Dietary oxalate and kidney stone formation.

Dietary oxalate is plant-derived and may be a component of vegetables, nuts, fruits, and grains. In normal individuals, approximately half of urinary oxalate is derived from the diet and half from endogenous synthesis. The amount of oxalate excreted in urine plays an important role in calcium oxalate stone formation. Large epidemiological cohort studies have demonstrated that urinary oxalate excretion is a continuous variable when indexed to stone risk. Thus, individuals with oxalate excretions >25 mg/day may benefit from a reduction of urinary oxalate output. The 24-h urine assessment may miss periods of transient surges in urinary oxalate excretion, which may promote stone growth and is a limitation of this analysis. In this review we describe the impact of dietary oxalate and its contribution to stone growth. To limit calcium oxalate stone growth, we advocate that patients maintain appropriate hydration, avoid oxalate-rich foods, and consume an adequate amount of calcium.

Hemoglobin-associated oxidative stress in the pericardial compartment of postoperative cardiac surgery patients.

Atherosclerosis and valvular heart disease often require treatment with corrective surgery to prevent future myocardial infarction, ischemic heart disease, and heart failure. Mechanisms underlying the development of the associated complications of surgery are multifactorial and have been linked to inflammation and oxidative stress, classically as measured in the blood or plasma of patients. Postoperative pericardial fluid (PO-PCF) has not been investigated in depth with respect to the potential to induce oxidative stress. This is important because cardiac surgery disrupts the integrity of the pericardial membrane surrounding the heart and causes significant alterations in the composition of the pericardial fluid (PCF). This includes contamination with hemolyzed blood and high concentrations of oxidized hemoglobin, which suggests that cardiac surgery results in oxidative stress within the pericardial space. Accordingly, we tested the hypothesis that PO-PCF is highly pro-oxidant and that the potential interaction between inflammatory cell-derived hydrogen peroxide with hemoglobin is associated with oxidative stress. Blood and PCF were collected from 31 patients at the time of surgery and postoperatively from 4 to 48 h after coronary artery bypass grafting, valve replacement, or valve repair (mitral or aortic). PO-PCF contained high concentrations of neutrophils and monocytes, which are capable of generating elevated amounts of superoxide and hydrogen peroxide through the oxidative burst. In addition, PO-PCF primed naive neutrophils resulting in an enhanced oxidative burst upon stimulation. The PO-PCF also contained increased concentrations of cell-free oxidized hemoglobin that was associated with elevated levels of F2α isoprostanes and prostaglandins, consistent with both oxidative stress and activation of cyclooxygenase. Lastly, protein analysis of the PO-PCF revealed evidence of protein thiol oxidation and protein carbonylation. We conclude that PO-PCF is highly pro-oxidant and speculate that it may contribute to the risk of postoperative complications.

The Bioenergetic Health Index: a new concept in mitochondrial translational research.

Bioenergetics has become central to our understanding of pathological mechanisms, the development of new therapeutic strategies and as a biomarker for disease progression in neurodegeneration, diabetes, cancer and cardiovascular disease. A key concept is that the mitochondrion can act as the 'canary in the coal mine' by serving as an early warning of bioenergetic crisis in patient populations. We propose that new clinical tests to monitor changes in bioenergetics in patient populations are needed to take advantage of the early and sensitive ability of bioenergetics to determine severity and progression in complex and multifactorial diseases. With the recent development of high-throughput assays to measure cellular energetic function in the small number of cells that can be isolated from human blood these clinical tests are now feasible. We have shown that the sequential addition of well-characterized inhibitors of oxidative phosphorylation allows a bioenergetic profile to be measured in cells isolated from normal or pathological samples. From these data we propose that a single value-the Bioenergetic Health Index (BHI)-can be calculated to represent the patient's composite mitochondrial profile for a selected cell type. In the present Hypothesis paper, we discuss how BHI could serve as a dynamic index of bioenergetic health and how it can be measured in platelets and leucocytes. We propose that, ultimately, BHI has the potential to be a new biomarker for assessing patient health with both prognostic and diagnostic value.

