Chronic kidney disease (CKD) is a progressive loss of renal function. The gradual decline in kidney function leads to an accumulation of toxins normally cleared by the kidneys, resulting in uremia. Uremic toxins are classified into three categories: free water-soluble low-molecular-weight solutes, protein-bound solutes, and middle molecules. CKD patients have increased risk of developing cardiovascular disease (CVD), due to an assortment of CKD-specific risk factors. The accumulation of uremic toxins in the circulation and in tissues is associated with the progression of CKD and its co-morbidities, including CVD. Although numerous uremic toxins have been identified to date and many of them are believed to play a role in the progression of CKD and CVD, very few toxins have been extensively studied. The pathophysiological mechanisms of uremic toxins must be investigated further for a better understanding of their roles in disease progression and to develop therapeutic interventions against uremic toxicity. This review discusses the renal and cardiovascular toxicity of uremic toxins indoxyl sulfate, p-cresyl sulfate, hippuric acid, TMAO, ADMA, TNF-α, and IL-6. A focus is also placed on potential therapeutic targets against uremic toxicity.

The comprehensiveness of data collected by "omics" modalities has demonstrated the ability to drastically transform our understanding of the molecular mechanisms of chronic, complex diseases such as musculoskeletal pathologies, how biomarkers are identified, and how therapeutic targets are developed. Standardization of protocols will enable comparisons between findings reported by multiple research groups and move the application of these technologies forward. Herein, we describe a protocol for parallel proteomic and metabolomic analysis of mouse intervertebral disc (IVD) tissues, building from the combined expertise of our collaborative team. This protocol covers dissection of murine IVD tissues, sample isolation, and data analysis for both proteomics and metabolomics applications. The protocol presented below was optimized to maximize the utility of a mouse model for "omics" applications, accounting for the challenges associated with the small starting quantity of sample due to small tissue size as well as the extracellular matrix-rich nature of the tissue.

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Background Curcumin is a commonly used herbal supplement with anti-inflammatory and anti-fibrotic properties. Animal studies and small human trials suggest curcumin reduces albuminuria in patients with chronic kidney disease. Micro-particle curcumin is a new, more bioavailable formulation of curcumin. Methods To determine whether micro-particle curcumin versus placebo slows the progression of albuminuric chronic kidney disease we conducted a randomized, double-blind, placebo-controlled trial with 6-month follow-up. We included adults with albuminuria (a random urine albumin-to-creatinine ratio >30 mg/mmol [265 mg/g] or a 24-hour urine collection with more than 300 mg of protein) and an estimated glomerular filtration rate (eGFR) between 15 and 60 ml/min per 1.73 m2 within the 3 months before randomization. We randomly allocated participants 1:1 to receive micro-particle curcumin capsules (90 mg/day) or matching placebo for 6 months. After randomization. The co-primary outcomes were the changes in albuminuria and the eGFR. Results We enrolled 533 participants, but 4/265 participants in the curcumin group and 15/268 in the placebo group withdrew consent or became ineligible. The 6-month change in albuminuria did not differ significantly between the curcumin and placebo groups (geometric mean ratio 0.94, 97.5% confidence interval [CI]: 0.82 to 1.08, P = .32). Similarly, the 6-month change in eGFR did not differ between groups (mean between-group difference -0.22 mL/min per 1.73m2, 97.5% CI: -1.38 to 0.95, P = 0.68). Conclusions Ninety milligrams of micro-particle curcumin daily did not slow the progression of albuminuric chronic kidney disease over six months. Trial Registration: ClinicalTrials.gov Identifier: NCT02369549

Background and Purpose: Cisplatin-induced nephrotoxicity manifests as acute kidney injury (AKI) in approximately one third of patients receiving cisplatin therapy. Current measures of AKI are inadequate in detecting AKI prior to significant renal injury, and better biomarkers are needed for early diagnosis of cisplatin-induced AKI. Experimental Approach: C57BL/6 and FVB/N mice were treated with a single intraperitoneal injection of cisplatin (15 mg kg-1) or saline. Plasma, urine, and kidney samples were collected prior to cisplatin injection and 24-, 48-, 72-, and 96-hours following cisplatin injection. Untargeted metabolomics was employed using liquid chromatography-mass spectrometry to identify early diagnostic biomarkers for cisplatin-induced AKI. Key Results: There was clear metabolic discrimination between saline and cisplatin-treated mice at all timepoints (day 1 to day 4). In total, 26 plasma, urine, and kidney metabolites were identified as exhibiting early alterations following cisplatin treatment. Several of the metabolites showing early alterations were associated with mitochondrial function and energetics, including intermediates of the tricarboxylic acid cycle, regulators of mitochondrial function and indicators of fatty acid β-oxidation dysfunction. Furthermore, several metabolites were derived from the gut microbiome. Conclusion and Implications: Our results highlight the detrimental effects of cisplatin on mitochondrial function and demonstrate potential involvement of the gut microbiome in the pathophysiology of cisplatin-induced AKI. Here we provide a panel of metabolites to guide future clinical studies of cisplatin-induced AKI and provide insight into potential mechanisms behind cisplatin nephrotoxicity.

