Substituted kynurenic acid derivatives as fluorophore-based probes for D- and L-amino acid oxidase assays and their in vitro application in eels.

High levels of D-amino acid oxidase (DAO) are associated with neurological and psychiatric disorders, while L-amino acid oxidase (LAO) exhibits antimicrobial and antitumor properties. The enzymatic conversion of the non-fluorescent kynurenine (KYN) into the endogenous weak fluorescent kynurenic acid (KYNA) by the action of DAO has previously been reported. However, the fluorescence of KYNA can be improved by changing the substituents on the aromatic rings. In this study, we prepared different 6-phenyl-substituted KYNA derivatives and investigated their fluorescence properties. Among them, 2-MePh-KYNA showed the maximum fluorescence quantum yield of 0.881 at 340 nm excitation and 418 nm emission wavelengths. The effects of solvent properties (dielectric constant, pKa, viscosity, and proticity) on the fluorescence intensity (FLI) of the KYNA derivatives were explored. The FLI of 2-MePh-KYNA was significantly large in protic solvents. Subsequently, 2-MePh-D-KYN and 2-MePh-L-KYN were prepared with high enantiopurity (>99.25%) for the enzymatic conversion. 2-MePh-D-KYN exhibited high sensitivity (∼19 times that of a commercial DAO substrate and ∼60 times that of the previously reported MeS-D-KYN) and high selectivity, as it was not cross-reactive towards LAO, while 2-MePh-L-KYN was also converted into 2-MePh-KYNA by LAO. Furthermore, the 2-MePh-D-KYN probe successfully detected DAO in eel liver, kidney, and heparin-anticoagulated plasma in the in vitro study.

Fumarate induces vesicular release of mtDNA to drive innate immunity.

Mutations in fumarate hydratase (FH) cause hereditary leiomyomatosis and renal cell carcinoma1. Loss of FH in the kidney elicits several oncogenic signalling cascades through the accumulation of the oncometabolite fumarate2. However, although the long-term consequences of FH loss have been described, the acute response has not so far been investigated. Here we generated an inducible mouse model to study the chronology of FH loss in the kidney. We show that loss of FH leads to early alterations of mitochondrial morphology and the release of mitochondrial DNA (mtDNA) into the cytosol, where it triggers the activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-TANK-binding kinase 1 (TBK1) pathway and stimulates an inflammatory response that is also partially dependent on retinoic-acid-inducible gene I (RIG-I). Mechanistically, we show that this phenotype is mediated by fumarate and occurs selectively through mitochondrial-derived vesicles in a manner that depends on sorting nexin 9 (SNX9). These results reveal that increased levels of intracellular fumarate induce a remodelling of the mitochondrial network and the generation of mitochondrial-derived vesicles, which allows the release of mtDNAin the cytosol and subsequent activation of the innate immune response.

The Na+,K+-ATPase in complex with beryllium fluoride mimics an ATPase phosphorylated state.

The Na+,K+-ATPase generates electrochemical gradients of Na+ and K+ across the plasma membrane via a functional cycle that includes various phosphoenzyme intermediates. However, the structure and function of these intermediates and how metal fluorides mimick them require further investigation. Here, we describe a 4.0 Å resolution crystal structure and functional properties of the pig kidney Na+,K+-ATPase stabilized by the inhibitor beryllium fluoride (denoted E2-BeFx). E2-BeFx is expected to mimic properties of the E2P phosphoenzyme, yet with unknown characteristics of ion and ligand binding. The structure resembles the E2P form obtained by phosphorylation from inorganic phosphate (Pi) and stabilized by cardiotonic steroids, including a low-affinity Mg2+ site near ion binding site II. Our anomalous Fourier analysis of the crystals soaked in Rb+ (a K+ congener) followed by a low-resolution rigid-body refinement (6.9-7.5 Å) revealed preocclusion transitions leading to activation of the dephosphorylation reaction. We show that the Mg2+ location indicates a site of initial K+ recognition and acceptance upon binding to the outward-open E2P state after Na+ release. Furthermore, using binding and activity studies, we find that the BeFx-inhibited enzyme is also able to bind ADP/ATP and Na+. These results relate the E2-BeFx complex to a transient K+- and ADP-sensitive E∗P intermediate of the functional cycle of the Na+,K+-ATPase, prior to E2P.

