Association of Coding Variants in Hydroxysteroid 17-beta Dehydrogenase 14 (HSD17B14) with Reduced Progression to End Stage Kidney Disease in Type 1 Diabetes.

BACKGROUND: Rare variants in gene coding regions likely have a greater impact on disease-related phenotypes than common variants through disruption of their encoded protein. We searched for rare variants associated with onset of ESKD in individuals with type 1 diabetes at advanced kidney disease stage. METHODS: Gene-based exome array analyses of 15,449 genes in five large incidence cohorts of individuals with type 1 diabetes and proteinuria were analyzed for survival time to ESKD, testing the top gene in a sixth cohort (n=2372/1115 events all cohorts) and replicating in two retrospective case-control studies (n=1072 cases, 752 controls). Deep resequencing of the top associated gene in five cohorts confirmed the findings. We performed immunohistochemistry and gene expression experiments in human control and diseased cells, and in mouse ischemia reperfusion and aristolochic acid nephropathy models. RESULTS: Protein coding variants in the hydroxysteroid 17-ß dehydrogenase 14 gene (HSD17B14), predicted to affect protein structure, had a net protective effect against development of ESKD at exome-wide significance (n=4196; P value=3.3 × 10-7). The HSD17B14 gene and encoded enzyme were robustly expressed in healthy human kidney, maximally in proximal tubular cells. Paradoxically, gene and protein expression were attenuated in human diabetic proximal tubules and in mouse kidney injury models. Expressed HSD17B14 gene and protein levels remained low without recovery after 21 days in a murine ischemic reperfusion injury model. Decreased gene expression was found in other CKD-associated renal pathologies. CONCLUSIONS: HSD17B14 gene is mechanistically involved in diabetic kidney disease. The encoded sex steroid enzyme is a druggable target, potentially opening a new avenue for therapeutic development.

PARK7 Protects Against Chronic Kidney Injury and Renal Fibrosis by Inducing SOD2 to Reduce Oxidative Stress.

Renal fibrosis is the final common pathway to chronic kidney diseases regardless of etiology. Parkinson disease protein 7 (PARK7) is a multifunctional protein involved in various cellular processes, but its pathophysiological role in kidneys remain largely unknown. Here, we have determined the role of PARK7 in renal fibrosis and have further elucidated the underlying mechanisms by using the in vivo mouse model of unilateral ureteric obstruction (UUO) and the in vitro model of transforming growth factor-b (TGFB1) treatment of cultured kidney proximal tubular cells. PARK7 decreased markedly in atrophic kidney tubules in UUO mice, and Park7 deficiency aggravated UUO-induced renal fibrosis, tubular cell apoptosis, ROS production and inflammation. In vitro, TGFB1 treatment induced fibrotic changes in renal tubular cells, which was accompanied by alterations of PARK7. Park7 knockdown exacerbated TGFB1-induced fibrotic changes, cell apoptosis and ROS production, whereas Park7 overexpression or treatment with ND-13 (a PARK7-derived peptide) attenuated these TGFB1-induced changes. Mechanistically, PARK7 translocated into the nucleus of renal tubular cells following TGFB1 treatment or UUO, where it induced the expression of SOD2, an antioxidant enzyme. Taken together, these results indicate that PARK7 protects against chronic kidney injury and renal fibrosis by inducing SOD2 to reduce oxidative stress in tubular cells.

MicroRNA-363-3p promotes apoptosis in response to cadmium-induced renal injury by down-regulating phosphoinositide 3-kinase expression.

We previously determined that specific microRNAs (miRNAs) are involved in renal pathophysiological occurrences induced by cadmium (Cd) in rats. This study expands our studies on miRNAs, determining their role in Cd-induced nephrotoxicity in occupational workers. We performed miRNA microarray analyses of blood and urine samples from patients diagnosed as occupational chronic Cd poisoning (OCCP) with abnormally elevated concentrations of urinary beta-2-microglobulin (U-ß2-MG), an indicator of tubular proteinuria. We also performed in vitro bioinformatics-based investigations of apoptosis-related genes targeted by miRNAs involved in the biological response to Cd exposure. We tested five differentially expressed miRNAs and determined a significant increase of sera miR-363-3p. Also, we determined that miR-363-3p increase is associated with phosphoinositide 3-kinase (PI3K) down-regulation and the suppressed proliferation and enhanced apoptosis of renal tubule epithelial cells. The obtained results suggest miR-363-3p involvement in the pathophysiology of Cd-induced renal injury in humans and maybe considered for possible interventional therapeutic strategies for Cd-associated kidney damage.

