Ulinastatin Attenuates LPS-Induced Inflammation and Inhibits Endoplasmic Reticulum Stress-Induced Apoptosis in Renal Tubular Epithelial Cells via Regulation of the TLR4/NF-κB and Nrf2/HO-1 Pathways.

Acute kidney injury (AKI) is one of the most common diseases in patients treated in intensive care units. This study was intended to explore the underlying mechanism by which ulinastatin (UTI) influenced the inflammation and apoptosis of renal tubular epithelial cells, HK-2.The effects of UTI on the cell viability of HK-2 cells were first measured by MTT and lactate dehydrogenase (LDH) detection kit. The apoptosis and inflammation of HK-2 cells were then determined by TUNEL, western blot, ELISA, and RT-qPCR. Then, the proteins in the Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf2)/Heme oxygenase 1 (HO-1) signaling pathways were measured by western blot for confirming the relationship between UTI and these pathways. Finally, Nrf-2 inhibitor ML385 and TLR4 activator CCL-34 were respectively used on LPS-induced HK-2 cells exposed to UTI for the conduction of gain-of-function and loss-of-function assays.UTI treatment boosted the cell viability of HK-2 cells damaged by LPS. Furthermore, UTI exposure cut down the apoptosis rate and inhibited the expression inflammatory factors of HK-2 cells induced by LPS. UTI treatment decreased the expression of proteins in the TLR4/NF-κB pathway, increased the HO-1 expression, and prompted the translocation of Nrf2 from the cytoplasm to the nucleus. The alleviated effects of UTI on inflammation and apoptosis LPS-induced HK-2 cells were abolished by ML385 and TLR4, respectively.UTI attenuates LPS-induced inflammation and inhibits endoplasmic reticulum stress-induced apoptosis in renal tubular epithelial cells by regulating TLR4/NF-κB and Nrf2/HO-1 pathways.

CPT1α maintains phenotype of tubules via mitochondrial respiration during kidney injury and repair.

Impaired energy metabolism in proximal tubular epithelial cells (PTECs) is strongly associated with various kidney diseases. Here, we characterized proximal tubular phenotype alternations during kidney injury and repair in a mouse model of folic acid nephropathy, in parallel, identified carnitine palmitoyltransferase 1α (CPT1α) as an energy stress response accompanied by renal tubular dedifferentiation. Genetic ablation of Cpt1α aggravated the tubular injury and interstitial fibrosis and hampered kidney repair indicate that CPT1α is vital for the preservation and recovery of tubular phenotype. Our data showed that the lipid accumulation and mitochondrial mass reduction induced by folic acid were persistent and became progressively more severe in PTECs without CPT1α. Interference of CPT1α reduced capacities of mitochondrial respiration and ATP production in PTECs, and further sensitized cells to folic acid-induced phenotypic changes. On the contrary, overexpression of CPT1α protected mitochondrial respiration and prevented against folic acid-induced tubular cell damage. These findings link CPT1α to intrinsic mechanisms regulating the mitochondrial respiration and phenotype of kidney tubules that may contribute to renal pathology during injury and repair.

p53 Deacetylation Alleviates Sepsis-Induced Acute Kidney Injury by Promoting Autophagy.