Bioenergetic profiles of peripheral mononuclear cells and systemic inflammation in women with Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS).

Inflammation is thought to contribute to the etiology of interstitial cystitis/bladder pain syndrome (IC/BPS). It is well-known that disruption in metabolism in immune cells contributes to inflammation in several inflammatory diseases. The purpose of this study was to investigate whether cellular bioenergetics is altered in monocytes and lymphocytes from women with IC/BPS, and if these alterations correlate with systemic inflammatory markers. Age and BMI matched adult healthy women (HS; n = 18) and women with IC/BPS (n = 18) were included in the study. Blood was collected to assess cellular bioenergetics in monocytes and lymphocytes using a Seahorse XF96 Analyzer and plasma cytokine levels were measured using Meso Scale Discovery immunoassays. The correlation between bioenergetic parameters, cytokines, and demographics was determined using Pearson correlation coefficients. Means of the two groups were compared using the two-group t-test. Patients with IC/BPS had reduced monocyte oxygen consumption rates and glycolytic rates compared to healthy subjects. In contrast, lymphocytes from these patients had increased oxygen consumption rates and glycolytic rates. Several cytokines and chemokines including Interferon-gamma (IFN-É£), tumor necrosis factor alpha (TNF-ɑ), Interleukin-6 (IL-6), Interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF) levels were significantly elevated in the plasma of patients with IC/BPS. However, Transforming growth factor (TGF-ß) and Interleukin-10 (IL-10) levels were significantly decreased in IC/BPS patients compared to HS. In addition, Interferon gamma (IFN-É£), TNF-ɑ, IL-8, and TGF-ß levels correlated with several bioenergetic parameters in monocytes or lymphocytes from healthy subjects. In contrast, TNF-ɑ and IL-8 correlated with bioenergetic parameters in monocytes from IC/BPS patients. Monocyte and lymphocyte cellular bioenergetics and plasma cytokine levels are different in patients with IC/PBS compared to HS. It appears that systemic inflammation is greater in this cohort which may negatively impact immune cell function. The relationship between cellular bioenergetics and inflammation in monocytes and lymphocytes could be important in understanding the pathogenesis of IC/PBS and warrants further investigation.

Dysfunctional mitochondrial bioenergetics and oxidative stress in Akita(+/Ins2)-derived ß-cells.

Insulin release from pancreatic ß-cells plays a critical role in blood glucose homeostasis, and ß-cell dysfunction leads to the development of diabetes mellitus. In cases of monogenic type 1 diabetes mellitus (T1DM) that involve mutations in the insulin gene, we hypothesized that misfolding of insulin could result in endoplasmic reticulum (ER) stress, oxidant production, and mitochondrial damage. To address this, we used the Akita(+/Ins2) T1DM model in which misfolding of the insulin 2 gene leads to ER stress-mediated ß-cell death and thapsigargin to induce ER stress in two different ß-cell lines and in intact mouse islets. Using transformed pancreatic ß-cell lines generated from wild-type Ins2(+/+) (WT) and Akita(+/Ins2) mice, we evaluated cellular bioenergetics, oxidative stress, mitochondrial protein levels, and autophagic flux to determine whether changes in these processes contribute to ß-cell dysfunction. In addition, we induced ER stress pharmacologically using thapsigargin in WT ß-cells, INS-1 cells, and intact mouse islets to examine the effects of ER stress on mitochondrial function. Our data reveal that Akita(+/Ins2)-derived ß-cells have increased mitochondrial dysfunction, oxidant production, mtDNA damage, and alterations in mitochondrial protein levels that are not corrected by autophagy. Together, these findings suggest that deterioration in mitochondrial function due to an oxidative environment and ER stress contributes to ß-cell dysfunction and could contribute to T1DM in which mutations in insulin occur.

Convergent mechanisms for dysregulation of mitochondrial quality control in metabolic disease: implications for mitochondrial therapeutics.

