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.

The E-liquid flavoring vanillin alters energy and autophagic pathways in human proximal tubule (HK-2) epithelial cells.

The use of flavored e-liquids in electronic nicotine delivery systems (ENDS) has become very popular in recent years, but effects of these products have not been well characterized outside the lung. In this study, acute exposure to the popular flavoring vanillin (VAN) was performed on human proximal tubule (HK-2) kidney cells. Cells were exposed to 0-1000 µM VAN for 24 or 48 h and cellular stress responses were determined. Mitochondrial viability using MTT assay showed a significant decrease between the control and 1000 µM group by 48 h. Seahorse XFp analysis showed significantly increased basal respiration, ATP production, and proton leak after 24 h exposure. By 48 h exposure, these parameters remained significantly increased in addition to non-mitochondrial respiration and maximal respiration. Glycolytic activity after 24 h exposure showed significant decreases in glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification. The autophagy markers microtubule-associated protein 1A/1B light chain 3 (LC3B-I and LC3B-II) were probed via western blotting. The ratio of LC3B-II/LC3B-I was significantly increased after 24 h exposure to VAN, but by 48 h this ratio significantly decreased. The mitophagy marker PINK1 showed an increasing trend at 24 h, and its downstream target Parkin was significantly increased between the control and 750 µM group only. Finally, the oxidative stress marker 4-HNE was significantly decreased after 48 h exposure to VAN. These results indicate that acute exposure to VAN in the kidney HK-2 model can induce energy and autophagic changes within the cell.

Curcumin prevents high glucose-induced stimulatory effects of renal cell secretome on fibroblast activation via mitigating intracellular free radicals and TGF-ß secretion.

Diabetic kidney disease (DKD) is a leading cause of kidney failure. However, the involvement of renal fibroblasts and their communications with renal epithelial cells during DKD remain poorly understood. We investigated the potential role of renal proximal tubular epithelial cells (PTECs) in renal fibroblast activation that might lead to DKD. Additionally, the protective effects of curcumin, a known antioxidant, against renal fibroblast activation induced by high glucose-treated PTECs were investigated. Secretome was collected from HK-2 PTECs under normal glucose, high glucose, high glucose pretreated/cotreated with curcumin, or osmotic control condition for 24 h. Such secretome was then used to treat BHK-21 renal fibroblasts for 24 h. BHK-21 cells treated with high glucose-induced secretome had increased levels of fibroblast activation markers, including spindle index, F-actin, α-smooth muscle actin (α-SMA), fibronectin, collagen I, matrix metalloproteinase-2 (MMP-2) and MMP-9, as compared with normal glucose and osmotic control conditions. However, all these increases were successfully mitigated by curcumin. In addition, high glucose markedly increased intracellular reactive oxygen species (ROS) and transforming growth factor-ß (TGF-ß) secretion, but did not affect the secretion of platelet-derived growth factor A (PDGFA) and interleukin-1ß (IL-1ß), in HK-2 renal cells as compared with normal glucose and osmotic control conditions. Both intracellular ROS and secreted TGF-ß levels were successfully mitigated by curcumin. Therefore, curcumin prevents the high glucose-induced stimulatory effects of renal cell secretome on fibroblast activation, at least in part, via mitigating intracellular ROS and TGF-ß secretion.

Kidney organoids reveal redundancy in viral entry pathways during ACE2-dependent SARS-CoV-2 infection.