Role of heme oxygenase in the regulation of the renal hemodynamics in a model of sex-dependent programmed hypertension by maternal diabetes.

Intrauterine programming of cardiovascular and renal function occurs in diabetes because of the adverse maternal environment. Heme oxygenase 1 (HO-1) and -2 (HO-2) exert vasodilatory and antioxidant actions, particularly in conditions of elevated HO-1 expression or deficient nitric oxide levels. We evaluated whether the activity of the heme-HO system is differentially regulated by oxidative stress in the female offspring of diabetic mothers, contributing to the improved cardiovascular function in comparison with males. Diabetes was induced in pregnant rats by a single dose of streptozotocin (STZ, 50 mg/kg ip) in late gestation. Three-month-old male offspring from diabetic mothers (MODs) exhibited higher blood pressure (BP), higher renal vascular resistance (RVR), worse endothelium-dependent response to acetylcholine (ACH), and an increased constrictor response to phenylephrine (PHE) compared with those in age-matched female offspring of diabetic mothers (FODs), which were abolished by chronic tempol (1 mM) treatment. In anesthetized animals, stannous mesoporphyrin (SnMP; 40 µmol/kg iv) administration, to inhibit HO activity, increased RVR in FODs and reduced glomerular filtration rate (GFR) in MODs, without altering these parameters in control animals. When compared with MODs, FODs showed lower nitrotirosyne levels and higher HO-1 protein expression in renal homogenates. Indeed, chronic treatment with tempol in MODs prevented elevations in nitrotyrosine levels and the acute renal hemodynamics response to SnMP. Then, maternal diabetes results in sex-specific hypertension and renal alterations associated with oxidative stress mainly in adult male offspring, which are reduced in the female offspring by elevation in HO-1 expression and lower oxidative stress levels.

[Intervention effects of drugs on GSH and SOD enzyme activity of rats kidney acutely poisoned by nickel carbonyl].

Objective: To evaluate the intervention effect of various drugs on glutathione (GSH) and superoxide dismutase (SOD) enzyme activity of rats kidney with acute nickel carbonyl poisoning. Methods: In January 2019, The 250 SPF male SD rats were randomly divided into normal control group (n=10) , poisoned group (n=40) and treatment groups (n=200) according to the random number table method. And the treatment groups were divided into methylprednisolone group (20 mg/kg) , DDC group (100 mg/kg) , sodium selenite group (10 µmol/kg) , Shenfu huiyang decoction group (0.25 ml) and methylprednisolone combined with DDC group (100 mg/kg) , with 40 mice in each group. Except for the normal control group, rats in the other groups were exposed to nickel carbonyl for 30 min, at 4 h and 30 h after exposure, the rats in each treatment group were intraperitoneally injected with corresponding drugs, and kidney tissues were collected 3 d and 7 d after administration, with 10 mice in each group. The activities of GSH and SOD in kidney were measured by enzyme-linked immunosorbent assay, and using electron microscopy observe ultrastructure changes. Results: Compared to the control group, the activities of GSH and SOD enzyme of poisoned group were significantly decreased at 3 d or 7 d after 4 h or 30 h exposure, and the difference was statistically significant (P=0.000, 0.031, 0.001, 0.033) , the epithelial nuclei of proximal convoluted tubules were pyknosis and lysosome hyperplasia in the cytoplasm. And compared to poisoned group, the activities of GSH and SOD enzyme of methylprednisolone+DDC group were significantly increased at treatment with 7 d after 4 h exposure, the difference was statistically significant (P=0.022, 0.000) , and the activities of GSH and SOD enzyme of methylprednisolone and enzyme of methylprednisolone+DDC group were significantly higher at 7 days than at 3 days, the difference was statistically significant (P=0.020, 0.017, 0.018, 0.033) . The results of electron microscopy showed that the cell nuclei and cytoplasmic organelles of proximal convolute tubule were almost restored to normal tissue level of both methylprednisolone group and methylprednisolone+DDC group. Conclusion: The methylprednisolone and methylprednisolone+DDC have obvious repair effect on renal enzyme activity level of rats with acute nickel carbonyl poisoning, and the treatment effect is better for a long time of medication.