Mitochondrial dysfunction is associated with a broad range of pathologies including diabetes, ethanol toxicity, metabolic syndrome and cardiac failure. It is now becoming clear that maintaining mitochondrial quality through a balance between biogenesis, reserve capacity and mitophagy is critical in determining the response to metabolic or xenobiotic stress. In diseases associated with metabolic stress, such as Type II diabetes and non-alcoholic and alcoholic steatosis, the mitochondria are subjected to multiple 'hits' such as hypoxia and oxidative and nitrative stress, which can overwhelm the mitochondrial quality control pathways. In addition, the underlying mitochondrial genetics that evolved to accommodate high-energy demand, low-calorie supply environments may now be maladapted to modern lifestyles (low-energy demand, high-calorie environments). The pro-oxidant and pro-inflammatory environment of a sedentary western lifestyle has been associated with modified redox cell signalling pathways such as steatosis, hypoxic signalling, inflammation and fibrosis. These data suggest that loss of mitochondrial quality control is intimately associated with the aberrant activation of redox cell signalling pathways under pathological conditions. In the present short review, we discuss evidence from alcoholic liver disease supporting this concept, the insights obtained from experimental models and the application of bioenergetic-based therapeutics in the context of maintaining mitochondrial quality.

Oxalate disrupts monocyte and macrophage cellular function via Interleukin-10 and mitochondrial reactive oxygen species (ROS) signaling.

Oxalate is a small compound found in certain plant-derived foods and is a major component of calcium oxalate (CaOx) kidney stones. Individuals that consume oxalate enriched meals have an increased risk of forming urinary crystals, which are precursors to CaOx kidney stones. We previously reported that a single dietary oxalate load induces nanocrystalluria and reduces monocyte cellular bioenergetics in healthy adults. The purpose of this study was to extend these investigations to identify specific oxalate-mediated mechanisms in monocytes and macrophages. We performed RNA-Sequencing analysis on monocytes isolated from healthy subjects exposed to a high oxalate (8 mmol) dietary load. RNA-sequencing revealed 1,198 genes were altered and Ingenuity Pathway Analysis demonstrated modifications in several pathways including Interleukin-10 (IL-10) anti-inflammatory cytokine signaling, mitochondrial metabolism and function, oxalic acid downstream signaling, and autophagy. Based on these findings, we hypothesized that oxalate induces mitochondrial and lysosomal dysfunction in monocytes and macrophages via IL-10 and reactive oxygen species (ROS) signaling which can be reversed with exogenous IL-10 or Mitoquinone (MitoQ; a mitochondrial targeted antioxidant). We exposed monocytes and macrophages to oxalate in an in-vitro setting which caused oxidative stress, a decline in IL-10 cytokine levels, mitochondrial and lysosomal dysfunction, and impaired autophagy in both cell types. Administration of exogenous IL-10 and MitoQ attenuated these responses. These findings suggest that oxalate impairs metabolism and immune response via IL-10 signaling and mitochondrial ROS generation in both monocytes and macrophages which can be potentially limited or reversed. Future studies will examine the benefits of these therapies on CaOx crystal formation and growth in vivo.

Hydroxyproline stimulates inflammation and reprograms macrophage signaling in a rat kidney stone model.

Meals rich in oxalate are associated with calcium oxalate (CaOx) kidney stone disease. Hydroxy-L-proline (HLP) is an oxalate precursor found in milk and collagen-containing foods. HLP has been shown to induce CaOx crystal formation in rodents. The purpose of this study was to evaluate the effect of HLP induced oxalate levels on inflammation and renal leukocytes during crystal formation. Male Sprague-Dawley rats (6-8 weeks old) were fed a control diet containing no oxalate for 3 days before being randomized to continue the control diet or 5% HLP for up to 28 days. Blood, 24 h urine, and kidneys were collected on Days 0, 7, 14, or 28. Urinary oxalate levels, crystal deposition, and renal macrophage markers were evaluated using ion chromatography-mass spectrometry, immunohistochemistry, and qRT-PCR. Renal leukocytes were assessed using flow cytometry and RNA-sequencing. HLP feeding increased urinary oxalate levels and renal crystal formation in animals within 7 days. HLP also increased renal macrophage populations on Days 14 and 28. Transcriptome analysis revealed that renal macrophages from animals fed HLP for 7 days were involved in inflammatory response and disease, stress response to LPS, oxidative stress, and immune cell trafficking. Renal macrophages isolated on Day 14 were involved in cell-mediated immunological pathways, ion homeostasis, and inflammatory response. Collectively, these findings suggest that HLP-mediated oxalate levels induce markers of inflammation, leukocyte populations, and reprograms signaling pathways in macrophages in a time-dependent manner. Additional studies investigating the significance of oxalate on renal macrophages could aid in our understanding of kidney stone formation.