AimCisplatin causes acute kidney injury (AKI) in approximately one third of patients. Serum creatinine and urinary output are poor markers of cisplatin‐induced AKI. Metabolomics was utilized to identify predictive or early diagnostic biomarkers of cisplatin‐induced AKI.MethodsThirty‐one adult head and neck cancer patients receiving cisplatin (dose ≥70 mg/m2) were recruited for metabolomics analysis. Urine and serum samples were collected prior to cisplatin (pre), 24–48 h after cisplatin (24–48 h) and 5–14 days (post) after cisplatin. Based on serum creatinine concentrations measured at the post timepoint, 11/31 patients were classified with clinical AKI. Untargeted metabolomics was performed using liquid chromatography‐mass spectrometry (LC‐MS).ResultsMetabolic discrimination was observed between “AKI” patients and “no AKI” patients at all timepoints. Urinary glycine, hippuric acid sulfate, 3‐hydroxydecanedioc acid and suberate were significantly different between AKI patients and no AKI patients prior to cisplatin infusion. Urinary glycine and hippuric acid sulfate were lower (−2.22‐fold and −8.85‐fold), whereas 3‐hydroxydecanedioc acid and suberate were higher (3.62‐fold and 1.91‐fold) in AKI patients relative to no AKI patients. Several urine and serum metabolites were found to be altered 24–48 h following cisplatin infusion, particularly metabolites involved with mitochondrial energetics.ConclusionsWe propose glycine, hippuric acid sulfate, 3‐hydroxydecanedioc acid and suberate as predictive biomarkers of predisposition to cisplatin‐induced AKI. Metabolites indicative of mitochondrial dysfunction may serve as early markers of subclinical AKI.

Congenital heart defects (CHDs) account for 1–5% of live births and are the leading cause of death in the first year life. Pregestational diabetes increases the risk for CHDs over five fold. We have recently shown that pregestational diabetes in mice induces CHDs in 58% of offspring. Exposure of the embryo to a hyperglycemic environment leads to oxidative stress. Reactive oxygen species (ROS) production results in oxidation of proteins vital for heart development, such as endothelial nitric oxide synthase (eNOS). Activity of eNOS is down‐regulated in diabetes as it becomes uncoupled, and its cofactors are oxidized, leading to decreased nitric oxide production and increased superoxide formation.Tetrahydrobiopterin (BH4) is a principal co‐factor required for eNOS dimer stabilization, and is an endogenous antioxidant. In states of oxidative stress, BH4 is itself oxidized leading to eNOS uncoupling. Treatment with BH4 has been shown to recouple eNOS and improve vascular endothelial function in diabetes. The aim of this study was to investigate the effects of BH4 on fetal heart development in mice with pregestational diabetes.Pregestational diabetes was induced by administering streptozotocin (STZ, 75 mg/kg, IP for 3 days) to adult female C57BL/6 mice. BH4 (Kuvan 10 mg/kg/day) was orally administered to the pregnant females via dissolution in peanut butter. Diabetic pregnant mice without BH4 treatment served as controls. Embryos were collected at E18.5 for histological analysis of cardiac morphology and coronary artery formation. Our data show that pregestational diabetes resulted in a spectrum of CHDs including atrial septal defect (ASD), ventricular septal defects (VSD), atrioventricular septal defect (AVSD), and double outlet right ventricle (DORV). Maternal diabetes also resulted in coronary artery malformations, including decreased left and right main coronary artery diameter, artery abundance, and smooth muscle content. Notably, BH4 treatment significantly decreased the incidence of CHDs from 59.4 to 26.7% and abrogated major CHDs such as VSD, AVSD and DORV. BH4 treatment also rescued coronary artery malformations.Furthermore, lineage tracing was performed with a global double fluorescent Mef2C‐Cre;mT/mG mouse, where anterior second heart field (SHF) progenitors are labeled with GFP. Fate mapping revealed significantly reduced numbers of GFP+ SHF progenitors contributing to the outflow tract cushions at E9.5, endocardial cushions at E12.5 and ventricular walls at E12.5, indicating defects in proliferation, migration and myocardialization induced by maternal diabetes. Finally, western blot analysis revealed low eNOS dimer to monomer ratio in the STZ‐induced diabetic E12.5 ventricles; this was reversed by BH4 treatment. These results suggest that eNOS uncoupling may be responsible for decreased SHF progenitor cell recruitment, thereby causing the CHDs observed.In conclusion, eNOS uncoupling contributes to the development of CHDs induced by pregestational diabetes in mice. BH4 treatment recouples eNOS and prevents CHDs, implicating it as a therapeutic target for the potential prevention of CHDs caused by pregestational diabetes in patients.Support or Funding InformationThis study was supported by an operating grant from the Canadian Institutes of Health Research (CIHR) to Q.F. (grant #MOP‐119600)