Fucoidan from Laminaria japonica Inhibits Expression of GLUT9 and URAT1 via PI3K/Akt, JNK and NF-κB Pathways in Uric Acid-Exposed HK-2 Cells.

This work aimed to investigate the effect of fucoidan (FPS) on urate transporters induced by uric acid (UA). The results showed that UA stimulated the expression of glucose transporter 9 (GLUT9) and urate transporter 1 (URAT1) in HK-2 cells, and FPS could reverse the effect. Moreover, UA could activate NF-κB, JNK and PI3K/Akt pathways, but both pathway inhibitors and FPS inhibited the UA-induced activation of these three pathways. These data suggested that FPS effectively inhibited the expression induction of reabsorption transporters URAT1 and GLUT9 by UA, through repressing the activation of NF-κB, JNK and PI3K/Akt signal pathways in HK-2 cells. The in vitro research findings support the in vivo results that FPS reduces serum uric acid content in hyperuricemia mice and rats through inhibiting the expression of URAT1 and GLUT9 in renal tubular epithelial cells. This study provides a theoretical basis for the application of FPS in the treatment of hyperuricemia.

The multitargeted kinase inhibitor KW-2449 ameliorates cisplatin-induced nephrotoxicity by targeting RIPK1-mediated necroptosis.

Cisplatin (cis-dichloro-diammine platinum, CDDP) is a well-known chemotherapeutic drug against a broad spectrum of human malignancies. However, the clinical utility of this effective chemotherapy agent is dose limited by its toxic side effects such as nephrotoxicity and ototoxicity. Necroptosis is a form of programmed necrotic cell death that is mediated by serine/threonine kinases, RIPK1 and RIPK3, together with MLKL. In this study, we identified that the multitargeted kinase inhibitor KW-2449 inhibited cisplatin-induced necroptosis, while potentiated cisplatin-induced apoptosis in cancer cells. Mechanistic studies indicated that KW-2449 directly inhibited RIPK1 kinase activity to block necroptosis. Oral administration of KW-2449 attenuated renal cell necrosis and reduced pro-inflammatory responses in mouse models of cisplatin-induced nephrotoxicity. Taken together, our study shows that KW-2449 is a novel necroptosis inhibitor by targeting RIPK1 kinase activity and has great clinic potential for the treatment of cisplatin-induced nephrotoxicity.

ROS-ERK Pathway as Dual Mediators of Cellular Injury and Autophagy-Associated Adaptive Response in Urinary Protein-Irritated Renal Tubular Epithelial Cells.

ERK, an extracellular signal-regulated protein kinase, is involved in various biological responses, such as cell proliferation and differentiation, cell morphology maintenance, cytoskeletal construction, apoptosis, and canceration of cells. In this study, we focused on ERK pathway on cellular injury and autophagy-associated adaptive response in urinary protein-irritated renal tubular epithelial cells and explored the potential mechanisms underlying it. By using antioxidants N-acetylcysteine and catalase, we found that ERK pathway was activated by a reactive oxygen species- (ROS-) dependent mechanism after exposure to urinary proteins. What is more, ERK inhibitor U0126 could decrease the release of neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), and the number of apoptotic cells induced by urinary proteins, indicating the damaging effects of ERK pathway in mediating cellular injury and apoptosis in HK-2 cells. Interestingly, we also found that the increased expression of microtubule-associated protein 1 light chain 3 (LC3)-II (a key marker of autophagy) and the decreased expression of p62 (autophagic substrate) induced by urinary proteins were reversed by U0126, suggesting autophagy was activated by ERK pathway. Furthermore, rapamycin reduced urinary protein-induced NGAL and KIM-1 secretion and cell growth inhibition, while chloroquine played the opposite effect, indicating that autophagy activation by ERK pathway was an adaptive response in the exposure to urinary proteins. Taken together, our results indicate that activated ROS-ERK pathway can induce cellular injury and in the meantime provide an autophagy-associated adaptive response in urinary protein-irritated renal tubular epithelial cells.