Targeting lysine-specific demethylase 1A inhibits renal epithelial-mesenchymal transition and attenuates renal fibrosis.

Lysine-specific histone demethylase 1 (LSD1) as the first identified histone/lysine demethylase regulates gene expression and protein functions in diverse diseases. In this study, we show that the expression of LSD1 is increased in mouse kidneys with unilateral ureteral obstruction (UUO) and in cultured NRK-52E cells undergoing TGF-ß1-induced epithelial-mesenchymal transition (EMT). Inhibition of LSD1 with its specific inhibitor ORY1001 attenuated renal EMT and fibrosis, which was associated with decreased the deposition of extracellular matrix proteins and the expression of fibrotic markers, including α-smooth muscle actin (α-SMA) and fibronectin, and the recovery of E-cadherin expression and decrease of N-cadherin expression in UUO kidneys and in NRK-52E cells induced with TGF-ß1. Targeting LSD1 also decreased the expression of Snail family transcriptional repressor 1 (Snail-1) and its interaction with LSD1 in UUO kidneys and in NRK-52E cells treated with TGF-ß1. In addition, we identified a novel LSD1-14-3-3ζ-PKCα axis in the regulation of the activation of AKT and Stat3 and then the activation of fibroblasts. This study suggests that LSD1 plays a critical role in regulation of renal EMT and fibrosis through activation of diverse signaling pathways and places an emphasis that LSD1 has potential as a therapeutic target for the treatment of renal fibrosis.

15-Lipoxygenase worsens renal fibrosis, inflammation, and metabolism in a murine model of ureteral obstruction.

15-Lipoxygenase (15-LO) is a nonheme iron-containing dioxygenase that has both pro- and anti-inflammatory roles in many tissues and disease states. 15-LO is thought to influence macrophage phenotype, and silencing 15-LO reduces fibrosis after acute inflammatory triggers. The goal of the present study was to determine whether altering 15-LO expression influences inflammation and fibrogenesis in a murine model of unilateral ureteral obstruction (UUO). C57BL/6J mice, 15-LO knockout (Alox15-/-) mice, and 15-LO transgenic overexpressing (15LOTG) mice were subjected UUO, and kidneys were analyzed at 3, 10, and 14 days postinjury. Histology for fibrosis, inflammation, cytokine quantification, flow cytometry, and metabolomics were performed on injured tissues and controls. PD146176, a specific 15-LO inhibitor, was used to complement experiments involving knockout animals. Compared with wild-type animals undergoing UUO, Alox15-/- mouse kidneys had less proinflammatory, profibrotic message along with less fibrosis and macrophage infiltration. PD146176 inhibited 15-LO and resulted in reduced fibrosis and macrophage infiltration similar to Alox15-/- mice. Flow cytometry revealed that Alox15-/- UUO-injured kidneys had a dynamic change in macrophage phenotype, with an early blunting of CD11bHiLy6CHi "M1" macrophages and an increase in anti-inflammatory CD11bHiLy6CInt "M2c" macrophages and reduced expression of the fractalkine receptor chemokine (C-X3-C motif) receptor 1. Many of these findings were reversed when UUO was performed on 15LOTG mice. Metabolomics analysis revealed that wild-type kidneys developed a glycolytic shift postinjury, while Alox15-/- kidneys exhibited increased oxidative phosphorylation. In conclusion, 15-LO manipulation by genetic or pharmacological means induces dynamic changes in the inflammatory microenvironment in the UUO model and appears to be critical in the progression of UUO-induced fibrosis.NEW & NOTEWORTHY 15-Lipoxygenase (15-LO) has both pro- and anti-inflammatory functions in leukocytes, and its role in kidney injury and repair is unexplored. Our study showed that 15-LO worsens inflammation and fibrosis in a rodent model of chronic kidney disease using genetic and pharmacological manipulation. Silencing 15-LO promotes an increase in M2c-like wound-healing macrophages in the kidney and alters kidney metabolism globally, protecting against anaerobic glycolysis after injury.

