Gut Dysbiosis in Experimental Kidney Disease: A Meta-Analysis of Rodent Repository Data.

SIGNIFICANCE STATEMENT: Alterations in gut microbiota contribute to the pathophysiology of a diverse range of diseases, leading to suggestions that chronic uremia may cause intestinal dysbiosis that contributes to the pathophysiology of CKD. Various small, single-cohort rodent studies have supported this hypothesis. In this meta-analysis of publicly available repository data from studies of models of kidney disease in rodents, cohort variation far outweighed any effect of experimental kidney disease on the gut microbiota. No reproducible changes in animals with kidney disease were seen across all cohorts, although a few trends observed in most experiments may be attributable to kidney disease. The findings suggest that rodent studies do not provide evidence for the existence of "uremic dysbiosis" and that single-cohort studies are unsuitable for producing generalizable results in microbiome research. BACKGROUND: Rodent studies have popularized the notion that uremia may induce pathological changes in the gut microbiota that contribute to kidney disease progression. Although single-cohort rodent studies have yielded insights into host-microbiota relationships in various disease processes, their relevance is limited by cohort and other effects. We previously reported finding metabolomic evidence that batch-to-batch variations in the microbiome of experimental animals are significant confounders in an experimental study. METHODS: To attempt to identify common microbial signatures that transcend batch variability and that may be attributed to the effect of kidney disease, we downloaded all data describing the molecular characterization of the gut microbiota in rodents with and without experimental kidney disease from two online repositories comprising 127 rodents across ten experimental cohorts. We reanalyzed these data using the DADA2 and Phyloseq packages in R, a statistical computing and graphics system, and analyzed data both in a combined dataset of all samples and at the level of individual experimental cohorts. RESULTS: Cohort effects accounted for 69% of total sample variance ( P <0.001), substantially outweighing the effect of kidney disease (1.9% of variance, P =0.026). We found no universal trends in microbial population dynamics in animals with kidney disease, but observed some differences (increased alpha diversity, a measure of within-sample bacterial diversity; relative decreases in Lachnospiraceae and Lactobacillus ; and increases in some Clostridia and opportunistic taxa) in many cohorts that might represent effects of kidney disease on the gut microbiota . CONCLUSIONS: These findings suggest that current evidence that kidney disease causes reproducible patterns of dysbiosis is inadequate. We advocate meta-analysis of repository data as a way of identifying broad themes that transcend experimental variation.

Impact of metabolic disorders on the structural, functional, and immunological integrity of the blood-brain barrier: Therapeutic avenues.

Mounting evidence has linked the metabolic disease to neurovascular disorders and cognitive decline. Using a murine model of a high-fat high-sugar diet mimicking obesity-induced type 2 diabetes mellitus (T2DM) in humans, we show that pro-inflammatory mediators and altered immune responses damage the blood-brain barrier (BBB) structure, triggering a proinflammatory metabolic phenotype. We find that disruption to tight junctions and basal lamina due to loss of control in the production of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) causes BBB impairment. Together the disruption to the structural and functional integrity of the BBB results in enhanced transmigration of leukocytes across the BBB that could contribute to an initiation of a neuroinflammatory response through activation of microglia. Using a humanized in vitro model of the BBB and T2DM patient post-mortem brains, we show the translatable applicability of our results. We find a leaky BBB phenotype in T2DM patients can be attributed to a loss of junctional proteins through changes in inflammatory mediators and MMP/TIMP levels, resulting in increased leukocyte extravasation into the brain parenchyma. We further investigated therapeutic avenues to reduce and restore the BBB damage caused by HFHS-feeding. Pharmacological treatment with recombinant annexin A1 (hrANXA1) or reversion from a high-fat high-sugar diet to a control chow diet (dietary intervention), attenuated T2DM development, reduced inflammation, and restored BBB integrity in the animals. Given the rising incidence of diabetes worldwide, understanding metabolic-disease-associated brain microvessel damage is vital and the proposed therapeutic avenues could help alleviate the burden of these diseases.

Uninephrectomy and class II PI3K-C2ß inactivation synergistically protect against obesity, insulin resistance and liver steatosis in mice.