Angiotensin receptors in the kidney and vasculature in hypertension and kidney disease.

Kidney disease, blood pressure determination, hypertension pathogenesis, and the renin-angiotensin system (RAS) are inextricably linked. Hence, understanding the RAS is pivotal to unraveling the pathophysiology of hypertension and the determinants to maintaining normal blood pressure. The RAS has been the subject of intense investigation for over a century. Moreover, medications that block the RAS are mainstay therapies in clinical medicine and have been shown to reduce morbidity and mortality in patients with diabetes, cardiovascular, and kidney diseases. The main effector peptide of the RAS is the interaction of the octapeptide- Ang II with its receptor. The type 1 angiotensin receptor (AT1R) is the effector receptor for Ang II. These G protein-coupled receptors (GPCRs) are ubiquitously expressed in a variety of cell lineages and tissues relevant to cardiovascular disease throughout the body. The advent of cell specific deletion of genes using Cre LoxP technology in mice has allowed for the identification of discreet actions of AT1Rs in blood pressure control and kidney disease. The kidney is one of the major targets of the RAS, which is responsible in maintaining fluid, electrolyte balance, and blood pressure. In this review we will discuss the role of AT1Rs in the kidney, vasculature, and immune cells and address their effects on hypertension and kidney disease.

NAM protects against cisplatin-induced acute kidney injury by suppressing the PARP1/p53 pathway.

Cisplatin is a commonly used anti-cancer drug, but it induces nephrotoxicity. As a water-soluble vitamin B family member, nicotinamide (NAM) was recently demonstrated to have beneficial effects for renal injury, but its underlying mechanism remains largely unclear. Here, we suggest that NAM may exert protective effects against cisplatin-induced acute kidney injury (AKI) mainly via suppressing the poly ADP-ribose polymerase 1 (PARP1)/p53 pathway. In our experiment, NAM protected against cisplatin-induced apoptosis both in cultured renal proximal tubular cells and AKI in mice. Mechanistically, NAM suppressed the expression and activation of p53, a known mediator of cisplatin-induced AKI. Upstream of p53, NAM attenuated the induction of γ-H2AX, a hallmark of DNA damage response. Interestingly, PARP1 was activated in cisplatin AKI and this activation was inhibited by NAM. Pharmacological inhibition of PARP1 with PJ34 significantly ameliorated p53 activation and cisplatin-induced cell death in RPTCs and AKI in mice. Thus, NAM may protect against cisplatin-induced AKI by suppressing the PARP1/p53 pathway.

PPAR-δ activation reduces cisplatin-induced apoptosis via inhibiting p53/Bax/caspase-3 pathway without modulating autophagy in murine renal proximal tubular cells.

BACKGROUND: Cisplatin-induced injury of renal proximal tubular cells results basically from increased apoptosis via mitochondrial damage, and is mitigated by appropriate enhancement of autophagy. Peroxisome proliferator-activated receptor-delta (PPAR-δ) reportedly protects against not only mitochondrial damages but also enhances autophagy. Thus, PPAR-δ may protect against cisplatin-induced kidney injury. METHODS: We examined the protective effects of PPAR-δ activation on cisplatin-induced cellular injury and their detailed mechanisms in a murine renal proximal tubular (mProx) cell line using GW0742, an authentic PPAR-δ activator. Cisplatin-induced cell damages were evaluated by TUNEL assay and immunoblot analyses for p53, 14-3-3, Bax, Bcl2, cytochrome C, and activated caspases. Autophagy status was examined by immunoblot analyses for p62 and LC3. RESULTS: GW0742 suppressed cisplatin-induced apoptosis of mProx cells by reducing the activation of caspase-3 via attenuating the phosphorylation of p53 and 14-3-3, mitochondrial Bax accumulation, cytochrome C release from mitochondria to the cytosol and ensuing cytosolic caspase-9 activation. In contrast, GW0742 did not diminish cisplatin-enhanced activation of caspases-8 or -12 as extrinsic or endothelium reticulum apoptotic pathways, respectively. The inhibitory effect of GW0742 on cisplatin-induced caspase-3 activation was significantly diminished by silencing of the PPAR-δ gene expression. GW0742 itself had no influence on starvation-stimulated or cisplatin-induced autophagy in mProx cells, suggesting that the protective effects were not mediated by autophagy modification. CONCLUSION: Our results indicate that GW0742 may serve as a candidate agent to mitigate cisplatin nephrotoxicity via inhibiting the mitochondrial apoptotic pathway considerably depending on PPAR-δ, without modulating autophagy.

