream target and verified the binding of early growth response-1 to a predicted sequence in the promoter region of the miR-20a-5p gene. Functionally, the miR-20a-5p mimic attenuated IRI and postischemic renal fibrosis, whereas the miR-20a-5p inhibitor delivery aggravated IRI and fibrosis. Importantly, delivery of the miR-20a-5p mimic or inhibitor in the donor kidneys attenuated or aggravated renal loss and structural damage in cold storage transplantation injury. Furthermore, our study identified miR-20a-5p as a negative regulator of acyl-CoA synthetase long-chain family member 4 (ACSL4) by targeting the 3' untranslated region of ACSL4 mRNA, thereby inhibiting ACSL4-dependent ferroptosis. Our results suggest a potential therapeutic application of miR-20a-5p in kidney transplantation through the inhibition of ACSL4-dependent ferroptosis.

Impact of hypoxia and reoxygenation on the extra/intracellular metabolome and on transporter expression in a human kidney proximal tubular cell line.

INTRODUCTION: Ischemia-reperfusion injury (IRI) induces several perturbations that alter immediate kidney graft function after transplantation and may affect long-term graft outcomes. Given the IRI-dependent metabolic disturbances previously reported, we hypothesized that proximal transporters handling endo/exogenous substrates may be victims of such lesions. OBJECTIVES: This study aimed to determine the impact of hypoxia/reoxygenation on the human proximal transport system through two semi-targeted omics analyses. METHODS: Human proximal tubular cells were cultured in hypoxia (6 or 24 h), each followed by 2, 24 or 48-h reoxygenation. We investigated the transcriptomic modulation of transporters. Using semi-targeted LC-MS/MS profiling, we characterized the extra/intracellular metabolome. Statistical modelling was used to identify significant metabolic variations. RESULTS: The expression profile of transporters was impacted during hypoxia (y + LAT1 and OCTN2), reoxygenation (MRP2, PEPT1/2, rBAT, and OATP4C1), or in both conditions (P-gp and GLUT1). The P-gp and GLUT1 transcripts increased (FC (fold change) = 2.93 and 4.11, respectively) after 2-h reoxygenation preceded by 24-h hypoxia. We observed a downregulation (FC = 0.42) of y+LAT1 after 24-h hypoxia, and of PEPT2 after 24-h hypoxia followed by 2-h reoxygenation (FC = 0.40). Metabolomics showed that hypoxia altered the energetic pathways. However, intracellular metabolic homeostasis and cellular exchanges were promptly restored after reoxygenation. CONCLUSION: This study provides insight into the transcriptomic response of the tubular transporters to hypoxia/reoxygenation. No correlation was found between the expression of transporters and the metabolic variations observed. Given the complexity of studying the global tubular transport systems, we propose that further studies focus on targeted transporters.

VNN1 contributes to the acute kidney injury-chronic kidney disease transition by promoting cellular senescence via affecting RB1 expression.

The mechanisms underlying acute kidney injury (AKI) and chronic kidney disease (CKD) progression include interstitial inflammation, cellular senescence, and oxidative stress (OS). Although vanin-1 (VNN1) plays an important role in OS, its contribution to the AKI-CKD transition remains unknown. Here, we explored the roles and mechanisms of VNN1 in the progression of the AKI-CKD transition. We observed that VNN1 expression was upregulated after ischemia/reperfusion (I/R) injury and high VNN1 expression levels were associated with poor renal repair after I/R injury. In VNN1 knockout (KO) mice, recovery of serum creatinine and blood urea nitrogen levels after I/R injury was accelerated and renal fibrosis was inhibited after severe I/R injury. Furthermore, in VNN1 KO mice, senescence of renal tubular cells was inhibited after severe I/R injury, as assessed by P16 expression and SA-ß-Gal assays. However, our results also revealed that VNN1 KO renal tubular cells did not resist senescence when OS was blocked. To elucidate the mechanism underlying VNN1-mediated regulation of senescence during the AKI-CKD transition, retinoblastoma 1 (RB1) was identified as a potential target. Our results suggest that the reduced senescence in VNN1 KO renal tubular cells was caused by suppressed RB1 expression and phosphorylation. Collectively, our results unveil a novel molecular mechanism by which VNN1 promotes AKI-CKD transition via inducing senescence of renal tubular cells by activating RB1 expression and phosphorylation after severe renal injury. The present study proposes a new strategy for designing therapies wherein VNN1 can be targeted to obstruct the AKI-CKD transition.