Uninephrectomy (UNx) in living kidney donors for transplantation is now routine clinical practice. While chronic kidney disease, due to bilateral kidney dysfunction, is associated with insulin resistance, liver steatosis, and type 2 diabetes, the metabolic impact of UNx remains unclear. To better understand the crosstalk between the kidney and insulin target tissues, we studied the metabolic consequences of UNx and the potential involvement of class II PI3K-C2ß, the inactivation of which has been reported to result in insulin sensitization. Mice underwent UNx or sham operation followed by either normal chow or high-fat diet (HFD). Seventeen weeks post-UNx, mice showed improved glucose tolerance, insulin sensitivity, and decreased HFD-induced liver steatosis. This was associated with an enhanced serum FGF21 and insulin-stimulated Akt signaling in the liver and muscle of both lean and obese mice. Remarkably, the combination of UNx and PI3K-C2ß inactivation protected against HFD-induced obesity and further potentiated the metabolic improvement observed in WT UNx mice correlating with a synergistic increase in metabolic tissues of (1) insulin-stimulated Akt signaling (2) FGFR1 and ßKlotho expression. We demonstrated a potential beneficial effect of kidney donation and more effectively with PI3K-C2ß inactivation to protect against metabolic disorders through a mutual insulin/FGF21 sensitization.

The MEK Inhibitor Trametinib Ameliorates Kidney Fibrosis by Suppressing ERK1/2 and mTORC1 Signaling.

BACKGROUND: During kidney fibrosis, a hallmark and promoter of CKD (regardless of the underlying renal disorder leading to CKD), the extracellular-regulated kinase 1/2 (ERK1/2) pathway, is activated and has been implicated in the detrimental differentiation and expansion of kidney fibroblasts. An ERK1/2 pathway inhibitor, trametinib, is currently used in the treatment of melanoma, but its efficacy in the setting of CKD and renal fibrosis has not been explored. METHODS: We investigated whether trametinib has antifibrotic effects in two mouse models of renal fibrosis-mice subjected to unilateral ureteral obstruction (UUO) or fed an adenine-rich diet-as well as in cultured primary human fibroblasts. We also used immunoblot analysis, immunohistochemical staining, and other tools to study underlying molecular mechanisms for antifibrotic effects. RESULTS: Trametinib significantly attenuated collagen deposition and myofibroblast differentiation and expansion in UUO and adenine-fed mice. We also discovered that in injured kidneys, inhibition of the ERK1/2 pathway by trametinib ameliorated mammalian target of rapamycin complex 1 (mTORC1) activation, another key profibrotic signaling pathway. Trametinib also inhibited the ERK1/2 pathway in cultured primary human renal fibroblasts stimulated by application of TGF-ß1, the major profibrotic cytokine, thereby suppressing downstream mTORC1 pathway activation. Additionally, trametinib reduced the expression of myofibroblast marker α-smooth muscle actin and the proliferation of renal fibroblasts, corroborating our in vivo data. Crucially, trametinib also significantly ameliorated renal fibrosis progression when administered to animals subsequent to myofibroblast activation. CONCLUSIONS: Further study of trametinib as a potential candidate for the treatment of chronic renal fibrotic diseases of diverse etiologies is warranted.

A novel model of reno-cardiac syndrome in the C57BL/ 6 mouse strain.

BACKGROUND: The end stage renal disease population has a 20 fold higher incidence of cardiovascular mortality compared to the overall population. The development of reno-cardiac syndrome in these patients will result in cardiovascular events to be the cause of 50% of fatalities. There is therefore a need to research improved therapeutic strategies to combat renal cardiac pathologies. Murine in vivo models contribute greatly to such research allowing for specific genetic modification and reduced miscellany, however there is currently no reliable model of reno-cardiac syndrome in the most common genetically modified mouse strain, the C57BL/6. In this study we have manipulated an established model of chronic renal disease using adenine infused diet and prolonged the course of its pathology achieving chronic renal failure and subsequent reno-cardiac syndrome in the C57BL/6 mouse. METHODS: Eight week-old male C57BL/ 6 mice were acclimatised for 7 days before administration of a 0.15% adenine diet or control diet for 20 weeks. Cardiac function was assessed in mice at week 20 by echocardiography. At experiment termination blood and urine samples were analysed biochemically and organ dysfunction/injury was determined using immunoblotting and immunohistochemistry. RESULTS: Administration of 0.15% adenine diet caused progressive renal failure resulting in reno-cardiac syndrome. At endpoint uraemia was confirmed by blood biochemistry which in the adenine fed mice showed significant increases in serum creatinine, urea, calcium (P < 0.0001) potassium (P < 0.05), and a significantly reduced glomerular filtration rate (P < 0.05). Reno-cardiac syndrome was confirmed by a significantly increased heart to body weight ratio (P < 0.0001) and echocardiography which showed significant reductions in percentage of ejection fraction, fractional shortening, fractional area change, (P < 0.0001) and an increase in left ventricular end diastolic volume (P < 0.05). Immunoblotting of kidney and heart tissue showed increased apoptosis (caspase 3) and fibrosis (fibronectin) and increases in the cardiac levels of phosphorylated Akt, and renal total Akt. Immunohistochemistry for α-SMA, collagen 1 and collagen 3 further confirmed fibrosis. CONCLUSIONS: We present a novel regimen of adenine diet which induces both chronic kidney disease and reno-cardiac syndrome in the C57/BL6 mouse strain. The non-surgical nature of this model makes it highly reproducible compared to other models currently available.