Recent studies have shown that autophagy upregulation can attenuate sepsis-induced acute kidney injury (SAKI). The tumor suppressor p53 has emerged as an autophagy regulator in various forms of acute kidney injury (AKI). Our previous studies showed that p53 acetylation exacerbated hemorrhagic shock-induced AKI and lipopolysaccharide (LPS)-induced endothelial barrier dysfunction. However, the role of p53-regulated autophagy in SAKI has not been examined and requires clarification. In this study, we observed the dynamic changes of autophagy in renal tubular epithelial cells (RTECs) and verified the protective effects of autophagy activation on SAKI. We also examined the changes in the protein expression, intracellular distribution (nuclear and cytoplasmic), and acetylation/deacetylation levels of p53 during SAKI following cecal ligation and puncture (CLP) or LPS treatment in mice and in a LPS-challenged human RTEC cell line (HK-2 cells). After sepsis stimulation, the autophagy levels of RTECs increased temporarily, followed by a sharp decrease. Autophagy inhibition was accompanied by an increased renal tubular injury score. By contrast, autophagy agonists could reduce renal tubular damage following sepsis. Surprisingly, the expression of p53 protein in both the renal cortex and HK-2 cells did not significantly change following sepsis stimulation. However, the translocation of p53 from the nucleus to the cytoplasm increased, and the acetylation of p53 was enhanced. In the mechanistic study, we found that the induction of p53 deacetylation, due to either the resveratrol/quercetin -induced activation of the deacetylase Sirtuin 1 (Sirt1) or the mutation of the acetylated lysine site in p53, promoted RTEC autophagy and alleviated SAKI. In addition, we found that acetylated p53 was easier to bind with Beclin1 and accelerated its ubiquitination-mediated degradation. Our study underscores the importance of deacetylated p53-mediated RTEC autophagy in future SAKI treatments.

Renal Tubular Epithelial TRPA1 Acts as An Oxidative Stress Sensor to Mediate Ischemia-Reperfusion-Induced Kidney Injury through MAPKs/NF-κB Signaling.

Oxidative stress and inflammation play important roles in the pathophysiology of acute kidney injury (AKI). Transient receptor potential ankyrin 1 (TRPA1) is a Ca2+-permeable ion channel that is sensitive to reactive oxygen species (ROS). The role of TRPA1 in AKI remains unclear. In this study, we used human and animal studies to assess the role of renal TRPA1 in AKI and to explore the regulatory mechanism of renal TRPA1 in inflammation via in vitro experiments. TRPA1 expression increased in the renal tubular epithelia of patients with AKI. The severity of tubular injury correlated well with tubular TRPA1 or 8-hydroxy-2'-deoxyguanosine expression. In an animal model, renal ischemia-reperfusion injury (IR) increased tubular TRPA1 expression in wild-type (WT) mice. Trpa1-/- mice displayed less IR-induced tubular injury, oxidative stress, inflammation, and dysfunction in kidneys compared with WT mice. In the in vitro model, TRPA1 expression increased in renal tubular cells under hypoxia-reoxygenation injury (H/R) conditions. We demonstrated that H/R evoked a ROS-dependent TRPA1 activation, which elevated intracellular Ca2+ level, increased NADPH oxidase activity, activated MAPK/NF-κB signaling, and increased IL-8. Renal tubular TRPA1 may serve as an oxidative stress sensor and a crucial regulator in the activation of signaling pathways and promote the subsequent transcriptional regulation of IL-8. These actions might be evident in mice with IR or patients with AKI.

Heme Oxygenase 1: A Defensive Mediator in Kidney Diseases.

The incidence of kidney disease is rising, constituting a significant burden on the healthcare system and making identification of new therapeutic targets increasingly urgent. The heme oxygenase (HO) system performs an important function in the regulation of oxidative stress and inflammation and, via these mechanisms, is thought to play a role in the prevention of non-specific injuries following acute renal failure or resulting from chronic kidney disease. The expression of HO-1 is strongly inducible by a wide range of stimuli in the kidney, consequent to the kidney's filtration role which means HO-1 is exposed to a wide range of endogenous and exogenous molecules, and it has been shown to be protective in a variety of nephropathological animal models. Interestingly, the positive effect of HO-1 occurs in both hemolysis- and rhabdomyolysis-dominated diseases, where the kidney is extensively exposed to heme (a major HO-1 inducer), as well as in non-heme-dependent diseases such as hypertension, diabetic nephropathy or progression to end-stage renal disease. This highlights the complexity of HO-1's functions, which is also illustrated by the fact that, despite the abundance of preclinical data, no drug targeting HO-1 has so far been translated into clinical use. The objective of this review is to assess current knowledge relating HO-1's role in the kidney and its potential interest as a nephroprotection agent. The potential therapeutic openings will be presented, in particular through the identification of clinical trials targeting this enzyme or its products.