SIRT1 attenuates sepsis-induced acute kidney injury via Beclin1 deacetylation-mediated autophagy activation.

Our previous studies showed that silent mating-type information regulation 2 homologue-1 (SIRT1, a deacetylase) upregulation could attenuate sepsis-induced acute kidney injury (SAKI). Upregulated SIRT1 can deacetylate certain autophagy-related proteins (Beclin1, Atg5, Atg7 and LC3) in vitro. However, it remains unclear whether the beneficial effect of SIRT1 is related to autophagy induction and the underlying mechanism of this effect is also unknown. In the present study, caecal ligation and puncture (CLP)-induced mice, and an LPS-challenged HK-2 cell line were established to mimic a SAKI animal model and a SAKI cell model, respectively. Our results demonstrated that SIRT1 activation promoted autophagy and attenuated SAKI. SIRT1 deacetylated only Beclin1 but not the other autophagy-related proteins in SAKI. SIRT1-induced autophagy and its protective effect against SAKI were mediated by the deacetylation of Beclin1 at K430 and K437. Moreover, two SIRT1 activators, resveratrol and polydatin, attenuated SAKI in CLP-induced septic mice. Our study was the first to demonstrate the important role of SIRT1-induced Beclin1 deacetylation in autophagy and its protective effect against SAKI. These findings suggest that pharmacologic induction of autophagy via SIRT1-mediated Beclin1 deacetylation may be a promising therapeutic approach for future SAKI treatment.

Bortezomib limits renal allograft interstitial fibrosis by inhibiting NF-κB/TNF-α/Akt/mTOR/P70S6K/Smurf2 pathway via IκBα protein stabilization.

Chronic allograft dysfunction is a major cause of late graft failure after kidney transplantation. One of the histological changes is interstitial fibrosis, which is associated with epithelial-mesenchymal transition. Bortezomib has been reported to prevent the progression of fibrosis in organs. We used rat renal transplantation model and human kidney 2 cell line treated with tumor necrosis factor-α (TNF-α) to examine their response to bortezomib. To explore the mechanism behind it, we assessed the previously studied TNF-α/protein kinase B (Akt)/Smad ubiquitin regulatory factor 2 (Smurf2) signaling and performed RNA sequencing. Our results suggested that bortezomib could attenuate the TNF-α-induced epithelial-mesenchymal transition and renal allograft interstitial fibrosis in vitro and in vivo. In addition to blocking Akt/mammalian target of rapamycin (mTOR)/p70S6 kinase/Smurf2 signaling, bortezomib's effect on the epithelial-mesenchymal transition was associated with inhibition of nuclear factor kappa B (NF-κB) pathway by stabilizing inhibitor of NF-κB. The study highlighted the therapeutic potential of bortezomib on renal allograft interstitial fibrosis. Such an effect may result from inhibition of NF-κB/TNF-α/Akt/mTOR/p70S6 kinase/Smurf2 signaling via stabilizing protein of inhibitor of NF-κB.

Role of the CTRP6/AMPK pathway in kidney fibrosis through the promotion of fatty acid oxidation.