With a high incidence of acute kidney injury among hospitalized COVID-19 patients, considerable attention has been focussed on whether SARS-CoV-2 specifically targets kidney cells to directly impact renal function, or whether renal damage is primarily an indirect outcome. To date, several studies have utilized kidney organoids to understand the pathogenesis of COVID-19, revealing the ability for SARS-CoV-2 to predominantly infect cells of the proximal tubule (PT), with reduced infectivity following administration of soluble ACE2. However, the immaturity of standard human kidney organoids represents a significant hurdle, leaving the preferred SARS-CoV-2 processing pathway, existence of alternate viral receptors, and the effect of common hypertensive medications on the expression of ACE2 in the context of SARS-CoV-2 exposure incompletely understood. Utilizing a novel kidney organoid model with enhanced PT maturity, genetic- and drug-mediated inhibition of viral entry and processing factors confirmed the requirement for ACE2 for SARS-CoV-2 entry but showed that the virus can utilize dual viral spike protein processing pathways downstream of ACE2 receptor binding. These include TMPRSS- and CTSL/CTSB-mediated non-endosomal and endocytic pathways, with TMPRSS10 likely playing a more significant role in the non-endosomal pathway in renal cells than TMPRSS2. Finally, treatment with the antihypertensive ACE inhibitor, lisinopril, showed negligible impact on receptor expression or susceptibility of renal cells to infection. This study represents the first in-depth characterization of viral entry in stem cell-derived human kidney organoids with enhanced PTs, providing deeper insight into the renal implications of the ongoing COVID-19 pandemic. IMPORTANCE: Utilizing a human iPSC-derived kidney organoid model with improved proximal tubule (PT) maturity, we identified the mechanism of SARS-CoV-2 entry in renal cells, confirming ACE2 as the sole receptor and revealing redundancy in downstream cell surface TMPRSS- and endocytic Cathepsin-mediated pathways. In addition, these data address the implications of SARS-CoV-2 exposure in the setting of the commonly prescribed ACE-inhibitor, lisinopril, confirming its negligible impact on infection of kidney cells. Taken together, these results provide valuable insight into the mechanism of viral infection in the human kidney.

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 400 mg/kg body weight MA. For the in vitro model, HK-2 human proximal tubular epithelial cells were treated with 2 mM or 5 mM MA for 24 h. 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.

Hemin mitigates contrast-induced nephropathy by inhibiting ferroptosis via HO-1/Nrf2/GPX4 pathway.

Contrast-induced nephropathy (CIN) is a common complication with adverse outcome after iodinated-contrast injection, yet still lacking effective medication. Heme oxygenase-1 (HO-1) has been reported to play an important role against renal injuries. Hemin, a HO-1 inducer and anti-porphyria medicine, may have a promising effect against CIN. In this study, we aim to investigate the effect of hemin on CIN model and the underlying molecular mechanisms in human proximal tubule epithelial cells (HK-2). To mimic a common condition in percutaneous coronary intervention (PCI) patients, CIN was induced by intravenous iopromide in high-fat fed diabetic rats. We found hemin, given right before iopromide, mitigated CIN with enhanced antioxidative capacity and reduced oxidative stress. HK-2 cells insulted by iopromide demonstrated decreased cell vitality and rising reactive oxygen species (ROS), which could also be inhibited by hemin. The effects of hemin involved a key molecule in ferroptosis, glutathione peroxidase (GPX4), whose down-expression by small interfering RNA (siRNA) reversed the effect of hemin on HK-2 cells. Furthermore, hemin's induction of GPX4 involved HO-1 and nuclear factor erythroid 2-related factor 2 (Nrf2). Either HO-1 or Nrf2 inhibitor prevented hemin's effect on GPX4 to a comparable extent, and over-expression of Nrf2 increased GPX4 expression. Moreover, intervention of ferroptosis inhibitor liproxstatin-1 also alleviated CIN in vivo. Therefore, we showed hemin mitigated CIN, inhibiting oxidative stress and ferroptosis, by upregulation of GPX4 via activation of HO-1/Nrf2. Hemin, as a clinical medicine, has a translational significance in treating CIN, and anti-ferroptosis is a potential therapeutic strategy for CIN.

Stx2 Induces Differential Gene Expression and Disturbs Circadian Rhythm Genes in the Proximal Tubule.

Shiga toxin-producing Escherichia coli (STEC) causes proximal tubular defects in the kidney. However, factors altered by Shiga toxin (Stx) within the proximal tubules are yet to be shown. We determined Stx receptor Gb3 in murine and human kidneys and confirmed the receptor expression in the proximal tubules. Stx2-injected mouse kidney tissues and Stx2-treated human primary renal proximal tubular epithelial cell (RPTEC) were collected and microarray analysis was performed. We compared murine kidney and RPTEC arrays and selected common 58 genes that are differentially expressed vs. control (0 h, no toxin-treated). We found that the most highly expressed gene was GDF15, which may be involved in Stx2-induced weight loss. Genes associated with previously reported Stx2 activities such as src kinase Yes phosphorylation pathway activation, unfolded protein response (UPR) and ribotoxic stress response (RSR) showed differential expressions. Moreover, circadian clock genes were differentially expressed, suggesting Stx2-induced renal circadian rhythm disturbance. Circadian rhythm-regulated proximal tubular Na+-glucose transporter SGLT1 (SLC5A1) was down-regulated, indicating proximal tubular functional deterioration, and mice developed glucosuria confirming proximal tubular dysfunction. Stx2 alters gene expression in murine and human proximal tubules through known activities and newly investigated circadian rhythm disturbance, which may result in proximal tubular dysfunctions.