Tissue xanthine oxidoreductase activity in a mouse model of aristolochic acid nephropathy.

Xanthine oxidoreductase (XOR) is a critical enzyme in purine metabolism and uric acid production, and its levels are reported to increase during stress, thereby promoting organ damage. Herein, we investigated the activity of XOR in a mouse model of aristolochic acid I (AA)-induced nephropathy, a type of nephrotoxic chronic kidney disease (CKD). A persistent decrease in renal function was observed in mice up to 4 weeks after 4 weeks of AA (2.5 mg kg-1 ) administration. Renal histology revealed an increase in tubular interstitial fibrosis over time. Although AA administration did not change XOR activity in the plasma, heart, liver, or muscle, XOR activity was persistently increased in renal tissue. Our results suggest that the renal tissue-specific increase in XOR activity is involved in the progression of tubulo-interstitial disorders, specifically fibrosis.

Renal tubule Cpt1a overexpression protects from kidney fibrosis by restoring mitochondrial homeostasis.

Chronic kidney disease (CKD) remains a major epidemiological, clinical, and biomedical challenge. During CKD, renal tubular epithelial cells (TECs) present a persistent inflammatory and profibrotic response. Fatty acid oxidation (FAO), the main source of energy for TECs, is reduced in kidney fibrosis and contributes to its pathogenesis. To determine whether gain of function in FAO (FAO-GOF) could protect from fibrosis, we generated a conditional transgenic mouse model with overexpression of the fatty acid shuttling enzyme carnitine palmitoyl-transferase 1A (CPT1A) in TECs. Cpt1a-knockin (CPT1A-KI) mice subjected to 3 models of renal fibrosis (unilateral ureteral obstruction, folic acid nephropathy [FAN], and adenine-induced nephrotoxicity) exhibited decreased expression of fibrotic markers, a blunted proinflammatory response, and reduced epithelial cell damage and macrophage influx. Protection from fibrosis was also observed when Cpt1a overexpression was induced after FAN. FAO-GOF restored oxidative metabolism and mitochondrial number and enhanced bioenergetics, increasing palmitate oxidation and ATP levels, changes that were also recapitulated in TECs exposed to profibrotic stimuli. Studies in patients showed decreased CPT1 levels and increased accumulation of short- and middle-chain acylcarnitines, reflecting impaired FAO in human CKD. We propose that strategies based on FAO-GOF may constitute powerful alternatives to combat fibrosis inherent to CKD.

DNase I Induces Other Endonucleases in Kidney Tubular Epithelial Cells by Its DNA-Degrading Activity.

Endonuclease-mediated DNA fragmentation is both an immediate cause and a result of apoptosis and of all other types of irreversible cell death after injury. It is produced by nine enzymes including DNase I, DNase 2, their homologs, caspase-activated DNase (CAD) and endonuclease G (EndoG). The endonucleases act simultaneously during cell death; however, regulatory links between these enzymes have not been established. We hypothesized that DNase I, the most abundant of endonucleases, may regulate other endonucleases. To test this hypothesis, rat kidney tubular epithelial NRK-52E cells were transfected with the DNase I gene or its inactive mutant in a pECFP expression vector, while control cells were transfected with the empty vector. mRNA expression of all nine endonucleases was studied using real-time RT-PCR; DNA strand breaks in endonuclease genes were determined by PCR and protein expression of the enzymes was measured by Western blotting and quantitative immunocytochemistry. Our data showed that DNase I, but not its inactive mutant, induces all other endonucleases at varying time periods after transfection, causes DNA breaks in endonuclease genes, and elevates protein expression of several endonucleases. This is the first evidence that endonucleases seem to be induced by the DNA-degrading activity of DNase I.

AMP-activated protein kinase contributes to cisplatin-induced renal epithelial cell apoptosis and acute kidney injury.