Acclimation to a High-Salt Diet Is Sex Dependent.

Background Premenopausal women are less likely to develop hypertension and salt-related complications than are men, yet the impact of sex on mechanisms regulating Na+ homeostasis during dietary salt challenges is poorly defined. Here, we determined whether female rats have a more efficient capacity to acclimate to increased dietary salt intake challenge. Methods and Results Age-matched male and female Sprague Dawley rats maintained on a normal-salt (NS) diet (0.49% NaCl) were challenged with a 5-day high-salt diet (4.0% NaCl). We assessed serum, urinary, skin, and muscle electrolytes; total body water; and kidney Na+ transporters during the NS and high-salt diet phases. During the 5-day high-salt challenge, natriuresis increased more rapidly in females, whereas serum Na+ and body water concentration increased only in males. To determine if females are primed to handle changes in dietary salt, we asked the question whether the renal endothelin-1 natriuretic system is more active in female rats, compared with males. During the NS diet, female rats had a higher urinary endothelin-1 excretion rate than males. Moreover, Ingenuity Pathway Analysis of RNA sequencing data identified the enrichment of endothelin signaling pathway transcripts in the inner medulla of kidneys from NS-fed female rats compared with male counterparts. Notably, in human subjects who consumed an Na+-controlled diet (3314-3668 mg/day) for 3 days, women had a higher urinary endothelin-1 excretion rate than men, consistent with our findings in NS-fed rats. Conclusions These results suggest that female sex confers a greater ability to maintain Na+ homeostasis during acclimation to dietary Na+ challenges and indicate that the intrarenal endothelin-1 natriuretic pathway is enhanced in women.

The mitochondria-targeted antioxidant mitoquinone protects against cold storage injury of renal tubular cells and rat kidneys.

The majority of kidneys used for transplantation are obtained from deceased donors. These kidneys must undergo cold preservation/storage before transplantation to preserve tissue quality and allow time for recipient selection and transport. However, cold storage (CS) can result in tissue injury, kidney discardment, or long-term renal dysfunction after transplantation. We have previously determined mitochondrial superoxide and other downstream oxidants to be important signaling molecules that contribute to CS plus rewarming (RW) injury of rat renal proximal tubular cells. Thus, this study's purpose was to determine whether adding mitoquinone (MitoQ), a mitochondria-targeted antioxidant, to University of Wisconsin (UW) preservation solution could offer protection against CS injury. CS was initiated by placing renal cells or isolated rat kidneys in UW solution alone (4 h at 4°C) or UW solution containing MitoQ or its control compound, decyltriphenylphosphonium bromide (DecylTPP) (1 µM in vitro; 100 µM ex vivo). Oxidant production, mitochondrial function, cell viability, and alterations in renal morphology were assessed after CS exposure. CS induced a 2- to 3-fold increase in mitochondrial superoxide generation and tyrosine nitration, partial inactivation of mitochondrial complexes, and a significant increase in cell death and/or renal damage. MitoQ treatment decreased oxidant production ~2-fold, completely prevented mitochondrial dysfunction, and significantly improved cell viability and/or renal morphology, whereas DecylTPP treatment did not offer any protection. These findings implicate that MitoQ could potentially be of therapeutic use for reducing organ preservation damage and kidney discardment and/or possibly improving renal function after transplantation.