Oxidized alkyl phospholipids stimulate sodium transport in proximal tubules via a nongenomic PPARγ-dependent pathway.

Oxidized phospholipids have been shown to exhibit pleiotropic effects in numerous biological contexts. For example, 1-O-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine (azPC), an oxidized phospholipid formed from alkyl phosphatidylcholines, is a peroxisome proliferator-activated receptor gamma (PPARγ) nuclear receptor agonist. Although it has been reported that PPARγ agonists including thiazolidinediones can induce plasma volume expansion by enhancing renal sodium and water retention, the role of azPC in renal transport functions is unknown. In the present study, we investigated the effect of azPC on renal proximal tubule (PT) transport using isolated PTs and kidney cortex tissues and also investigated the effect of azPC on renal sodium handling in vivo. We showed using a microperfusion technique that azPC rapidly stimulated Na+/HCO3- cotransporter 1 (NBCe1) and luminal Na+/H+ exchanger (NHE) activities in a dose-dependent manner at submicromolar concentrations in isolated PTs from rats and humans. The rapid effects (within a few minutes) suggest that azPC activates NBCe1 and NHE via nongenomic signaling. The stimulatory effects were completely blocked by specific PPARγ antagonist GW9662, ERK kinase inhibitor PD98059, and CD36 inhibitor sulfosuccinimidyl oleate. Treatment with an siRNA against PPAR gamma completely blocked the stimulation of both NBCe1 and NHE by azPC. Moreover, azPC induced ERK phosphorylation in rat and human kidney cortex tissues, which were completely suppressed by GW9662 and PD98059 treatments. These results suggest that azPC stimulates renal PT sodium-coupled bicarbonate transport via a CD36/PPARγ/mitogen-activated protein/ERK kinase/ERK pathway. We conclude that the stimulatory effects of azPC on PT transport may be partially involved in volume expansion.

Adenine overload induces ferroptosis in human primary proximal tubular epithelial cells.

The pathogenesis of crystal nephropathy involves deposition of intratubular crystals, tubular obstruction and cell death. The deposition of 8-dihydroxyadenine (DHA) crystals within kidney tubules, for instance, is caused by a hereditary deficiency of adenine phosphoribosyl transferase in humans or adenine overload in preclinical models. However, the downstream pathobiological patterns of tubular cell attrition in adenine/DHA-induced nephropathy remain poorly understood. In this study, we investigated: (i) the modes of adenine-induced tubular cell death in an experimental rat model and in human primary proximal tubular epithelial cells (PTEC); and (ii) the therapeutic effect of the flavonoid baicalein as a novel cell death inhibitor. In a rat model of adenine diet-induced crystal nephropathy, significantly elevated levels of tubular iron deposition and lipid peroxidation (4-hydroxynonenal; 4-HNE) were detected. This phenotype is indicative of ferroptosis, a novel form of regulated necrosis. In cultures of human primary PTEC, adenine overload-induced significantly increased mitochondrial superoxide levels, mitochondrial depolarisation, DNA damage and necrotic cell death compared with untreated PTEC. Molecular interrogation of adenine-stimulated PTEC revealed a significant reduction in the lipid repair enzyme glutathione peroxidase 4 (GPX4) and the significant increase in 4-HNE compared with untreated PTEC, supporting the concept of ferroptotic cell death. Moreover, baicalein treatment inhibited ferroptosis in adenine-stimulated PTEC by selectively modulating the mitochondrial antioxidant enzyme superoxide dismutase 2 (SOD2) and thus, suppressing mitochondrial superoxide production and DNA damage. These data identify ferroptosis as the primary pattern of PTEC necrosis in adenine-induced nephropathy and establish baicalein as a potential therapeutic tool for the clinical management of ferroptosis-associated crystal nephropathies (e.g., DHA nephropathy, oxalate nephropathy).

Unexpected Enhancement of Cytotoxicity of Cisplatin in a Rat Kidney Proximal Tubular Cell Line Overexpressing Mitochondrial Glutathione Transport Activity.