Antioxidant enzyme peroxiredoxin 5 regulates cyst growth and ciliogenesis via modulating Plk1 stability.

Oxidative stress is emerging as a contributing factor to the homeostasis in cystic diseases. However, the role antioxidant enzymes play in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD) remains elusive. Peroxiredoxin 5 (Prdx5) is an antioxidant enzyme that catalyzes the reduction of H2 O2 and alkyl hydroperoxide and plays an important role in different biological processes. In this study, we show that Prdx5 is downregulated in a PKD mutant mouse model and ADPKD patient kidneys. Knockdown of Prdx5 resulted in the formation of cysts in a three-dimensional mouse inner medullar collecting duct (IMCD) cell Matrigel culture system. The mechanisms of Prdx5 deficiency mediated cyst growth include: (1) induction of oxidative stress as indicated by increased mRNA expression of heme oxygenase-1, an oxidant stress marker; (2) activation of Erk, S6 and mTORC1, which contribute to cystic renal epithelial cell proliferation and cyst growth; (3) abnormal centrosome amplification and multipolar spindle formation which result in genome instability; (4) upregulation of Polo-like kinase 1 (Plk1) and Aurora kinase A, important mitotic kinases involved in cell proliferation and ciliogenesis; (5) impaired formation of primary cilia in mouse IMCD3 and retinal pigment epithelial cells, which could be rescued by inhibiting Plk1 activity; and (6) restraining the effect of Wnt3a and Wnt5a ligands on primary cilia in mouse IMCD3 cells, while regulating the activity of the canonical and non-canonical Wnt signaling in a separate cilia independent mechanism, respectively. Importantly, we found that targeting Plk1 with its inhibitor, volasertib, delayed cyst growth in Pkd1 conditional knockout mouse kidneys. Together, these findings indicate that Prdx5 is an important antioxidant that regulates cyst growth via diverse mechanisms, in particular, the Prdx5-Plk1 axis, and that induction and activation of Prdx5, alone or together with inhibition of Plk1, represent a promising strategy for combatting ADPKD.

Sex- and age-specific regulation of ACE2: Insights into severe COVID-19 susceptibility.

Aged males disproportionately succumb to increased COVID-19 severity, hospitalization, and mortality compared to females. Angiotensin-converting enzyme 2 (ACE2) and transmembrane protease, serine 2 (TMPRSS2) facilitate SARS-CoV-2 viral entry and may have sexually dimorphic regulation. As viral load dictates disease severity, we investigated the expression, protein levels, and activity of ACE2 and TMPRSS2. Our data reveal that aged males have elevated ACE2 in both mice and humans across organs. We report the first comparative study comprehensively investigating the impact of sex and age in murine and human levels of ACE2 and TMPRSS2, to begin to elucidate the sex bias in COVID-19 severity.

Ectodomain shedding by ADAM proteases as a central regulator in kidney physiology and disease.

Besides its involvement in blood and bone physiology, the kidney's main function is to filter substances and thereby regulate the electrolyte composition of body fluids, acid-base balance and toxin removal. Depending on underlying conditions, the nephron must undergo remodeling and cellular adaptations. The proteolytic removal of cell surface proteins via ectodomain shedding by A Disintegrin and Metalloproteases (ADAMs) is of importance for the regulation of cell-cell and cell-matrix adhesion of renal cells. ADAM10 controls glomerular and tubule development in a Notch1 signaling-dependent manner and regulates brush border composition. ADAM17 regulates the renin angiotensin system and is together with ADAM10 involved in calcium phosphate homeostasis. In kidney disease ADAMs, especially ADAM17 contribute to inflammation through their involvement in IL-6 trans-signaling, Notch-, epithelial growth factor receptor-, and tumor necrosis factor α signaling. ADAMs are interesting drug targets to reduce the inflammatory burden, defective cell adhesion and impaired signaling pathways in kidney diseases.