Oxalate Alters Cellular Bioenergetics, Redox Homeostasis, Antibacterial Response, and Immune Response in Macrophages.

Individuals with calcium oxalate (CaOx) kidney stones can have secondarily infected calculi which may play a role in the development of recurrent urinary tract infection (UTI). Uropathogenic Escherichia coli (UPEC) is the most common causative pathogen of UTIs. Macrophages play a critical role in host immune defense against bacterial infections. Our previous study demonstrated that oxalate, an important component of the most common type of kidney stone, impairs monocyte cellular bioenergetics and redox homeostasis. The objective of this study was to investigate whether oxalate compromises macrophage metabolism, redox status, anti-bacterial response, and immune response. Monocytes (THP-1, a human monocytic cell line) were exposed to sodium oxalate (soluble oxalate; 50 µM) for 48 hours prior to being differentiated into macrophages. Macrophages were subsequently exposed to calcium oxalate crystals (50 µM) for 48 hours followed by UPEC (MOI 1:2 or 1:5) for 2 hours. Peritoneal macrophages and bone marrow-derived macrophages (BMDM) from C57BL/6 mice were also exposed to oxalate. THP-1 macrophages treated with oxalate had decreased cellular bioenergetics, mitochondrial complex I and IV activity, and ATP levels compared to control cells. In addition, these cells had a significant increase in mitochondrial and total reactive oxygen species levels, mitochondrial gene expression, and pro-inflammatory cytokine (i.e. Interleukin-1ß, IL-1ß and Interleukin-6, IL-6) mRNA levels and secretion. In contrast, oxalate significantly decreased the mRNA levels and secretion of the anti-inflammatory cytokine, Interleukin-10 (IL-10). Further, oxalate increased the bacterial burden of primary macrophages. Our findings demonstrate that oxalate compromises macrophage metabolism, redox homeostasis, and cytokine signaling leading to a reduction in anti-bacterial response and increased infection. These data highlight a novel role of oxalate on macrophage function.

Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis.

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) crystals. Crystalluria occurs when urine becomes supersaturated with minerals (e.g., calcium, oxalate, phosphate) and precedes kidney stone formation. Standard methods to assess crystalluria in stone formers include microscopy, filtration, and centrifugation. However, these methods primarily detect microcrystals and not nanocrystals. Nanocrystals have been suggested to be more harmful to kidney epithelial cells than microcrystals in vitro. Here, we describe the ability of Nanoparticle Tracking analysis (NTA) to detect human urinary nanocrystals. Healthy adults were fed a controlled oxalate diet prior to drinking an oxalate load to stimulate urinary nanocrystals. Urine was collected for 24 hours before and after the oxalate load. Samples were processed and washed with ethanol to purify samples. Urinary nanocrystals were stained with the calcium binding fluorophore, Fluo-4 AM. After staining, the size and count of nanocrystals were determined using NTA. The findings from this study show NTA can efficiently detect nanocrystalluria in healthy adults. These findings suggest NTA could be a valuable early detection method of nanocrystalluria in patients with kidney stone disease.

Dietary Oxalate Loading Impacts Monocyte Metabolism and Inflammatory Signaling in Humans.

Diet has been associated with several metabolic diseases and may impact immunity. Increased consumption of meals with high oxalate content may stimulate urinary calcium oxalate (CaOx) crystals, which are precursors to CaOx kidney stones. We previously reported that CaOx stone formers have decreased monocyte cellular bioenergetics compared to healthy participants and oxalate reduces monocyte metabolism and redox status in vitro. The purpose of this study was to investigate whether dietary oxalate loading impacts monocyte cellular bioenergetics, mitochondrial complex activity, and inflammatory signaling in humans. Healthy participants (n = 40; 31.1 ± 1.3 years) with a BMI of 24.9 ± 0.6 kg/m2 consumed a controlled low oxalate diet for 3 days before drinking a blended preparation of fruits and vegetables containing a large amount of oxalate. Blood and urine were collected before (pre-oxalate) and for 5 h after the oxalate load to assess urinary oxalate levels, monocyte cellular bioenergetics and mitochondrial complex activity, and plasma cytokine/chemokine levels. Urinary oxalate levels significantly increased in post-oxalate samples compared to pre-oxalate samples. Monocyte cellular bioenergetics, mitochondrial complex I activity, and plasma cytokine and chemokine levels were altered to varying degrees within the study cohort. We demonstrate for the first time that dietary oxalate loading may impact monocyte metabolism and immune response in a cohort of healthy adults, but these response are variable. Further studies are warranted to understand oxalate mediated mechanisms on circulating monocytes and how this potentially influences CaOx kidney stone formation. Clinical Trial Registration: ClinicalTrials.gov, identifier NCT03877276.