IκB Kinase Inhibitor Attenuates Sepsis-Induced Cardiac Dysfunction in CKD.

Patients with CKD requiring dialysis have a higher risk of sepsis and a 100-fold higher mortality rate than the general population with sepsis. The severity of cardiac dysfunction predicts mortality in patients with sepsis. Here, we investigated the effect of preexisting CKD on cardiac function in mice with sepsis and whether inhibition of IκB kinase (IKK) reduces the cardiac dysfunction in CKD sepsis. Male C57BL/6 mice underwent 5/6 nephrectomy, and 8 weeks later, they were subjected to LPS (2 mg/kg) or sepsis by cecal ligation and puncture (CLP). Compared with sham operation, nephrectomy resulted in significant increases in urea and creatinine levels, a small (P<0.05) reduction in ejection fraction (echocardiography), and increases in the cardiac levels of phosphorylated IκBα, Akt, and extracellular signal-regulated kinase 1/2; nuclear translocation of the NF-κB subunit p65; and inducible nitric oxide synthase (iNOS) expression. When subjected to LPS or CLP, compared with sham-operated controls, CKD mice exhibited exacerbation of cardiac dysfunction and lung inflammation, greater increases in levels of plasma cytokines (TNF-α, IL-1ß, IL-6, and IL-10), and greater increases in the cardiac levels of phosphorylated IKKα/ß and IκBα, nuclear translocation of p65, and iNOS expression. Treatment of CKD mice with an IKK inhibitor (IKK 16; 1 mg/kg) 1 hour after CLP or LPS administration attenuated these effects. Thus, preexisting CKD aggravates the cardiac dysfunction caused by sepsis or endotoxemia in mice; this effect may be caused by increased cardiac NF-κB activation and iNOS expression.

Elevated hepatic 11ß-hydroxysteroid dehydrogenase type 1 induces insulin resistance in uremia.

Insulin resistance and associated metabolic sequelae are common in chronic kidney disease (CKD) and are positively and independently associated with increased cardiovascular mortality. However, the pathogenesis has yet to be fully elucidated. 11ß-Hydroxysteroid dehydrogenase type 1 (11ßHSD1) catalyzes intracellular regeneration of active glucocorticoids, promoting insulin resistance in liver and other metabolic tissues. Using two experimental rat models of CKD (subtotal nephrectomy and adenine diet) which show early insulin resistance, we found that 11ßHSD1 mRNA and protein increase in hepatic and adipose tissue, together with increased hepatic 11ßHSD1 activity. This was associated with intrahepatic but not circulating glucocorticoid excess, and increased hepatic gluconeogenesis and lipogenesis. Oral administration of the 11ßHSD inhibitor carbenoxolone to uremic rats for 2 wk improved glucose tolerance and insulin sensitivity, improved insulin signaling, and reduced hepatic expression of gluconeogenic and lipogenic genes. Furthermore, 11ßHSD1(-/-) mice and rats treated with a specific 11ßHSD1 inhibitor (UE2316) were protected from metabolic disturbances despite similar renal dysfunction following adenine experimental uremia. Therefore, we demonstrate that elevated hepatic 11ßHSD1 is an important contributor to early insulin resistance and dyslipidemia in uremia. Specific 11ßHSD1 inhibitors potentially represent a novel therapeutic approach for management of insulin resistance in patients with CKD.

Monomeric eNAMPT in the development of experimental diabetes in mice: a potential target for type 2 diabetes treatment.