Purification of Angiotensin-Converting Enzyme (ACE) from Sheep Kidney and Inhibition Effect of Reduced Nicotinamide Adenine Dinucleotide (NADH) on Purified ACE Activity.

Angiotensin-converting enzyme (ACE, EC 3.4.15.1) is a significant enzyme that regulates blood pressure. ACE inhibitors are often used in the treatment of hypertension. In this work, ACE was purified and characterized in one step with affinity chromatography from sheep kidneys. ACE was 10305-fold purified and specific activity was 19,075 EU/mg protein. The molecular weight and purity of ACE were found with SDS-PAGE and observed two bands at about 60 kDa and 70 kDa on the gel. The effects of reduced nicotinamide adenine dinucleotide (NADH), an antioxidant compound, on purified ACE activity were also researched. NADH on ACE activity showed an inhibition effect. The inhibition type of NADH was determined to be non-competitive inhibition by the Lineweaver-Burk chart and IC50 and Ki values for NADH were 244.33 and 175.08 µM, respectively. These results suggest that antioxidant substances might be efficient in preventing hypertension.

Concurrent changes in thermal tolerance thresholds and cellular heat stress response reveals novel molecular signatures and markers of high temperature acclimation in rainbow trout.

The objective of this study was to better understand the molecular mechanisms which regulate acclimatory responses and thermal safety margins of rainbow trout (Oncorhynchus mykiss) at temperatures above physiological optimum. For this, we investigated the time course of changes in critical thermal tolerance thresholds and associated hepatic and renal transcript abundance of molecular markers related to cellular stress response, during high temperature acclimation. The experimental fish were initially acclimated to 17 °C and later exposed to a gradually raised elevated temperature regime (22 °C) for a period of 30 days. CTmax, CTmin and mRNA expression of candidate markers were examined before the thermal challenge (T0) and over the time-course (days) of high temperature exposure (T1, T3, T7, T15 and T30). With respect to organismal response, CTmax was significantly elevated at T3, but the degree of gain in heat tolerance was not persistent. Contrarily, we observed a gradual loss in cold tolerance with highest CTmin estimate at T30. Based on the time-course of mRNA expression, the studied markers could be categorized into those which were persistently elevated (hsp70a, hsp70b, hspa5, hsp90a, hsp90b, stip1 and serpinh1 in kidney and hsp90b in liver); those which concurred with changes in CTmin (hspbp1, hsp90b, stip1, gr1, hif1a, hyou1, tnfa and tlr5 in kidney); and those which concurred with changes in CTmax (hsp90a, serpinh1, tlr5 and lmo2 in liver). Apparently, transcriptional changes in kidney and liver reflected CTmin and CTmax trend, respectively. Expression profile of stip1 and tlr5 suggest that they are potential novel markers which could reflect thermal limits in rainbow trout. Hepatic metabolic markers were either initially elevated (alt, glud, g6pase1) or down-regulated at different time-points (ast2, gls1, fas, cpt1b, mtor), linked to gluconeogenesis and metabolic depression, respectively. Whereas, growth-axis markers showed no significant differences. Overall, this time-course analysis has revealed potential associations in organismal and tissue-specific cellular response to high temperature acclimation in a thermally sensitive coldwater ectotherm.

Depth of the Steroid Core Location Determines the Mode of Na,K-ATPase Inhibition by Cardiotonic Steroids.

Cardiotonic steroids (CTSs) are specific inhibitors of Na,K-ATPase (NKA). They induce diverse physiological effects and were investigated as potential drugs in heart diseases, hypertension, neuroinflammation, antiviral and cancer therapy. Here, we compared the inhibition mode and binding of CTSs, such as ouabain, digoxin and marinobufagenin to NKA from pig and rat kidneys, containing CTSs-sensitive (α1S) and -resistant (α1R) α1-subunit, respectively. Marinobufagenin in contrast to ouabain and digoxin interacted with α1S-NKA reversibly, and its binding constant was reduced due to the decrease in the deepening in the CTSs-binding site and a lower number of contacts between the site and the inhibitor. The formation of a hydrogen bond between Arg111 and Asp122 in α1R-NKA induced the reduction in CTSs' steroid core deepening that led to the reversible inhibition of α1R-NKA by ouabain and digoxin and the absence of marinobufagenin's effect on α1R-NKA activity. Our results elucidate that the difference in signaling, and cytotoxic effects of CTSs may be due to the distinction in the deepening of CTSs into the binding side that, in turn, is a result of a bent-in inhibitor steroid core (marinobufagenin in α1S-NKA) or the change of the width of CTSs-binding cavity (all CTSs in α1R-NKA).