The TGF-ß/HDAC7 axis suppresses TCA cycle metabolism in renal cancer.

Mounting evidence points to alterations in mitochondrial metabolism in renal cell carcinoma (RCC). However, the mechanisms that regulate the TCA cycle in RCC remain uncharacterized. Here, we demonstrate that loss of TCA cycle enzyme expression is retained in RCC metastatic tissues. Moreover, proteomic analysis demonstrates that reduced TCA cycle enzyme expression is far more pronounced in RCC relative to other tumor types. Loss of TCA cycle enzyme expression is correlated with reduced expression of the transcription factor PGC-1α, which is also lost in RCC tissues. PGC-1α reexpression in RCC cells restores the expression of TCA cycle enzymes in vitro and in vivo and leads to enhanced glucose carbon incorporation into TCA cycle intermediates. Mechanistically, TGF-ß signaling, in concert with histone deacetylase 7 (HDAC7), suppresses TCA cycle enzyme expression. Our studies show that pharmacologic inhibition of TGF-ß restores the expression of TCA cycle enzymes and suppresses tumor growth in an orthotopic model of RCC. Taken together, this investigation reveals a potentially novel role for the TGF-ß/HDAC7 axis in global suppression of TCA cycle enzymes in RCC and provides insight into the molecular basis of altered mitochondrial metabolism in this malignancy.

Sex differences in redox homeostasis in renal disease.

Sex differences in redox signaling in the kidney present new challenges and opportunities for understanding the physiology and pathophysiology of the kidney. This review will focus on reactive oxygen species, immune-related signaling pathways and endothelin-1 as potential mediators of sex-differences in redox homeostasis in the kidney. Additionally, this review will highlight male-female differences in redox signaling in several major cardiovascular and renal disorders namely acute kidney injury, diabetic nephropathy, kidney stone disease and salt-sensitive hypertension. Furthermore, we will discuss the contribution of redox signaling in the pathogenesis of postmenopausal hypertension and preeclampsia.

Dietary Oxalate Induces Urinary Nanocrystals in Humans.

INTRODUCTION: Crystalluria is thought to be associated with kidney stone formation and can occur when urine becomes supersaturated with calcium, oxalate, and phosphate. The principal method used to identify urinary crystals is microscopy, with or without a polarized light source. This method can detect crystals above 1 µm in diameter (microcrystals). However, analyses of calcium oxalate kidney stones have indicated that crystallite components in these calculi are 50-100 nm in diameter. Recent studies have suggested that nanocrystals (<200 nm) elicit more injury to renal cells compared to microcrystals. The purpose of this study was to determine whether (i) urinary nanocrystals can be detected and quantified by nanoparticle tracking analysis (NTA, a high-resolution imaging technology), (ii) early-void urine samples from healthy subjects contain calcium nanocrystals, and (iii) a dietary oxalate load increases urinary nanocrystal formation. METHODS: Healthy subjects consumed a controlled low-oxalate diet for 3 days before a dietary oxalate load. Urinary crystals were isolated by centrifugation and assessed using NTA before and 5 hours after the oxalate load. The morphology and chemical composition of crystals was assessed using electron microscopy, Fourier-transform infrared spectroscopy (FTIR), and ion chromatography-mass spectrometry (IC-MS). RESULTS: Urinary calcium oxalate nanocrystals were detected in pre-load samples and increased substantially following the oxalate load. CONCLUSION: These findings indicate that NTA can quantify urinary nanocrystals and that meals rich in oxalate can promote nanocrystalluria. NTA should provide valuable insight about the role of nanocrystals in kidney stone formation.