AIMS/HYPOTHESIS: Serum extracellular nicotinamide phosphoribosyltransferase (eNAMPT) concentrations are elevated in type 2 diabetes. However, the relationship between abnormally elevated serum eNAMPT and type 2 diabetes pathophysiology is unclear. eNAMPT circulates in functionally and structurally distinct monomeric and dimeric forms. Dimeric eNAMPT promotes NAD biosynthesis. The role of eNAMPT-monomer is unclear but it may have NAD-independent proinflammatory effects. However, studies of eNAMPT in type 2 diabetes have not distinguished between monomeric and dimeric forms. Since type 2 diabetes is characterised by chronic inflammation, we hypothesised a selective NAD-independent role for eNAMPT-monomer in type 2 diabetes. METHODS: Two mouse models were used to examine the role of eNAMPT-monomer in type 2 diabetes; (1) a mouse model of diabetes fed a high-fat diet (HFD) for 10 weeks received i.p. injections with an anti-monomeric-eNAMPT antibody; and (2) lean non-diabetic mice received i.p. injections with recombinant monomeric eNAMPT daily for 14 days. RESULTS: Serum monomeric eNAMPT levels were elevated in HFD-fed mouse models of diabetes, whilst eNAMPT-dimer levels were unchanged. eNAMPT-monomer neutralisation in HFD-fed mice resulted in lower blood glucose levels, amelioration of impaired glucose tolerance (IGT) and whole-body insulin resistance, improved pancreatic islet function, and reduced inflammation. These effects were maintained for at least 3 weeks post-treatment. eNAMPT-monomer administration induced a diabetic phenotype in mice, characterised by elevated blood glucose, IGT, impaired pancreatic insulin secretion and the presence of systemic and tissue inflammation, without changes in NAD levels. CONCLUSIONS/INTERPRETATION: We demonstrate that elevation of monomeric-eNAMPT plays an important role in the pathogenesis of diet-induced diabetes via proinflammatory mechanisms. These data provide proof-of-concept evidence that the eNAMPT-monomer represents a potential therapeutic target for type 2 diabetes.

Endothelial Angiogenesis and Barrier Function in Response to Thrombin Require Ca2+ Influx through the Na+/Ca2+ Exchanger.

Thrombin acts on the endothelium by activating protease-activated receptors (PARs). The endothelial thrombin-PAR system becomes deregulated during pathological conditions resulting in loss of barrier function and a pro-inflammatory and pro-angiogenic endothelial phenotype. We reported recently that the ion transporter Na(+)/Ca(2+) exchanger (NCX) operating in the Ca(2+)-influx (reverse) mode promoted ERK1/2 activation and angiogenesis in vascular endothelial growth factor-stimulated primary human vascular endothelial cells. Here, we investigated whether Ca(2+) influx through NCX was involved in ERK1/2 activation, angiogenesis, and endothelial barrier dysfunction in response to thrombin. Reverse-mode NCX inhibitors and RNAi-mediated NCX1 knockdown attenuated ERK1/2 phosphorylation in response to thrombin or an agonist of PAR-1, the main endothelial thrombin receptor. Conversely, promoting reverse-mode NCX by suppressing Na(+)-K(+)-ATPase activity enhanced ERK1/2 activation. Reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced primary human vascular endothelial cell angiogenesis, quantified as proliferation and tubular differentiation. Reverse-mode NCX inhibitors or NCX1 knockdown preserved barrier integrity upon thrombin stimulation in vitro. Moreover, the reverse-mode NCX inhibitor SEA0400 suppressed Evans' blue albumin extravasation to the lung and kidneys and attenuated edema formation and ERK1/2 activation in the lungs of mice challenged with a peptide activator of PAR-1. Mechanistically, thrombin-induced ERK1/2 activation required NADPH oxidase 2-mediated reactive oxygen species (ROS) production, and reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced ROS production. We propose that reverse-mode NCX is a novel mechanism contributing to thrombin-induced angiogenesis and hyperpermeability by mediating ERK1/2 activation in a ROS-dependent manner. Targeting reverse-mode NCX could be beneficial in pathological conditions involving unregulated thrombin signaling.

Ischemic conditioning protects the uremic heart in a rodent model of myocardial infarction.