In previous studies, we identified the two principal transporters that mediate the uptake of glutathione (GSH) from cytoplasm into the mitochondrial matrix of rat kidney proximal tubular cells. We hypothesized that genetic modulation of transporter expression could markedly alter susceptibility of renal proximal tubular cells to a broad array of oxidants and mitochondrial toxicants. Indeed, we previously showed that overexpression of either of these transporters resulted in diminished susceptibility to several chemicals. In the present work, we investigated the influence of overexpression of the mitochondrial 2-oxoglutarate carrier (OGC) in NRK-52E cells on the cytotoxicity of the antineoplastic drug cisplatin. In contrast to previous results showing that overexpression of the mitochondrial OGC provided substantial protection of NRK-52E cells from injury due to several toxicants, we found a remarkable enhancement of cellular injury from exposure to cisplatin as compared to wild-type NRK-52E cells. Despite the oxidative stress that cisplatin is known to cause in the renal proximal tubule, the increased concentrations of mitochondrial GSH associated with OGC overexpression likely resulted in increased delivery of cisplatin to molecular targets and increased cellular injury rather than the typical protection observed in the previous work.

Hepatic processing of mercuric ions facilitates delivery to renal proximal tubules.

Mercury (Hg) is a toxic heavy metal to which humans are exposed on a regular basis. Hg has a high affinity for thiol-containing biomolecules with the majority of Hg in blood being bound to albumin. The current study tested the hypothesis that circulating Hg-albumin complexes are taken up into hepatocytes and processed to form Hg-glutathione (GSH) conjugates (GSH-Hg-GSH). Subsequently, GSH-Hg-GSH conjugates are exported from hepatocytes into blood via multidrug resistance transporters (MRP) 3 and 5. To test this hypothesis, the portal vein and hepatic artery in Wistar rats were ligated to prevent delivery of Hg to the liver. Ligated and control rats were injected with HgCl2 or GSH-Hg-GSH (containing radioactive Hg) and the disposition of Hg was assessed in various organs. Renal accumulation of Hg was reduced significantly in ligated rats exposed to HgCl2. In contrast, when rats were exposed to GSH-Hg-GSH, the renal accumulation of Hg was similar in control and ligated rats. Experiments using HepG2 cells indicate that Hg-albumin conjugates are taken up by hepatocytes and additional experiments using inside-out membrane vesicles showed that MRP3 and MRP5 mediate the export of GSH-Hg-GSH from hepatocytes. These data are the first to show that Hg-albumin complexes are processed within hepatocytes to form GSH-Hg-GSH, which is, in part, exported back into blood via MRP3 and MRP5 for eventual excretion in urine.

Foodborne fluoxetine impacts the immune response in rainbow trout (Oncorhynchus mykkis).

The aim of this study was to reveal the effects of foodborne fluoxetine on morphological and condition profile, hematological profile, biochemical and oxidative stress indices on juvenile rainbow trout. The study was performed according to OECD Guideline No. 215. Fluoxetine was incorporated into Biomar 921 pellets at a dose of 0.047 mg/kg (environmental concentration), 0.577 mg/kg and 6.7 mg/kg. There was statistically significant change in hematological profile, including an increasing trend in neutrophil/lymphocyte ratio and a decreasing trend in the number of lymphocytes. Measurements of oxidative stress indicated decreased activity of the detoxifying enzyme glutathione-S-transferase in the liver and kidney. However, the measurement of GR, GPx, CAT, SOD activity, and TBARS showed no changes. Histopathological examination revealed damage to the proximal tubules of caudal kidney in exposed groups. This study confirms that fluoxetine has a significant effect on immune response.

(-)-α-Bisabolol as a protective agent against epithelial renal cytotoxicity induced by amphotericin B.

INTRODUCTION: Amphotericin B (AmB), used for systemic fungal infections, has a limited clinical application because of its high nephrotoxicity. Natural antioxidant and anti-inflammatory substances have been widely studied for protection against drug-induced nephrotoxicity. α-Bisabolol (BIS) has demonstrated a nephroprotective effect on both in vitro and in vivo models. AIMS: The aim of this work was to evaluate the effect of BIS against AmB-induced nephrotoxicity in vitro. MATERIAL AND METHODS: LLC-MK2 cells were pre- and post-treated with non-toxic BIS concentrations and/or AmB IC50 (13.97 µM). Cell viability was assessed by MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] assay. Flow cytometry analyses were used to assess cell death mechanism, production of reactive oxidative stress (ROS) and mitochondrial transmembrane potential. Kidney Injury Molecule-1 (KIM-1) levels were measured via ELISA. KEY FINDINGS: The present work showed that BIS pretreatment (125; 62.5 and 31.25 µM) increased cell viability when compared to the group treated only with AmB IC50. AmB treatment induced both necrosis (7-AAD-labeled cells) and late apoptosis (AnxV-labeled). BIS was able to prevent the occurrence of these events. These effects were associated with a decrease of ROS accumulation, improving transmembrane mitochondrial potential and protecting against tubular cell damage, highlighted by the inhibition of KIM-1 release after BIS treatment. SIGNIFICANCE: BIS presented a potential effect on model of renal cytotoxicity induced by AmB, bringing perspectives for the research of new nephroprotective agents.