Cisplatin, a commonly used anticancer drug, has been shown to induce acute kidney injury, which limits its clinical use in cancer treatment. Emerging evidence has suggested that AMP-activated protein kinase (AMPK), which functions as a cellular energy sensor, is activated by various cellular stresses that deplete cellular ATP. However, the potential role of AMPK in cisplatin-induced apoptosis of renal tubular epithelial cells has not been studied. In this study, we demonstrated that cisplatin activates AMPK (Thr172 phosphorylation) in cultured renal tubular epithelial cells in a time-dependent manner, which was associated with p53 phosphorylation. Compound C, a selective AMPK inhibitor, suppressed cisplatin-induced AMPK activation, p53 phosphorylation, Bax induction, and caspase 3 activation. Furthermore, silencing AMPK expression by siRNA attenuated cisplatin-induced p53 phosphorylation, Bax induction, and caspase 3 activation. In a mouse model of cisplatin-induced kidney injury, compound C inhibited p53 phosphorylation, Bax expression, caspase 3 activation, and apoptosis, protecting the kidney from injury and dysfunction. Taken together, these results suggest that the AMPK-p53-Bax signaling pathway plays a crucial role in cisplatin-induced tubular epithelial cell apoptosis.

Protective role of DJ-1 in endotoxin-induced acute kidney injury.

Acute kidney injury (AKI) is a frequent complication of sepsis and an important cause of morbidity and mortality worldwide. A cornerstone of sepsis-associated AKI is dysregulated inflammation, leading to increased tissue oxidative stress and free radical formation, which leads to multiple forms of cell death. DJ-1 is a peroxiredoxin protein with multiple functions, including its ability to control cellular oxidative stress. Although DJ-1 is expressed prominently by renal tubules, its role in AKI has not been investigated. In the present study, we examined the effect of DJ-1 deficiency in a murine model of endotoxin-induced AKI. Endotoxemia induced greater kidney injury in DJ-1-deficient mice. Furthermore, DJ-1 deficiency increased renal oxidative stress associated with increased renal tubular apoptosis and with expression of death domain-associated protein (DAXX). Similar to the in vivo model, in vitro experiments using a medullary collecting duct cell line (mIMCD3) and cytotoxic serum showed that serum obtained from wild-type mice resulted in increased expression of s100A8/s100A9, DAXX, and apoptosis in DJ-1-deficient mIMCD3 cells. Our findings demonstrate a novel renal protective role for renal tubular DJ-1 during endotoxemia through control of oxidative stress, renal inflammation, and DAXX-dependent apoptosis.

Rutaecarpine derivative Cpd-6c alleviates acute kidney injury by targeting PDE4B, a key enzyme mediating inflammation in cisplatin nephropathy.

Acute kidney injury (AKI), characterized by a rapid decline in renal function, is triggered by an acute inflammatory response that leads to kidney damage. An effective treatment for AKI is lacking. Using in vitro and in vivo AKI models, our laboratory has identified a series of anti-inflammatory molecules and their derivatives. In the current study, we identified the protective role of rutaecarpine (Ru) on renal tubules. We obtained a series of 3-aromatic sulphonamide-substituted Ru derivatives exhibiting enhanced renoprotective and anti-inflammatory function. We identified Compound-6c(Cpd-6c) as having the best activity and examined its protective effect against cisplatin nephropathy both in vivo and in vitro in cisplatin-stimulated tubular epithelial cells (TECs). Our results showed that Cpd-6c restored renal function more effectively than Ru, as evidenced by reduced blood urea nitrogen and serum creatinine levels in mice. Cpd-6c alleviated tubular injury, as shown by PAS staining and molecular analysis of kidney injury molecule-1 (KIM-1), with both prevention and treatment protocols in cisplatin-treated mice. Moreover, Cpd-6c decreased kidney inflammation, oxidative stress and programmed cell death. These results have also been confirmed in cisplatin-treated TECs. Using web-prediction algorithms, molecular docking, and cellular thermal shift assay (CETSA), we identified phosphodiesterase 4B (PDE4B) as a Cpd-6c target. In addition, we firstly found that PDE4B was up-regulated significantly in the serum of AKI patients. After identifying the function of PDE4B in cisplatin-treated tubular epithelial cells by siRNA transfection or PDE4 inhibitor rolipram, we showed that Cpd-6c treatment did not protect against cisplatin-induced injury in PDE4B knockdown TECs, thus indicating that Cpd-6c exerts its renoprotective and anti-oxidative effects via the PDE4B-dependent pathway. Collectively, Cpd-6c might serve as a potential therapeutic agent for AKI and PDE4B may be highly involved in the initiation and progression of AKI.