BACKGROUND: Outcomes after acute myocardial infarction in patients with chronic kidney disease are extremely poor. Ischemic conditioning techniques are among the most powerful cytoprotective strategies discovered to date. However, experimental data suggest that comorbidity may attenuate the protective effects of ischemic conditioning. METHODS AND RESULTS: We conducted investigations into the effects of chronic uremia on myocardial infarct size and the protective effects of ischemic preconditioning (IPC), remote ischemic preconditioning, and ischemic postconditioning in 2 rodent models of chronic uremia. In addition, a limited investigation into the signaling mechanisms involved in cardioprotection after IPC was performed in both uremic and nonuremic animals. Myocardial infarct size was increased in uremic animals, but all 3 conditioning strategies (IPC, remote IPC, ischemic postconditioning) proved highly efficacious in reducing myocardial infarct size (relative reduction, 86%, 39%, and 65% [P<0.005, P<0.05, and P<0.05], respectively). Moreover, some protocols (IPC and ischemic postconditioning) appeared to be more effective in uremic than in sham (nonuremic) animals. Analysis of the signaling mechanisms revealed that components of both the reperfusion injury salvage kinase and survivor activating factor enhancement pathways were similarly upregulated in both uremic and nonuremic animals after an IPC stimulus. CONCLUSION: Conditioning strategies may present the best opportunity to improve outcomes for patients with chronic kidney disease after an acute coronary syndrome.

Erythropoietin attenuates acute kidney dysfunction in murine experimental sepsis by activation of the ß-common receptor.

The ß-common receptor (ßcR) plays a pivotal role in the nonhematopoietic tissue-protective effects of erythropoietin (EPO). Here we determined whether EPO reduces the acute kidney injury (AKI) caused by sepsis and whether this effect is mediated by the ßcR. In young (2 months old) C57BL/6 wild-type and ßcR knockout mice, lipopolysaccharide caused a significant increase in serum urea and creatinine, hence AKI. This AKI was not associated with any overt morphological alterations in the kidney and was attenuated by EPO given 1 h after lipopolysaccharide in wild-type but not in ßcR knockout mice. In the kidneys of endotoxemic wild-type mice, EPO enhanced the phosphorylation of Akt, glycogen synthase kinase-3ß, and endothelial nitric oxide synthase, and inhibited the activation of nuclear factor-κB. All these effects of EPO were lost in ßcR knockout mice. Since sepsis is more severe in older animals or patients, we tested whether EPO was renoprotective in 8-month-old wild-type and ßcR knockout mice that underwent cecal ligation and puncture. These older mice developed AKI at 24 h, which was attenuated by EPO treatment 1 h post cecal ligation and puncture in wild-type mice but not in ßcR knockout mice. Thus, activation of the ßcR by EPO is essential for the observed reduction in AKI in either endotoxemic young mice or older mice with polymicrobial sepsis, and for the activation of well-known signaling pathways by EPO.

Evidence of a causal and modifiable relationship between kidney function and circulating trimethylamine N-oxide.

The host-microbiota co-metabolite trimethylamine N-oxide (TMAO) is linked to increased cardiovascular risk but how its circulating levels are regulated remains unclear. We applied "explainable" machine learning, univariate, multivariate and mediation analyses of fasting plasma TMAO concentration and a multitude of phenotypes in 1,741 adult Europeans of the MetaCardis study. Here we show that next to age, kidney function is the primary variable predicting circulating TMAO, with microbiota composition and diet playing minor, albeit significant, roles. Mediation analysis suggests a causal relationship between TMAO and kidney function that we corroborate in preclinical models where TMAO exposure increases kidney scarring. Consistent with our findings, patients receiving glucose-lowering drugs with reno-protective properties have significantly lower circulating TMAO when compared to propensity-score matched control individuals. Our analyses uncover a bidirectional relationship between kidney function and TMAO that can potentially be modified by reno-protective anti-diabetic drugs and suggest a clinically actionable intervention for decreasing TMAO-associated excess cardiovascular risk.

Dexamethasone ameliorates renal ischemia-reperfusion injury.