CTRP6, a newly identified adiponectin analogue, has been shown to be involved in inflammation, diabetes and cardiovascular diseases. Recently, increasing evidence has shown that CTRP6 plays a critical role in fibrotic diseases, such as myocardial fibrosis and skin fibrosis. FAO, an important energy source for kidney proximal tubular cells, also participates in the process of fibrosis. Therefore, our study aimed to investigate the effect of CTRP6 on mediating FAO in kidney fibrosis and the underlying associated mechanism. Firstly, the activity of CTRP6 and the key enzymes of FAO (CPT1A, ACOX1) were tested in vivo and vitro. Next, the regulatory effect of CTRP6/AMPK on FAO was accessed in animal models and in cell lines. Additionally, we explored the effect of exogenous recombinant CTRP6 on renal tubular epithelial cell differentiation. Decreased CTRP6 and p-AMPK were detected in UUO-induced kidney fibrosis and in TGF-ß1-treated HK-2 cells. We also observed that defective FAO occurred during kidney fibrosis. Moreover, the human CTRP6 peptide could inhibit the ECM deposition and promote the phosphorylation of AMPK by promoting FAO. However, the inhibitory effects of CTRP6 on TGF-ß1-induced ECM deposition and the protective effects of CTRP6 on FAO could be abolished by compound C, a selective inhibitor of AMPK. Compound C also reversed the CTRP6-mediated upregulation of p-AMPK. The mediation of FAO by CTRP6 plays a key role in kidney fibrosis by regulating TGF-ß1-induced renal tubular epithelial cell differentiation by promoting FAO, which is mediated via AMPK activation.

Pharmacological inhibition of Vanin-1 is not protective in models of acute and chronic kidney disease.

Oxidative stress is a key concept in basic, translational, and clinical research to understand the pathophysiology of various disorders, including cardiovascular and renal diseases. Although attempts to directly reduce oxidative stress with redox-active substances have until now largely failed to prove clinical benefit, indirect approaches to combat oxidative stress enzymatically have gained further attention as potential therapeutic strategies. The pantetheinase Vanin-1 is expressed on kidney proximal tubular cells, and its reaction product cysteamine is described to negatively affect redox homeostasis by inhibiting the replenishment of cellular antioxidative glutathione stores. Vanin-1-deficient mice were shown to be protected against oxidative stress damage. The aim of this study was to elucidate whether pharmacological inhibition of Vanin-1 protects mice from oxidative stress-related acute or chronic kidney injury as well. By studying renal ischemia-reperfusion injury in Col4α3-/- (Alport syndrome) mice and in vitro hypoxia-reoxygenation in human proximal tubular cells we found that treatment with a selective and potent Vanin-1 inhibitor resulted in ample inhibition of enzymatic activity in vitro and in vivo. However, surrogate parameters of metabolic and redox homeostasis were only partially and insufficiently affected. Consequently, apoptosis and reactive oxygen species level in tubular cells as well as overall kidney function and fibrotic processes were not improved by Vanin-1 inhibition. We thus conclude that Vanin-1 functionality in the context of cardiovascular diseases needs further investigation and the biological relevance of pharmacological Vanin-1 inhibition for the treatment of kidney diseases remains to be proven.

Protein disulfide isomerase inhibition impairs Keap1/Nrf2 signaling and mitochondrial function and induces apoptosis in renal proximal tubular cells.

Renal proximal tubular apoptosis plays a critical role in kidney health and disease. However, cellular molecules that trigger renal apoptosis remain elusive. Here, we evaluated the effect of inhibiting protein disulfide isomerase (PDI), a critical thioredoxin chaperone protein, on apoptosis as well as the underlying mechanisms in human renal proximal tubular (HK2) cells. HK2 cells were transfected with PDI-specific siRNA in the absence and presence of an antioxidant, tempol. PDI siRNA transfection resulted in a decrease of ~70% in PDI protein expression and enzyme activity. PDI inhibition increased caspase-3 activity and induced profound cell apoptosis. Mitochondrial function, as assessed by mitochondrial cytochrome c levels, mitochondrial membrane potential, oxygen consumption, and ATP levels, was significantly reduced in PDI-inhibited cells. Also, PDI inhibition caused nuclear factor erythroid 2-related factor 2 (Nrf2; a redox-sensitive transcription factor) cytoplasmic sequestration, decreased superoxide dismutase and glutathione-S-transferase activities, and increased oxidative stress. In PDI-inhibited cells, tempol reduced apoptosis, caspase-3 activity, and oxidative stress and also restored Nrf2 nuclear translocation and mitochondrial function. Silencing Nrf2 in the cells abrogated the beneficial effect of tempol, whereas Kelch-like ECH-associated protein 1 (an Nrf2 regulatory protein) silencing protected cells from PDI inhibitory effects. Collectively, our data indicate that PDI inhibition diminishes Nrf2 nuclear translocation, causing oxidative stress that further triggers mitochondrial dysfunction and renal cell apoptosis. This study suggests an important role for PDI in renal cell apoptosis involving Nrf2 and mitochondrial dysfunction.