Semaporin3A inhibitor ameliorates renal fibrosis through the regulation of JNK signaling.

Renal fibrosis is the common pathological pathway in progressive renal diseases. In the present study, we analyzed the roles of semaphorin 3 A (SEMA3A) on renal fibrosis and the effect of SEMA3A inhibitor (SEMA3A-I) using a unilateral ureteral obstruction (UUO) mouse model. Expression of SEMA3A in the proximal tubulus and neuropilin-1, a recepor of SEMA3A, in fibloblast and tubular cells were increased in UUO kidneys. The expression of myofibroblast marker tenascin-C and fibronection as well as renal fibrosis were increased in UUO kidneys, all of which were ameliorated by SEMA3A-I. In addition, the JNK signaling pathway, known as the target of SEMA3A signaling, was activated in proximal tubular cells and fibroblast cells after UUO surgery, and SEMA3A-I significantly attenuated the activation. In vitro, treatments with SEMA3A as well as transforming growth factor-ß1 (TGF-ß1) in human proximal tubular cells lost epithelial cell characteristics, and SEMA3A-I significantly ameliorated this transformation. The JNK inhibitor SP600125 partially reversed SEMA3A and TGF-ß1-induced cell transformation, indicating that JNK signaling is involved in SEMA3A-induced renal fibrosis. In addition, treatment with SEMA3A in fibroblast cells activated expression of tenascin-C, collagen type I, and fibronection, indicating that SEMA3A may accelerate renal fibrosis through the activation of fibroblast cells. Analysis of human data revealed the positive correlation between urinary SEMA3A and urinary N-acetyl-ß-d-glucosaminidase, indicating the association between SEMA3A and tubular injury. In conclusion, SEMA3A signaling is involved in renal fibrosis through the JNK signaling pathway and SEMA3A-I might be a therapeutic option for protecting from renal fibrosis.NEW & NOTEWORTHY Renal fibrosis is the common pathological pathway in the progression of renal diseases. This study, using a unilateral ureteral obstruction (UUO) mouse model, indicated increased semaphorin3A (SEMA3A) signaling in renal tubular cells as well as fibroblast cells under UUO surgery, and SEMA3A inhibitor ameliorated UUO-induced renal fibrosis through the regulation of JNK signaling. The study proposes the potential therapeutic option of SEMA3A inhibitor to treat renal fibrosis.

Chronic activation of AMP-activated protein kinase leads to early-onset polycystic kidney phenotype.

AMP-activated protein kinase (AMPK) plays a key role in the cellular response to low energy stress and has emerged as an attractive therapeutic target for tackling metabolic diseases. Whilst significant progress has been made regarding the physiological role of AMPK, its function in the kidney remains only partially understood. We use a mouse model expressing a constitutively active mutant of AMPK to investigate the effect of AMPK activation on kidney function in vivo. Kidney morphology and changes in gene and protein expression were monitored and serum and urine markers were measured to assess kidney function in vivo. Global AMPK activation resulted in an early-onset polycystic kidney phenotype, featuring collecting duct cysts and compromised renal function in adult mice. Mechanistically, the cystic kidneys had increased cAMP levels and ERK activation, increased hexokinase I (Hk I) expression, glycogen accumulation and altered expression of proteins associated with autophagy. Kidney tubule-specific activation of AMPK also resulted in a polycystic phenotype, demonstrating that renal tubular AMPK activation caused the cystogenesis. Importantly, human autosomal dominant polycystic kidney disease (ADPKD) kidney sections revealed similar protein localisation patterns to that observed in the murine cystic kidneys. Our findings show that early-onset chronic AMPK activation leads to a polycystic kidney phenotype, suggesting dysregulated AMPK signalling is a contributing factor in cystogenesis.