Anti-fibrotic effect of 6-bromo-indirubin-3'-oxime (6-BIO) via regulation of activator protein-1 (AP-1) and specificity protein-1 (SP-1) transcription factors in kidney cells.

PAI-1 and CTGF are overexpressed in kidney diseases and cause fibrosis of the lungs, liver, and kidneys. We used a rat model of unilateral ureteral obstruction (UUO) to investigate whether 6-BIO, a glycogen synthase kinase-3ß inhibitor, attenuated fibrosis by inhibiting PAI-1 and CTGF in vivo. Additionally, TGFß-induced cellular fibrosis was observed in vitro using the human kidney proximal tubular epithelial cells (HK-2), and rat interstitial fibroblasts (NRK49F). Expression of fibrosis-related proteins and signaling molecules such as PAI-1, CTGF, TGFß, αSMA, SMAD, and MAPK were determined in HK-2 and NRK49F cells using immunoblotting. To identify the transcription factors that regulate the expression of PAI-1 and CTGF the promoter activities of AP-1 and SP-1 were analyzed using luciferase assays. Confocal microscopy was used to observe the co-localization of AP-1 and SP-1 to PAI-1 and CTGF. Expression of PAI-1, CTGF, TGFß, and α-SMA increased in UUO model as well as in TGFß-treated HK-2 and NRK49F cells. Furthermore, UUO and TGFß treatment induced the activation of P-SMAD2/3, SMAD4, P-ERK 1/2, P-P38, and P-JNK MAPK signaling pathways. PAI-1, CTGF, AP-1 and SP-1 promoter activity increased in response to TGFß treatment. However, treatment with 6-BIO decreased the expression of proteins and signaling pathways associated with fibrosis in UUO model as well as in TGFß-treated HK-2 and NRK49F cells. Moreover, 6-BIO treatment attenuated the expression of PAI-1 and CTGF as well as the promoter activities of AP-1 and SP-1, thereby regulating the SMAD and MAPK signaling pathways, and subsequently exerting anti-fibrotic effects on kidney cells.

The polypeptide antibiotic polymyxin B acts as a pro-inflammatory irritant by preferentially targeting macrophages.

Polymyxin B (PMB) is an essential antibiotic active against multidrug-resistant bacteria, such as multidrug-resistant Pseudomonas aeruginosa (MDRP). However, the clinical use of PMB is limited, because PMB causes serious side effects, such as nephrotoxicity and neurotoxicity, probably due to its cytotoxic activity. However, cytotoxic mechanisms of PMB are poorly understood. In this study, we found that macrophages are particularly sensitive to PMB, when compared with other types of cells, including fibroblasts and proximal tubule (PT) cells. Of note, PMB-induced necrosis of macrophages allowed passive release of high mobility group box 1 (HMGB1). Moreover, upon exposure of PMB to macrophages, the innate immune system mediated by the NLR family pyrin domain containing 3 (NLRP3) inflammasome that promotes the release of pro-inflammatory cytokines such as interleukin-1ß (IL-1ß) was stimulated. Interestingly, PMB-induced IL-1ß release occurred in the absence of the pore-forming protein gasdermin D (GSDMD), which supports the idea that PMB causes plasma membrane rupture accompanying necrosis. Emerging evidence has suggested that both HMGB1 and IL-1ß released from macrophages contribute to excessive inflammation that promote pathogenesis of various diseases, including nephrotoxicity and neurotoxicity. Therefore, these biochemical properties of PMB in macrophages may be associated with the induction of the adverse organ toxicity, which provides novel insights into the mechanisms of PMB-related side effects.

Nephrotoxic Effects of Paraoxon in Three Rat Models of Acute Intoxication.