Management of a Preterm Infant with Renal Tubular Dysgenesis: A Case Report and Review of the Literature.

Renal tubular dysgenesis (RTD) is the absence or poor development of the renal proximal tubules caused by gene mutations in the renin-angiotensin system. Although RTD has been considered fatal, improving neonatal intensive care management has enhanced survival outcomes. However, little has been reported on the survival of extremely preterm infants. This study reports the survival of an extremely preterm infant with RTD and discusses the appropriate management of RTD by reviewing the literature. A female infant weighing 953 g was delivered at 27 weeks' gestation by Cesarean section because of oligohydramnios. She exhibited severe persistent pulmonary hypertension, severe systemic hypotension, and renal dysfunction shortly after birth. Respiratory management was successfully undertaken using nitric oxide inhalation and high-frequency oscillatory ventilation. Desmopressin was effective in maintaining her blood pressure and urinary output. She was diagnosed with RTD based on genetic testing, which revealed a compound heterozygous mutation in the angiotensin-converting enzyme gene in exon 18 (c.2689delC; p.Pro897fs) and exon 20 (c.3095dupT; p.Leu1032fs). At 2 years, she started receiving oral fludrocortisone for treating persistently high serum creatinine levels, which was attributed to nephrogenic diabetes insipidus caused by RTD. Subsequently, her urine output decreased, and renal function was successfully maintained. Currently, there is no established treatment for RTD. Considering cases reported to date, treatment with vasopressin and fludrocortisone appears to be most effective for survival and maintenance of renal function in patients with RTD. This study presents the successful management of RTD using this strategy in an extremely preterm infant.

Acid Loading Unmasks Glucose Homeostatic Instability in Proximal-Tubule-Targeted Insulin/Insulin-Like-Growth-Factor-1 Receptor Dual Knockout Mice.

BACKGROUND/AIMS: Metabolic syndrome and type 2 diabetes are associated with some degree of acidosis. Acidosis has also been shown to upregulate renal gluconeogenesis. Whether impaired insulin or insulin-like-growth factor 1 receptor (IGF1) signaling alter this relationship is not known. Our aim was to determine the effects of deletion of insulin and IGF1 receptors (Insr and Igf1r) from renal proximal tubule (PT) on the gluconeogenic response to acidosis. METHODS: We developed a mouse model with PT-targeted dual knockout (KO) of the Insr/Igf1r by driving Cre-recombinase with the gamma-glutamyl transferase (gGT) promoter. Male and female mice were maintained as control or acidotic by treatment with NH4Cl in the drinking water for 1-week. RESULTS: Acidosis in both genotypes increased renal expression of phosphoenolpyruvate carboxykinase (PEPCK) and fructose-1-bisphosphatase (FBP1), but not glucose-6-phosphatase catalytic subunit (G6PC), which showed significantly lower expression in the KO regardless of treatment. Several differences between KO and WT suggested a protective role for insulin/IGF1 receptor signaling in maintaining relative euglycemia in the face of acidosis. First, the increase in FBP1 with acid was greater in the KO (significant interactive term). Secondly, proximal-tubule-associated FOXO1 and AKT overall protein levels were suppressed by acid loading in the KO, but not in the WT. Robust intact insulin signaling would be needed to reduce gluconeogenesis in PT. Third, phosphorylated FOXO1 (pS256) levels were markedly reduced by acid loading in the KO PT, but not in the WT. This reduction would support greater gluconeogenesis. Fourth, the sodium-glucose cotransporter (SGLT1) was increased by acid loading in the KO kidney, but not the WT. While this would not necessarily affect gluconeogenesis, it could result in increased circulatory glucose via renal reabsorption. Reduced susceptibility to glucose-homeostatic dysregulation in the WT could potentially relate to the sharp (over 50%) reduction in renal levels of sirtuin-1 (SIRT1), which deacetylates and regulates transcription of a number of genes. This reduction was absent in the KO. CONCLUSION: Insulin resistance of the kidney may increase whole-body glucose instability a major risk factor for morbidity in diabetes. High dietary acid loads provide a dilemma for the kidney, as ammoniagenesis liberates α-ketoglutarate, which is a substrate for gluconeogenesis. We demonstrate an important role for insulin and/or IGF1 receptor signaling in the PT to facilitate this process and reduce excursions in blood glucose. Thus, medications and lifestyle changes that improve renal insulin sensitivity may also provide added benefit in type 2 diabetes especially when coupled with metabolic acidosis.