Calorie Restriction Protects against Contrast-Induced Nephropathy via SIRT1/GPX4 Activation.

Calorie restriction (CR) extends lifespan and increases resistance to multiple forms of stress, including renal ischemia-reperfusion (I/R) injury. However, whether CR has protective effects on contrast-induced nephropathy (CIN) remains to be determined. In this study, we evaluated the therapeutic effects of CR on CIN and investigated the potential mechanisms. CIN was induced by the intravenous injection of iodinated contrast medium (CM) iopromide (1.8 g/kg) into Sprague Dawley rats with normal food intake or 40% reduced food intake, 4 weeks prior to iopromide administration. We found that CR was protective of CIN, assessed by renal structure and function. CM increased apoptosis, reactive oxygen species (ROS), and inflammation in the renal outer medulla, which were decreased by CR. The silent information regulator 1 (SIRT1) participated in the protective effect of CR on CIN, by upregulating glutathione peroxidase 4 (GPX4), a regulator of ferroptosis, because this protective effect was reversed by EX527, a specific SIRT1 antagonist. Our study showed that CR protected CIN via SIRT1/GPX4 activation. CR may be used to mitigate CIN.

VEGFR3 tyrosine kinase inhibition aggravates cisplatin nephrotoxicity.

Expansion of renal lymphatic networks, or lymphangiogenesis (LA), is well recognized during development and is now being implicated in kidney diseases. Although LA is associated with multiple pathological conditions, very little is known about its role in acute kidney injury. The purpose of this study was to evaluate the role of LA in a model of cisplatin-induced nephrotoxicity. LA is predominately regulated by vascular endothelial growth factor (VEGF)-C and VEGF-D, ligands that exert their function through their cognate receptor VEGF receptor 3 (VEGFR3). We demonstrated that use of MAZ51, a selective VEGFR3 inhibitor, caused significantly worse structural and functional kidney damage in cisplatin nephrotoxicity. Apoptotic cell death and inflammation were also increased in MAZ51-treated animals compared with vehicle-treated animals following cisplatin administration. Notably, MAZ51 caused significant upregulation of intrarenal phospho-NF-κB, phospho-JNK, and IL-6. Cisplatin nephrotoxicity is associated with vascular congestion due to endothelial dysfunction. Using three-dimensional tissue cytometry, a novel approach to explore lymphatics in the kidney, we detected significant vascular autofluorescence attributed to erythrocytes in cisplatin alone-treated animals. Interestingly, no such congestion was detected in MAZ51-treated animals. We found increased renal vascular damage in MAZ51-treated animals, whereby MAZ51 caused a modest decrease in the endothelial markers endomucin and von Willebrand factor, with a modest increase in VEGFR2. Our findings identify a protective role for de novo LA in cisplatin nephrotoxicity and provide a rationale for the development of therapeutic approaches targeting LA. Our study also suggests off-target effects of MAZ51 on the vasculature in the setting of cisplatin nephrotoxicity.NEW & NOTEWORTHY Little is known about injury-associated LA in the kidney and its role in the pathophysiology of acute kidney injury (AKI). Observed exacerbation of cisplatin-induced AKI after LA inhibition was accompanied by increased medullary damage and cell death in the kidney. LA inhibition also upregulated compensatory expression of LA regulatory proteins, including JNK and NF-κB. These data support the premise that LA is induced during AKI and lymphatic expansion is a protective mechanism in cisplatin nephrotoxicity.

Involvement of endoplasmic reticulum stress-activated PERK-eIF2α-ATF4 signaling pathway in T-2 toxin-induced apoptosis of porcine renal epithelial cells.