Adenosine kinase inhibition attenuates ischemia reperfusion-induced acute kidney injury.

Acute kidney injury (AKI) has a high morbidity and mortality, and there is no targeted treatment yet. One of the main causes of AKI is ischemia-reperfusion (IR). Increased release of adenosine under stress and hypoxia exerts anti-inflammatory and antioxidant effects. Adenosine kinase (ADK) is an important enzyme that eliminates adenosine in cells, and can maintain low adenosine concentration in cells. Our previous studies have shown that pretreatment of adenosine kinase inhibitor ABT-702 could markedly attenuate cisplatin-induced nephrotoxicity both in vivo and in vitro. This study is designed to investigate the effect of ADK inhibition on IR-induced AKI. The results showed that ADK expression was positively correlated with the degree of renal tubular injury, which suggested that the degree of ADK inhibition reflected the severity of acute tubular necrosis. In vivo, ADK inhibitor could reduce IR-induced renal injury, which might play a protective role by increasing tissue adenosine level, inhibiting oxidative stress, and reducing cell apoptosis. In HK2 cells, cobaltous dichloride (CoCl2) increased the level of oxidative stress, up-regulated the production of pro-inflammatory factor, and induced apoptosis, ADK inhibition could alleviate the above damaging effects. Moreover, the anti-apoptotic effect exerted by ADK inhibition was independent of inosine. In summary, our results support the idea that ADK inhibition has protective effects on IR-induced AKI. Adenosine kinase inhibition might provide a new target for AKI prevention and treatment.

Therapeutic Targeting of Aristolochic Acid Induced Uremic Toxin Retention, SMAD 2/3 and JNK/ERK Pathways in Tubulointerstitial Fibrosis: Nephroprotective Role of Propolis in Chronic Kidney Disease.

The nephrotoxicity of aristolochic acids (AAs), p-cresyl sulfate (PCS) and indoxyl sulfate (IS) were well-documented, culminating in tubulointerstitial fibrosis (TIF), advanced chronic kidney disease (CKD) and fatal urothelial cancer. Nonetheless, information regarding the attenuation of AAs-induced nephropathy (AAN) and uremic toxin retention is scarce. Propolis is a versatile natural product, exerting anti-oxidant, anti-cancer and anti-fibrotic properties. We aimed to evaluate nephroprotective effects of propolis extract (PE) in a murine model. AAN was developed to retain circulating PCS and IS using C57BL/6 mice, mimicking human CKD. The kidney sizes/masses, renal function indicators, plasma concentrations of PCS/IS, tissue expressions of TIF, α-SMA, collagen IaI, collagen IV and signaling pathways in transforming growth factor-ß (TGF-ß) family were analyzed among the control, PE, AAN, and AAN-PE groups. PE ameliorated AAN-induced renal atrophy, renal function deterioration, TIF, plasma retention of PCS and IS. PE also suppressed α-SMA expression and deposition of collagen IaI and IV in the fibrotic epithelial-mesenchymal transition. Notably, PE treatment in AAN model inhibited not only SMAD 2/3-dependent pathways but also SMAD-independent JNK/ERK activation in the signaling cascades of TGF-ß family. Through disrupting fibrotic epithelial-mesenchymal transition and TGF-ß signaling transduction pathways, PE improves TIF and thereby facilitates renal excretion of PCS and IS in AAN. In light of multi-faced toxicity of AAs, PE may be capable of developing a new potential drug to treat CKD patients exposed to AAs.

Tubule-Specific Mst1/2 Deficiency Induces CKD via YAP and Non-YAP Mechanisms.