Multiparametric assessment of mitochondrial respiratory inhibition in HepG2 and RPTEC/TERT1 cells using a panel of mitochondrial targeting agrochemicals.

Evidence is mounting for the central role of mitochondrial dysfunction in several pathologies including metabolic diseases, accelerated ageing, neurodegenerative diseases and in certain xenobiotic-induced organ toxicity. Assessing mitochondrial perturbations is not trivial and the outcomes of such investigations are dependent on the cell types used and assays employed. Here we systematically investigated the effect of electron transport chain (ETC) inhibitors on multiple mitochondrial-related parameters in two human cell types, HepG2 and RPTEC/TERT1. Cells were exposed to a broad range of concentrations of 20 ETC-inhibiting agrochemicals and capsaicin, consisting of inhibitors of NADH dehydrogenase (Complex I, CI), succinate dehydrogenase (Complex II, CII) and cytochrome bc1 complex (Complex III, CIII). A battery of tests was utilised, including viability assays, lactate production, mitochondrial membrane potential (MMP) and the Seahorse bioanalyser, which simultaneously measures extracellular acidification rate [ECAR] and oxygen consumption rate [OCR]. CI inhibitors caused a potent decrease in OCR, decreased mitochondrial membrane potential, increased ECAR and increased lactate production in both cell types. Twenty-fourhour exposure to CI inhibitors decreased viability of RPTEC/TERT1 cells and 3D spheroid-cultured HepG2 cells in the presence of glucose. CI inhibitors decreased 2D HepG2 viability only in the absence of glucose. CII inhibitors had no notable effects in intact cells up to 10 µM. CIII inhibitors had similar effects to the CI inhibitors. Antimycin A was the most potent CIII inhibitor, with activity in the nanomolar range. The proposed CIII inhibitor cyazofamid demonstrated a mitochondrial uncoupling signal in both cell types. The study presents a comprehensive example of a mitochondrial assessment workflow and establishes measurable key events of ETC inhibition.

Dietary fructose enhances angiotensin II-stimulated Na+ transport via activation of PKC-α in renal proximal tubules.

Angiotensin II (ANG II) stimulates proximal nephron transport via activation of classical protein kinase C (PKC) isoforms. Acute fructose treatment stimulates PKC and dietary fructose enhances ANG II's ability to stimulate Na+ transport, but the mechanisms are unclear. We hypothesized that dietary fructose enhances ANG II's ability to stimulate renal proximal tubule Na+ reabsorption by augmenting PKC-α activation and increases in intracellular Ca2+. We measured total and isoform-specific PKC activity, basal and ANG II-stimulated oxygen consumption, a surrogate of Na+ reabsorption, and intracellular Ca2+ in proximal tubules from rats given either 20% fructose in their drinking water (fructose group) or tap water (control group). Total PKC activity was measured by ELISA. PKC-α, PKC-ß, and PKC-γ activities were assessed by measuring particulate-to-soluble ratios. Intracelluar Ca2+ was measured using fura 2. ANG II stimulated total PKC activity by 53 ± 15% in the fructose group but not in the control group (-15 ± 11%, P < 0.002). ANG II stimulated PKC-α by 0.134 ± 0.026 but not in the control group (-0.002 ± 0.020, P < 0.002). ANG II increased PKC-γ activity by 0.008 ± 0.003 in the fructose group but not in the control group (P < 0.046). ANG II did not stimulate PKC-ß in either group. ANG II increased Na+ transport by 454 ± 87 nmol·min-1·mg protein-1 in fructose group, and the PKC-α/ß inhibitor Gö6976 blocked this increase (-96 ± 205 nmol·min-1·mg protein-1, P < 0.045). ANG II increased intracellular Ca2+ by 148 ± 53 nM in the fructose group but only by 43 ± 10 nM in the control group (P < 0.035). The intracellular Ca2+ chelator BAPTA blocked the ANG II-induced increase in Na+ transport in the fructose group. We concluded that dietary fructose enhances ANG II's ability to stimulate renal proximal tubule Na+ reabsorption by augmenting PKC-α activation via elevated increases in intacellular Ca2+.