T-2 toxin is a highly toxic trichothecene that can induce toxic effects in a variety of organs and tissues, but the pathogenesis of its nephrotoxicity has not been elucidated. In this study, we assessed the involvement of protein kinase RNA-like ER kinase (PERK)-mediated endoplasmic reticulum (ER) stress and apoptosis in PK-15 cells cultured at different concentrations of T-2 toxin. Cell viability, antioxidant capacity, intracellular calcium (Ca2+) content, apoptotic rate, levels of ER stress, and apoptosis-related proteins were studied. T-2 toxin inhibited cell proliferation; increased the apoptosis rate; and was accompanied by increased cleaved caspase-3 expression, altered intracellular oxidative stress marker levels, and intracellular Ca2+ overloading. The ER stress inhibitor 4-phenylbutyrate (4-PBA) and PERK selective inhibitor GSK2606414 prevented the decrease of cell activity and apoptosis caused by T-2 toxin. The altered expression of glucose regulatory protein 78 (GRP78), C/EBP homologous protein (CHOP), and caspase-12 proved that ER stress was involved in cell injury triggered by T-2 toxin. T-2 toxin activated the phosphorylation of PERK and the alpha subunit of eukaryotic initiation factor 2 (eIF2α) and upregulated the activating transcription factor 4 (ATF4), thereby triggering ER stress via the GRP78/PERK/CHOP signaling pathway. This study provides a new perspective for understanding the nephrotoxicity of T-2 toxin.

Inhibition effect of nicotinamide (vitamin B3) and reduced glutathione (GSH) peptide on angiotensin-converting enzyme activity purified from sheep kidney.

Angiotensin-converting enzyme (ACE, EC 3.4.15.1) plays a significant role in blood pressure regulation and inhibition of this enzyme is one of the significant drug targets for the treatment of hypertension. In this work, ACE was purified from sheep kidneys with the affinity chromatography method in one step. The purity and molecular weight of ACE were designated using the SDS-PAGE method and observed two bands at around 60 kDa and 70 kDa on the gel. The effects of nicotinamide (vitamin B3) and reduced glutathione (GSH) peptide on purified ACE were researched. Nicotinamide and GSH peptide on purified ACE showed an inhibition effect. IC50 values for nicotinamide and GSH were calculated as 14.3 µM and 7.3 µM, respectively. Type of inhibition and Ki values for nicotinamide and GSH from the Lineweaver-Burk graph were determined. The type of inhibition for nicotinamide and GSH was determined as non-competitive inhibition. Ki value was calculated as 15.4 µM for nicotinamide and 6.7 µM for GSH. Also, GSH peptide showed higher inhibitory activity on ACE activity than nicotinamide. In this study, it was concluded that nicotinamide and GSH peptide compounds, which show an inhibition effect on ACE activity, may have both protective and therapeutic effects against hypertension.

Physiological and pathophysiological roles of acidic mammalian chitinase (CHIA) in multiple organs.

Acidic mammalian chitinase (CHIA) belongs to the 18-glycosidase family and is expressed in epithelial cells and certain immune cells (such as neutrophils and macrophages) in various organs. Under physiological conditions, as a hydrolase, CHIA can degrade chitin-containing pathogens, participate in Type 2 helper T (Th2)-mediated inflammation, and enhance innate and adaptive immunity to pathogen invasion. Under pathological conditions, such as rhinitis, ocular conjunctivitis, asthma, chronic atrophic gastritis, type 2 diabetes, and pulmonary interstitial fibrosis, CHIA expression is significantly changed. In addition, studies have shown that CHIA has an anti-apoptotic effect, promotes epithelial cell proliferation and maintains organ integrity, and these effects are not related to chitinase degradation. CHIA can also be used as a biomolecular marker in diseases such as chronic atrophic gastritis, dry eye, and acute kidney damage caused by sepsis. Analysis of the authoritative TCGA database shows that CHIA expression in gastric adenocarcinoma, liver cancer, renal clear cell carcinoma and other tumors is significantly downregulated compared with that in normal tissues, but the specific mechanism is unclear. This review is based on all surveys conducted to date and summarizes the expression patterns and functional diversity of CHIA in various organs. Understanding the physiological and pathophysiological relevance of CHIA in multiple organs opens new possibilities for disease treatment.