In the setting of renal ischemia-reperfusion injury (IRI), the effect and mechanism of action of glucocorticoids are not well understood. In rat renal IRI, a single dose of dexamethasone administered before ischemia, or at the onset of reperfusion, ameliorated biochemical and histologic acute kidney injury after 24 h. Dexamethasone upregulated Bcl-xL, downregulated ischemia-induced Bax, inhibited caspase-9 and caspase-3 activation, and reduced apoptosis and necrosis of proximal tubular cells. In addition, dexamethasone decreased the number of infiltrating neutrophils and ICAM-1. We observed the protective effect of dexamethasone in neutrophil-depleted mice, suggesting a neutrophil-independent mechanism. In vitro, dexamethasone protected human kidney proximal tubular (HK-2) cells during serum starvation and IRI-induced apoptosis, but inhibition of MEK 1/2 abolished its anti-apoptotic effects in these conditions. Dexamethasone stimulated rapid and transient phosphorylation of ERK 1/2, which required the presence of the glucocorticoid receptor and was independent of transcriptional activity. In summary, in the setting of renal ischemia-reperfusion injury, dexamethasone directly protects against kidney injury by a receptor-dependent, nongenomic mechanism.

Immunohistochemistry of Kidney a-SMA, Collagen 1, and Collagen 3, in A Novel Mouse Model of Reno-cardiac Syndrome.

Cardiorenal syndrome defines a synergistic pathology of the heart and kidneys where failure of one organ causes failure in the other. The incidence of cardiovascular mortality caused by this syndrome, is 20 fold higher in the end stage renal disease (ESRD) population compared to the population as a whole thus necessitating the need for improved therapeutic strategies to combat reno-cardiac pathologies. Murine in vivo models play a major role in such research permitting precise genetic modification thus reducing miscellany, however presently there is no steadfast model of reno-cardiac syndrome in the most common genetically modified mouse strain, the C57BL/6 mouse. In this study we have modified an established model of chronic renal disease using adenine diet and extended the associated pathology achieving chronic renal failure and consequent reno-cardiac syndrome in the C57BL/6 mouse. Eight week-old male C57BL/6 mice were acclimatized for 7 days before administration of a 0.15% adenine diet or control diet for 20 weeks after which the experiment was terminated and blood, urine and organs were collected and analyzed biochemically and by immunohistochemistry. Administration of 0.15% adenine diet caused progressive renal failure resulting in a reno-cardiac syndrome confirmed by a significantly increased heart to body weight ratio (P < 0.0001). Blood biochemistry showed that adenine fed mice had significantly increased serum creatinine, urea (P < 0.0001), and a significantly reduced glomerular filtration rate (P < 0.05), while immunohistochemistry of the kidneys for α-SMA, collagen 1 and collagen 3 showed severe fibrosis. We present a novel regimen of adenine diet which induces both chronic kidney disease and reno-cardiac syndrome in the C57BL/6 mouse strain. The non-surgical nature of this model makes it highly reproducible compared to other models currently available.

Ergocalciferol improves endothelial vasodilatory and vasoconstrictor function in an in vivo model of mild uraemia.

Endothelial dysfunction and vitamin D deficiency are prevalent in patients with cardiovascular disease (CVD) and chronic kidney disease (CKD). Both are risk factors for cardiovascular events in patients with CKD. No studies have investigated the effect of nutritional forms of vitamin D on endothelial function in earlier stages of CKD, when vascular endothelium may be more amenable to this therapy. We studied the effect of ergocalciferol in a pre-clinical model of mild uraemia. Male Wistar rats underwent either a 5/6th nephrectomy or sham surgery. Four weeks after the final stage of the surgery, these two groups were randomly allocated to placebo or an oral dose of 1000 iu of ergocalcfierol at day 7 and 2 pre sacrifice. Vascular responses to acetylcholine, Spermine NONOate and phenylephrine were determined in aortic rings. Blood pressure, calcium, phosphate and parathyroid hormone were measured in all groups. Ergocalciferol significantly improved the endothelium-dependent responses to acetylcholine and overcame the blunting of the contractile response to phenylephrine seen in uraemic animals. Ergocalciferol improved the contractile response to potassium chloride in uraemic, but not sham animals. All effects occurred independently of changes to calcium, phosphate, parathyroid hormone and systolic blood pressure. There were no differences in endothelium-independent relaxation to Spermine NONOate. In summary, in a model of mild uraemia, ergocalciferol improved vasodilator and vasoconstrictor tone independently of blood pressure and bone mineral parameters suggesting a direct effect of ergocalciferol on the endothelium.

Batch effect exerts a bigger influence on the rat urinary metabolome and gut microbiota than uraemia: a cautionary tale.