Role of SET7/9 in the progression of ischemic renal injury in diabetic and non-diabetic rats.

SET domain with lysine methyltransferase 7/9 (Set7/9), a histone lysine methyltransferase (HMT), recently suggested to exert a critical role among kidney disorders, whereas its role in diabetes associated IRI co-morbidity remains complete elusive. The present study aimed to understand the role of SET7/9 and histone methylation in regulation of inflammatory signaling under IRI in diabetes mellitus and non-diabetic rats. Our results demonstrated that IRI caused renal dysfunction via increased blood urea nitrogen (BUN) levels in ND and DM rats. The NF-κB mediated inflammatory cascade like increased p-NF-κB, reduced IκBα levels followed by enhanced leukocyte infiltration as shown by increased MCP-1 expressions. IRI results in increased histone H3 methylation at lysine 4 and 36 (H3K4Me2, H3K36Me2), and decreased histone H3 methylation at lysine 9. Additionally, IRI increased the protein and mRNA expression of H3K4Me2 specific histone methyltransferase-SET7/9 in DM and ND rats. The abovementioned results remain prominent in DM rats compared to ND rats followed by IRI. Further, treatment with a novel SET7/9 inhibitor; cyproheptadine, significantly improved renal functioning via reducing the BUN levels in ND and DM rats. Hence, this study demonstrated the role of SET7/9 in mediating active transcription via H3K4Me2, ultimately regulated the NFκB-mediated inflammatory cascade. Therefore, SET7/9 can be explored as novel target for drug development against IRI under DM and ND conditions.

Protein Kinase C-δ Mediates Kidney Tubular Injury in Cold Storage-Associated Kidney Transplantation.

BACKGROUND: Kidney injury associated with cold storage is a determinant of delayed graft function and the long-term outcome of transplanted kidneys, but the underlying mechanism remains elusive. We previously reported a role of protein kinase C-δ (PKCδ) in renal tubular injury during cisplatin nephrotoxicity and albumin-associated kidney injury, but whether PKCδ is involved in ischemic or transplantation-associated kidney injury is unknown. METHODS: To investigate PKCδ's potential role in injury during cold storage-associated transplantation, we incubated rat kidney proximal tubule cells in University of Wisconsin (UW) solution at 4°C for cold storage, returning them to normal culture medium at 37°C for rewarming. We also stored kidneys from donor mice in cold UW solution for various durations, followed by transplantation into syngeneic recipient mice. RESULTS: We observed PKCδ activation in both in vitro and in vivo models of cold-storage rewarming or transplantation. In the mouse model, PKCδ was activated and accumulated in mitochondria, where it mediated phosphorylation of a mitochondrial fission protein, dynamin-related protein 1 (Drp1), at serine 616. Drp1 activation resulted in mitochondrial fission or fragmentation, accompanied by mitochondrial damage and tubular cell death. Deficiency of PKCδ in donor kidney ameliorated Drp1 phosphorylation, mitochondrial damage, tubular cell death, and kidney injury during cold storage-associated transplantation. PKCδ deficiency also improved the repair and function of the renal graft as a life-supporting kidney. An inhibitor of PKCδ, δV1-1, protected kidneys against cold storage-associated transplantation injury. CONCLUSIONS: These results indicate that PKCδ is a key mediator of mitochondrial damage and renal tubular injury in cold storage-associated transplantation and may be an effective therapeutic target for improving renal transplant outcomes.