Contribution of Dietary Oxalate and Oxalate Precursors to Urinary Oxalate Excretion.

Kidney stone disease is increasing in prevalence, and the most common stone composition is calcium oxalate. Dietary oxalate intake and endogenous production of oxalate are important in the pathophysiology of calcium oxalate stone disease. The impact of dietary oxalate intake on urinary oxalate excretion and kidney stone disease risk has been assessed through large cohort studies as well as smaller studies with dietary control. Net gastrointestinal oxalate absorption influences urinary oxalate excretion. Oxalate-degrading bacteria in the gut microbiome, especially Oxalobacter formigenes, may mitigate stone risk through reducing net oxalate absorption. Ascorbic acid (vitamin C) is the main dietary precursor for endogenous production of oxalate with several other compounds playing a lesser role. Renal handling of oxalate and, potentially, renal synthesis of oxalate may contribute to stone formation. In this review, we discuss dietary oxalate and precursors of oxalate, their pertinent physiology in humans, and what is known about their role in kidney stone disease.

PRDM16 suppresses HIF-targeted gene expression in kidney cancer.

Analysis of transcriptomic data demonstrates extensive epigenetic gene silencing of the transcription factor PRDM16 in renal cancer. We show that restoration of PRDM16 in RCC cells suppresses in vivo tumor growth. RNaseq analysis reveals that PRDM16 imparts a predominantly repressive effect on the RCC transcriptome including suppression of the gene encoding semaphorin 5B (SEMA5B). SEMA5B is a HIF target gene highly expressed in RCC that promotes in vivo tumor growth. Functional studies demonstrate that PRDM16's repressive properties, mediated by physical interaction with the transcriptional corepressors C-terminal binding proteins (CtBP1/2), are required for suppression of both SEMA5B expression and in vivo tumor growth. Finally, we show that reconstitution of RCC cells with a PRDM16 mutant unable to bind CtBPs nullifies PRDM16's effects on both SEMA5B repression and tumor growth suppression. Collectively, our data uncover a novel epigenetic basis by which HIF target gene expression is amplified in kidney cancer and a new mechanism by which PRDM16 exerts its tumor suppressive effects.

Alteration of renal respiratory Complex-III during experimental type-1 diabetes.

BACKGROUND: Diabetes has become the single most common cause for end-stage renal disease in the United States. It has been established that mitochondrial damage occurs during diabetes; however, little is known about what initiates mitochondrial injury and oxidant production during the early stages of diabetes. Inactivation of mitochondrial respiratory complexes or alteration of their critical subunits can lead to generation of mitochondrial oxidants, mitochondrial damage, and organ injury. Thus, one goal of this study was to determine the status of mitochondrial respiratory complexes in the rat kidney during the early stages of diabetes (5-weeks post streptozotocin injection). METHODS: Mitochondrial complex activity assays, blue native gel electrophoresis (BN-PAGE), Complex III immunoprecipitation, and an ATP assay were performed to examine the effects of diabetes on the status of respiratory complexes and energy levels in renal mitochondria. Creatinine clearance and urine albumin excretion were measured to assess the status of renal function in our model. RESULTS: Interestingly, of all four respiratory complexes only cytochrome c reductase (Complex-III) activity was significantly decreased, whereas two Complex III subunits, Core 2 protein and Rieske protein, were up regulated in the diabetic renal mitochondria. The BN-PAGE data suggested that Complex III failed to assemble correctly, which could also explain the compensatory upregulation of specific Complex III subunits. In addition, the renal F0F1-ATPase activity and ATP levels were increased during diabetes. CONCLUSION: In summary, these findings show for the first time that early (and selective) inactivation of Complex-III may contribute to the mitochondrial oxidant production which occurs in the early stages of diabetes.