BACKGROUND: The serine/threonine kinases MST1 and MST2 are core components of the Hippo pathway, which has been found to be critically involved in embryonic kidney development. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are the pathway's main effectors. However, the biologic functions of the Hippo/YAP pathway in adult kidneys are not well understood, and the functional role of MST1 and MST2 in the kidney has not been studied. METHODS: We used immunohistochemistry to examine expression in mouse kidneys of MST1 and MST2, homologs of Hippo in Drosophila. We generated mice with tubule-specific double knockout of Mst1 and Mst2 or triple knockout of Mst1, Mst2, and Yap. PCR array and mouse inner medullary collecting duct cells were used to identify the primary target of Mst1/Mst2 deficiency. RESULTS: MST1 and MST2 were predominantly expressed in the tubular epithelial cells of adult kidneys. Deletion of Mst1/Mst2 in renal tubules increased activity of YAP but not TAZ. The kidneys of mutant mice showed progressive inflammation, tubular and glomerular damage, fibrosis, and functional impairment; these phenotypes were largely rescued by deletion of Yap in renal tubules. TNF-α expression was induced via both YAP-dependent and YAP-independent mechanisms, and TNF-α and YAP amplified the signaling activities of each other in the tubules of kidneys with double knockout of Mst1/Mst2. CONCLUSIONS: Our findings show that tubular Mst1/Mst2 deficiency leads to CKD through both the YAP and non-YAP pathways and that tubular YAP activation induces renal fibrosis. The pathogenesis seems to involve the reciprocal stimulation of TNF-α and YAP signaling activities.

SerpinB2 Regulates Immune Response in Kidney Injury and Aging.

BACKGROUND: Expression of SerpinB2, a regulator of inflammatory processes, has been described in the context of macrophage activation and cellular senescence. Given that mechanisms for these processes interact and can shape kidney disease, it seems plausible that SerpinB2 might play a role in renal aging, injury, and repair. METHODS: We subjected SerpinB2 knockout mice to ischemia-reperfusion injury or unilateral ureteral obstruction. We performed phagocyte depletion to study SerpinB2's role beyond the effects of macrophages and transplanted bone marrow from knockout mice to wild-type mice and vice versa to dissect cell type-dependent effects. Primary tubular cells and macrophages from SerpinB2 knockout and wild-type mice were used for functional studies and transcriptional profiling. RESULTS: Cultured senescent tubular cells, kidneys of aged mice, and renal stress models exhibited upregulation of SerpinB2 expression. Functionally, lack of SerpinB2 in aged knockout mice had no effect on the magnitude of senescence markers but associated with enhanced kidney damage and fibrosis. In stress models, inflammatory cell infiltration was initially lower in knockout mice but later increased, leading to an accumulation of significantly more macrophages. SerpinB2 knockout tubular cells showed significantly reduced expression of the chemokine CCL2. Macrophages from knockout mice exhibited reduced phagocytosis and enhanced migration. Macrophage depletion and bone marrow transplantation experiments validated the functional relevance of these cell type-specific functions of SerpinB2. CONCLUSIONS: SerpinB2 influences tubule-macrophage crosstalk by supporting tubular CCL2 expression and regulating macrophage phagocytosis and migration. In mice, SerpinB2 expression seems to be needed for coordination and timely resolution of inflammation, successful repair, and kidney homeostasis during aging. Implications of SerpinB2 in human kidney disease deserve further exploration.

Cisplatin nephrotoxicity is induced via poly(ADP-ribose) polymerase activation in adult zebrafish and mice.