BACKGROUND: Rodent models are invaluable for studying biological processes in the context of whole organisms. The reproducibility of such research is based on an assumption of metabolic similarity between experimental animals, controlled for by breeding and housing strategies that minimise genetic and environmental variation. Here, we set out to demonstrate the effect of experimental uraemia on the rat urinary metabolome and gut microbiome but found instead that the effect of vendor shipment batch was larger in both areas than that of uraemia. RESULTS: Twenty four Wistar rats obtained from the same commercial supplier in two separate shipment batches underwent either subtotal nephrectomy or sham procedures. All animals undergoing subtotal nephrectomy developed an expected uraemic phenotype. The urinary metabolome was studied using 1H-NMR spectroscopy and found to vary significantly between animals from different batches, with substantial differences in concentrations of a broad range of substances including lactate, acetate, glucose, amino acids, amines and benzoate derivatives. In animals from one batch, there was a complete absence of the microbiome-associated urinary metabolite hippurate, which was present in significant concentrations in animals from the other batch. These differences were so prominent that we would have drawn quite different conclusions about the effect of uraemia on urinary phenotype depending on which batch of animals we had used. Corresponding differences were seen in the gut microbiota between animals in different batches when assessed by the sequencing of 16S rRNA gene amplicons, with higher alpha diversity and different distributions of Proteobacteria subtaxa and short-chain fatty acid producing bacteria in the second batch compared to the first. Whilst we also demonstrated differences in both the urinary metabolome and gut microbiota associated with uraemia, these effects were smaller in size than those associated with shipment batch. CONCLUSIONS: These results challenge the assumption that experimental animals obtained from the same supplier are metabolically comparable, and provide metabolomic evidence that batch-to-batch variations in the microbiome of experimental animals are significant confounders in an experimental study. We discuss strategies for reducing such variability and the need for transparency in research publications about the supply of experimental animals.

Generation of endogenous hydrogen sulfide by cystathionine gamma-lyase limits renal ischemia/reperfusion injury and dysfunction.

The generation of endogenous hydrogen sulfide may either limit or contribute to the degree of tissue injury caused by ischemia/reperfusion. A total of 74 male Wistar rats were used to investigate the effects of endogenous and exogenous hydrogen sulfide in renal ischemia/reperfusion. Administration of the irreversible cystathionine gamma-lyase (CSE) inhibitor, dL-propargylglycine, prevented the recovery of renal function after 45 min ischemia and 72 h reperfusion. The hydrogen sulfide donor sodium hydrosulfide attenuated the (renal, tubular, and glomerular) dysfunction and injury caused by 45 min ischemia and 6 h reperfusion. Western blot analysis of kidneys taken at 30 min reperfusion showed that sodium hydrosulfide significantly attenuated phosphorylation of mitogen-activated protein kinases (p-38, c-JUN N-terminal protein kinase 1/2, and extracellular signal-regulated kinase 1/2) and activation of nuclear factor-kappaB. At 6 h reperfusion, sodium hydrosulfide significantly attenuated the histological score for acute tubular necrosis, the activation of caspase-3 and Bid, the decline in the expression of anti-apoptotic Bcl-2, and the expression of nuclear factor-kappaB-dependent proteins (inducible nitric oxide synthase, cyclo-oxygenase-2, and intercellular adhesion molecule-1). These findings suggest that (1) the synthesis of endogenous hydrogen sulfide by CSE is essential to protect the kidney against ischemia/reperfusion injury and dysfunction and aids in the recovery of renal function following ischemia/reperfusion, (2) hydrogen sulfide generated by sodium hydrosulfide reduces ischemia/reperfusion injury and dysfunction, and morphological changes of the kidney, and (3) the observed protective effects of hydrogen sulfide are due to both anti-apoptotic and anti-inflammatory effects.

Glucolipotoxicity initiates pancreatic ß-cell death through TNFR5/CD40-mediated STAT1 and NF-κB activation.