The delayed effects of acute intoxication by organophosphates (OPs) are poorly understood, and the various experimental animal models often do not take into account species characteristics. The principal biochemical feature of rodents is the presence of carboxylesterase in blood plasma, which is a target for OPs and can greatly distort their specific effects. The present study was designed to investigate the nephrotoxic effects of paraoxon (O,O-diethyl O-(4-nitrophenyl) phosphate, POX) using three models of acute poisoning in outbred Wistar rats. In the first model (M1, POX2x group), POX was administered twice at doses 110 µg/kg and 130 µg/kg subcutaneously, with an interval of 1 h. In the second model (M2, CBPOX group), 1 h prior to POX poisoning at a dose of 130 µg/kg subcutaneously, carboxylesterase activity was pre-inhibited by administration of specific inhibitor cresylbenzodioxaphosphorin oxide (CBDP, 3.3 mg/kg intraperitoneally). In the third model (M3), POX was administered subcutaneously just once at doses of LD16 (241 µg/kg), LD50 (250 µg/kg), and LD84 (259 µg/kg). Animal observation and sampling were performed 1, 3, and 7 days after the exposure. Endogenous creatinine clearance (ECC) decreased in 24 h in the POX2x group (p = 0.011). Glucosuria was observed in rats 24 h after exposure to POX in both M1 and M2 models. After 3 days, an increase in urinary excretion of chondroitin sulfate (CS, p = 0.024) and calbindin (p = 0.006) was observed in rats of the CBPOX group. Morphometric analysis revealed a number of differences most significant for rats in the CBPOX group. Furthermore, there was an increase in the area of the renal corpuscles (p = 0.0006), an increase in the diameter of the lumen of the proximal convoluted tubules (PCT, p = 0.0006), and narrowing of the diameter of the distal tubules (p = 0.001). After 7 days, the diameter of the PCT lumen was still increased in the nephrons of the CBPOX group (p = 0.0009). In the M3 model, histopathological and ultrastructural changes in the kidneys were revealed after the exposure to POX at doses of LD50 and LD84. Over a period from 24 h to 3 days, a significant (p = 0.018) expansion of Bowman's capsule was observed in the kidneys of rats of both the LD50 and LD84 groups. In the epithelium of the proximal tubules, stretching of the basal labyrinth, pycnotic nuclei, and desquamation of microvilli on the apical surface were revealed. In the epithelium of the distal tubules, partial swelling and destruction of mitochondria and pycnotic nuclei was observed, and nuclei were displaced towards the apical surface of cells. After 7 days of the exposure to POX, an increase in the thickness of the glomerular basement membrane (GBM) was observed in the LD50 and LD84 groups (p = 0.019 and 0.026, respectively). Moreover, signs of damage to tubular epithelial cells persisted with blockage of the tubule lumen by cellular detritus and local destruction of the surface of apical cells. Comparison of results from the three models demonstrates that the nephrotoxic effects of POX, evaluated at 1 and 3 days, appear regardless of prior inhibition of carboxylesterase activity.

PM2.5 Induces Early Epithelial Mesenchymal Transition in Human Proximal Tubular Epithelial Cells through Activation of IL-6/STAT3 Pathway.

Particulate matter exposure has been known as a potential risk for the global burden of disease, such as respiratory and cardiovascular diseases. Accumulating evidence suggests that PM2.5 (particulate matter with a diameter less than 2.5 µm) is associated with increased risk of kidney disease, but the mechanisms underlying the renal injury caused by PM2.5 remain to be elucidated. This study investigated the effects of PM2.5 on human proximal tubular epithelial (HK-2) cells by monolayer and 3D spheroid cultures and explored the potential mechanisms. The typical morphology of HK-2 cells showed epithelial-mesenchymal transition (EMT), resulting in reduced adhesion and enhanced migration after PM2.5 exposure, and was accompanied by decreased E-cadherin expression and increased vimentin and α-SMA expressions. Exposure to PM2.5 in the HK-2 cells could lead to an increase in interleukin-6 (IL-6) levels and cause the activation of signal transducer and activator of transcription 3 (STAT3), which is involved in EMT features of HK-2 cells. Furthermore, blocking IL-6/STAT3 signaling by an IL-6 neutralizing antibody or STAT3 inhibitor was sufficient to reverse PM2.5-induced EMT characteristics of the HK-2 cells. Our study suggests that PM2.5 could induce early renal tubule cell injury, contributing to EMT change, and the induction of IL-6/STAT3 pathway may play an important role in this process.