Cisplatin is a well-known chemotherapy medication used to treat numerous cancers. However, treatment with cisplatin in cancer therapy has major side effects, such as nephrotoxic acute kidney injury. Adult vertebrate kidneys are commonly used as models of cisplatin-induced nephrotoxic acute kidney injury. Embryonic zebrafish kidney is more simplified and is composed simply of two nephrons and thus is an excellent model for the investigation of cisplatin nephrotoxicity. Here, we developed a novel model to induce cisplatin nephrotoxicity in adult zebrafish and demonstrated that intraperitoneal injection of cisplatin caused a decline in kidney proximal tubular function based on fluorescein-labeled dextran uptake and alkaline phosphatase staining. We also showed that cisplatin induced histological injury of the kidney tubules, quantified by tubular injury scores on the periodic acid-Schiff-stained kidney sections. As shown in a mouse model of cisplatin-induced nephrotoxicity, the activation of poly(ADP-ribose) polymerase (PARP), an enzyme implicated in cisplatin-induced cell death, was markedly increased after cisplatin injection in adult zebrafish. Furthermore, pharmacological inhibition of PARP using a specific PARP inhibitor PJ 34 hydrochloride (PJ34) or 3-aminobenzamide ameliorated kidney proximal tubular functional and histological damages in cisplatin-injected adult zebrafish kidneys. Administration of a combination of PARP inhibitors PJ34 and 3-aminobenzamide additively protected renal function and histology in zebrafish and mouse models of cisplatin nephrotoxicity. In conclusion, these data suggest that adult zebrafish are not only suitable for drug screening and genetic manipulation but also useful as a simplified but powerful model to study the pathophysiology of cisplatin nephrotoxicity and establish new therapies for treating human kidney diseases.

DsbA-L deficiency exacerbates mitochondrial dysfunction of tubular cells in diabetic kidney disease.

Excessive mitochondrial fission has been identified as the central pathogenesis of diabetic kidney disease (DKD), but the precise mechanisms remain unclear. Disulfide-bond A oxidoreductase-like protein (DsbA-L) is highly expressed in mitochondria in tubular cells of the kidney, but its pathophysiological role in DKD is unknown. Our bioinformatics analysis showed that tubular DsbA-L mRNA levels were positively associated with eGFR but negatively associated with Scr and 24h-proteinuria in CKD patients. Furthermore, the genes that were coexpressed with DsbA-L were mainly enriched in mitochondria and were involved in oxidative phosphorylation. In vivo, knockout of DsbA-L exacerbated diabetic mice tubular cell mitochondrial fragmentation, oxidative stress and renal damage. In vitro, we found that DsbA-L was localized in the mitochondria of HK-2 cells. High glucose (HG, 30 mM) treatment decreased DsbA-L expression followed by increased mitochondrial ROS (mtROS) generation and mitochondrial fragmentation. In addition, DsbA-L knockdown exacerbated these abnormalities, but this effect was reversed by overexpression of DsbA-L. Mechanistically, under HG conditions, knockdown DsbA-L expression accentuated JNK phosphorylation in HK-2 cells. Furthermore, administration of a JNK inhibitor (SP600125) or the mtROS scavenger MitoQ significantly attenuated JNK activation and subsequent mitochondrial fragmentation in DsbA-L-knockdown HK-2 cells. Additionally, the down-regulation of DsbA-L also amplified the gene and protein expression of mitochondrial fission factor (MFF) via the JNK pathway, enhancing its ability to recruit DRP1 to mitochondria. Taken together, these results link DsbA-L to alterations in mitochondrial dynamics during tubular injury in the pathogenesis of DKD and unveil a novel mechanism by which DsbA-L modifies mtROS/JNK/MFF-related mitochondrial fission.

Insights into the heparan sulphate-dependent externalisation of transglutaminase-2 (TG2) in glucose-stimulated proximal-like tubular epithelial cells.