Type 2 diabetes is a chronic metabolic disorder, where failure to maintain normal glucose homoeostasis is associated with, and exacerbated by, obesity and the concomitant-elevated free fatty acid concentrations typically found in these patients. Hyperglycaemia and hyperlipidaemia together contribute to a decline in insulin-producing ß-cell mass through activation of the transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription (STAT)-1. There are however a large number of molecules potentially able to modulate NF-κB and STAT1 activity, and the mechanism(s) by which glucolipotoxicity initially induces NF-κB and STAT1 activation is currently poorly defined. Using high-density microarray analysis of the ß-cell transcritptome, we have identified those genes and proteins most sensitive to glucose and fatty acid environment. Our data show that of those potentially able to activate STAT1 or NF-κB pathways, tumour necrosis factor receptor (TNFR)-5 is the most highly upregulated by glucolipotoxicity. Importantly, our data also show that the physiological ligand for TNFR5, CD40L, elicits NF-κB activity in ß-cells, whereas selective knockdown of TNFR5 ameliorates glucolipotoxic induction of STAT1 expression and NF-κB activity. This data indicate for the first time that TNFR5 signalling has a major role in triggering glucolipotoxic islet cell death.

The effect of uraemia on the duration of arrhythmias in the context of cardioprotective ischaemic conditioning strategies.

BACKGROUND: Sudden cardiac death is a leading cause of death in patients with chronic kidney disease and end stage renal disease. Ischaemic conditioning strategies confer profound myocardial protection and, in the absence of uraemia, have been reported to reduce myocardial dysrhythmias. Recent data confirms that ischaemic conditioning can protect the uraemic heart. However, the effect of uraemia on myocardial arrhythmogenesis in the context of ischaemia-reperfusion injury and whether ischaemic conditioning can modulate this is unknown. OBJECTIVE: We investigated the effect of underling chronic uraemia on the duration of arrhythmias in the context of cardioprotective ischaemic conditioning strategies. METHODS: We examined the effect of chronic uraemia on arrhythmias occurring in the context of myocardial ischaemia-reperfusion injury and the ability of ischaemic preconditioning (IPC), remote ischaemic preconditioning (RIPC) and ischaemic postconditioning (iPOST) to suppress arrhythmogenesis in uraemic and non-uraemic animals. RESULTS: IPC led to a reduction in the frequency and duration of arrhythmias occurring during ischaemia and reperfusion. Neither RIPC nor iPOST affected the duration or frequency of ischaemic or reperfusion arrhythmias. Underlying uraemia did not alter the frequency or duration of ischaemic arrhythmias in any of the experiments however it was associated with a reduction in reperfusion arrhythmia duration in the IPC and iPOST experiments. CONCLUSIONS: These studies demonstrate that underlying chronic uraemia does not reduce the threshold for arrhythmia timing or duration resulting from myocardial ischaemia-reperfusion and underlying uraemia did not alter the effects of these cardioprotective ischaemic conditioning strategies in the context of arrhythmia duration. SUMMARY: This novel work in a rodent model of chronic uraemia establishes that underlying uraemia does not increase the susceptibility to myocardial ischaemia-reperfusion induced arrhythmias. When compared with the non-uraemic heart, the uraemic heart has a similar response to the effects of ischaemic conditioning strategies in terms of their effect on arrhythmia timing and duration.

Glucose induces protein targeting to glycogen in hepatocytes by fructose 2,6-bisphosphate-mediated recruitment of MondoA to the promoter.

In the liver, a high glucose concentration activates transcription of genes encoding glucose 6-phosphatase and enzymes for glycolysis and lipogenesis by elevation in phosphorylated intermediates and recruitment of the transcription factor ChREBP (carbohydrate response element binding protein) and its partner, Mlx, to gene promoters. A proposed function for this mechanism is intracellular phosphate homeostasis. In extrahepatic tissues, MondoA, the paralog of ChREBP, partners with Mlx in transcriptional induction by glucose. We tested for glucose induction of regulatory proteins of the glycogenic pathway in hepatocytes and identified the glycogen-targeting proteins, G(L) and PTG (protein targeting to glycogen), as being encoded by Mlx-dependent glucose-inducible genes. PTG induction by glucose was MondoA dependent but ChREBP independent and was enhanced by forced elevation of fructose 2,6-bisphosphate and by additional xylitol-derived metabolites. It was counteracted by selective depletion of fructose 2,6-bisphosphate with a bisphosphatase-active kinase-deficient variant of phosphofructokinase 2/fructosebisphosphatase 2, which prevented translocation of MondoA to the nucleus and recruitment to the PTG promoter. We identify a novel role for MondoA in the liver and demonstrate that elevated fructose 2,6-bisphosphate is essential for recruitment of MondoA to the PTG promoter. Phosphometabolite activation of MondoA and ChREBP and their recruitment to target genes is consistent with a mechanism for gene regulation to maintain intracellular phosphate homeostasis.