Drug Repurposing in Rare Diseases: An Integrative Study of Drug Screening and Transcriptomic Analysis in Nephropathic Cystinosis.

Diagnosis and cure for rare diseases represent a great challenge for the scientific community who often comes up against the complexity and heterogeneity of clinical picture associated to a high cost and time-consuming drug development processes. Here we show a drug repurposing strategy applied to nephropathic cystinosis, a rare inherited disorder belonging to the lysosomal storage diseases. This approach consists in combining mechanism-based and cell-based screenings, coupled with an affordable computational analysis, which could result very useful to predict therapeutic responses at both molecular and system levels. Then, we identified potential drugs and metabolic pathways relevant for the pathophysiology of nephropathic cystinosis by comparing gene-expression signature of drugs that share common mechanisms of action or that involve similar pathways with the disease gene-expression signature achieved with RNA-seq.

Aldehyde-driven transcriptional stress triggers an anorexic DNA damage response.

Endogenous DNA damage can perturb transcription, triggering a multifaceted cellular response that repairs the damage, degrades RNA polymerase II and shuts down global transcription1-4. This response is absent in the human disease Cockayne syndrome, which is caused by loss of the Cockayne syndrome A (CSA) or CSB proteins5-7. However, the source of endogenous DNA damage and how this leads to the prominent degenerative features of this disease remain unknown. Here we find that endogenous formaldehyde impedes transcription, with marked physiological consequences. Mice deficient in formaldehyde clearance (Adh5-/-) and CSB (Csbm/m; Csb is also known as Ercc6) develop cachexia and neurodegeneration, and succumb to kidney failure, features that resemble human Cockayne syndrome. Using single-cell RNA sequencing, we find that formaldehyde-driven transcriptional stress stimulates the expression of the anorexiogenic peptide GDF15 by a subset of kidney proximal tubule cells. Blocking this response with an anti-GDF15 antibody alleviates cachexia in Adh5-/-Csbm/m mice. Therefore, CSB provides protection to the kidney and brain against DNA damage caused by endogenous formaldehyde, while also suppressing an anorexic endocrine signal. The activation of this signal might contribute to the cachexia observed in Cockayne syndrome as well as chemotherapy-induced anorectic weight loss. A plausible evolutionary purpose for such a response is to ensure aversion to genotoxins in food.

New Alkoxy- Analogues of Epoxyeicosatrienoic Acids Attenuate Cisplatin Nephrotoxicity In Vitro via Reduction of Mitochondrial Dysfunction, Oxidative Stress, Mitogen-Activated Protein Kinase Signaling, and Caspase Activation.

The usage of cisplatin, a highly potent chemotherapeutic, is limited by its severe nephrotoxicity. Arachidonic acid (ARA)-derived epoxyeicosatrienoic acids (EETs) and soluble epoxide hydrolase (sEH) inhibitors were shown to ameliorate this dose-limiting side effect, but both approaches have some pharmacological limitations. Analogues of EETs are an alternative avenue with unique benefits, but the current series of analogues face concerns regarding their structure and mimetic functionality. Hence, in this study, regioisomeric mixtures of four new ARA alkyl ethers were synthesized, characterized, and assessed as EET analogues against the concentration- and time-dependent toxicities of cisplatin in porcine proximal tubular epithelial cells. All four ether groups displayed bioisostere activity, ranging from marginal for methoxy- (1), good for n-propoxy- (4), and excellent for ethoxy- (2) and i-propoxy- (3). Compounds 2 and 3 displayed cytoprotective effects comparable to that of an EET regioisomeric mixture (5) against high, acute cisplatin exposures but were more potent against low to moderate, chronic exposures. Compounds 2 and 3 (and 5) acted through stabilization of the mitochondrial transmembrane potential and attenuation of reactive oxygen species, leading to reduced phosphorylation of mitogen-activated protein kinases p38 and JNK and decreased activation of caspase-9 and caspase-3. This study demonstrates that alkoxy- groups are potent and more metabolically stable bioisostere alternatives to the epoxide within EETs that enable sEH-independent activity. It also illustrates the potential of ether-based mimics of EETs and other epoxy fatty acids as promising nephroprotective agents to tackle the clinically relevant side effect of cisplatin without compromising its antineoplastic function.