The extracellular matrix crosslinking enzyme transglutaminase 2 (TG2) is highly implicated in tissue fibrosis that precedes end-stage kidney failure. TG2 is unconventionally secreted through extracellular vesicles in a way that depends on the heparan sulphate (HS) proteoglycan syndecan-4 (Sdc4), the deletion of which reduces experimental kidney fibrosis as a result of lower extracellular TG2 in the tubule-interstitium. Here we establish a model of TG2 externalisation in NRK-52E tubular epithelial cells subjected to glucose stress. HS-binding TG2 mutants had reduced extracellular TG2 in transfected NRK-52E, suggesting that TG2-externalisation depends on an intact TG2 heparin binding site. Inhibition of N-ethylmaleimide sensitive factor (NSF) vesicle-fusing ATPase, which was identified in the recently elucidated TG2 kidney membrane-interactome, led to significantly lower TG2-externalisation, thus validating the involvement of membrane fusion in TG2 secretion. As cyclin-G-associated kinase (GAK) had emerged as a further TG2-partner in the fibrotic kidney, we investigated whether glucose-induced TG2-externalisation was accompanied by TG2 phosphorylation in consensus sequences of cyclin-dependent kinase (CDK). Glucose stress led to intense TG2 phosphorylation in serine/threonine CDK-target. TG2 phosphorylation by tyrosine kinases was also increased by glucose. Although the precise role of glucose-induced TG2 phosphorylation is unknown, these novel data suggest that phosphorylation may be involved in TG2 membrane-trafficking.

Blockade of the Adenosine A3 Receptor Attenuates Caspase 1 Activation in Renal Tubule Epithelial Cells and Decreases Interleukins IL-1ß and IL-18 in Diabetic Rats.

Diabetic nephropathy (DN) is the main cause of end-stage renal disease, which remains incurable. The progression of DN is associated with progressive and irreversible renal fibrosis and also high levels of adenosine. Our aim was to evaluate the effects of ADORA3 antagonism on renal injury in streptozotocin-induced diabetic rats. An ADORA3 antagonist that was administered in diabetic rats greatly inhibited the levels of inflammatory interleukins IL-1ß and IL-18, meanwhile when adenosine deaminase was administered, there was a non-selective attenuation of the inflammatory mediators IL-1ß, IL-18, IL-6, and induction of IL-10. The ADORA3 antagonist attenuated the high glucose-induced activation of caspase 1 in HK2 cells in vitro. Additionally, ADORA3 antagonisms blocked the increase in caspase 1 and the nuclear localization of NFκB in the renal tubular epithelium of diabetic rats, both events that are involved in regulating the production and activation of IL-1ß and IL-18. The effects of the A3 receptor antagonist resulted in the attenuation of kidney injury, as evidenced by decreased levels of the pro-fibrotic marker α-SMA at histological levels and the restoration of proteinuria in diabetic rats. We conclude that ADORA3 antagonism represents a potential therapeutic target that mechanistically works through the selective blockade of the NLRP3 inflammasome.

A study of sirolimus and mTOR kinase inhibitor in a hypomorphic Pkd1 mouse model of autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (PKD) is characterized by cyst formation and growth, which are partially driven by abnormal proliferation of tubular cells. Proproliferative mechanistic target of rapamycin (mTOR) complexes 1 and 2 (mTORC1 and mTORC2) are activated in the kidneys of mice with PKD. Sirolimus indirectly inhibits mTORC1. Novel mTOR kinase inhibitors directly inhibit mTOR kinase, resulting in the inhibition of mTORC1 and mTORC2. The aim of the present study was to determine the effects of sirolimus versus the mTOR kinase inhibitor torin2 on cyst growth and kidney function in the Pkd1 p.R3277C (Pkd1RC/RC) mouse model, a hypomorphic Pkd1 model orthologous to the human condition, and to determine the effects of sirolimus versus torin2 on mTORC1 and mTORC2 signaling in PKD1-/- cells and in the kidneys of Pkd1RC/RC mice. In vitro, both inhibitors reduced mTORC1 and mTORC2 phosphorylated substrates and negatively impacted cellular metabolic activity, as measured by MTT assay. Pkd1RC/RC mice were treated with sirolimus or torin2 from 50 to 120 days of age. Torin2 was as effective as sirolimus in decreasing cyst growth and improving loss of kidney function. Both sirolimus and torin2 decreased phosphorylated S6 protein, phosphorylated eukaryotic translation initiation factor 4E-binding protein 1, phosphorylated Akt, and proliferation in Pkd1RC/RC kidneys. In conclusion, torin2 and sirolimus were equally effective in decreasing cyst burden and improving kidney function and mediated comparable effects on mTORC1 and mTORC2 signaling and proliferation in the Pkd1RC/RC kidney.