Normothermic Ex-vivo Kidney Perfusion in a Porcine Auto-Transplantation Model Preserves the Expression of Key Mitochondrial Proteins: An Unbiased Proteomics Analysis.

Normothermic ex-vivo kidney perfusion (NEVKP) results in significantly improved graft function in porcine auto-transplant models of donation after circulatory death injury compared with static cold storage (SCS); however, the molecular mechanisms underlying these beneficial effects remain unclear. We performed an unbiased proteomics analysis of 28 kidney biopsies obtained at three time points from pig kidneys subjected to 30 min of warm ischemia, followed by 8 h of NEVKP or SCS, and auto-transplantation. 70/6593 proteins quantified were differentially expressed between NEVKP and SCS groups (false discovery rate < 0.05). Proteins increased in NEVKP mediated key metabolic processes including fatty acid ß-oxidation, the tricarboxylic acid cycle, and oxidative phosphorylation. Comparison of our findings with external datasets of ischemia-reperfusion and other models of kidney injury confirmed that 47 of our proteins represent a common signature of kidney injury reversed or attenuated by NEVKP. We validated key metabolic proteins (electron transfer flavoprotein subunit beta and carnitine O-palmitoyltransferase 2, mitochondrial) by immunoblotting. Transcription factor databases identified members of the peroxisome proliferator-activated receptors (PPAR) family of transcription factors as the upstream regulators of our dataset, and we confirmed increased expression of PPARA, PPARD, and RXRA in NEVKP with reverse transcription polymerase chain reaction. The proteome-level changes observed in NEVKP mediate critical metabolic pathways. These effects may be coordinated by PPAR-family transcription factors and may represent novel therapeutic targets in ischemia-reperfusion injury.

Untargeted metabolomics reveals N, N, N-trimethyl-L-alanyl-L-proline betaine (TMAP) as a novel biomarker of kidney function.

The diagnosis and prognosis of chronic kidney disease (CKD) currently relies on very few circulating small molecules, which can vary by factors unrelated to kidney function. In end-stage renal disease (ESRD), these same small molecules are used to determine dialysis dose and dialytic clearance. Therefore, we aimed to identify novel plasma biomarkers to estimate kidney function in CKD and dialytic clearance in ESRD. Untargeted metabolomics was performed on plasma samples from patients with a single kidney, non-dialysis CKD, ESRD and healthy controls. For ESRD patients, pre- and post-dialysis plasma samples were obtained from several dialysis modalities. Metabolomics analysis revealed over 400 significantly different features in non-dialysis CKD and ESRD plasma compared to controls while less than 35 features were significantly altered in patients with a single kidney. N,N,N-trimethyl-L-alanyl-L-proline betaine (TMAP, AUROC = 0.815) and pyrocatechol sulfate (AUROC = 0.888) outperformed creatinine (AUROC = 0.745) in accurately identifying patients with a single kidney. Several metabolites accurately predicted ESRD; however, when comparing pre-and post-hemodialysis, TMAP was the most robust biomarker of dialytic clearance for all modalities (AUROC = 0.993). This study describes TMAP as a novel potential biomarker of kidney function and dialytic clearance across several hemodialysis modalities.

The effect of chronic kidney disease on CYP2B expression and activity in male Wistar rats.

Chronic kidney disease (CKD) is characterized by progressive reduction in kidney function over time. CKD affects greater than 10% of the population and its incidence is on the rise due to the growing prevalence of its risk factors. Previous studies demonstrated CKD alters nonrenal clearance of drugs in addition to reducing renal clearance. We assessed the function and expression of hepatic CYP2B enzymes using a rat model of CKD. CKD was induced in Wistar rats by supplementing their chow with adenine and confirmed through the detection of elevated uremic toxins in plasma. Liver enzymes AST and ALT were unchanged by the adenine diet. Bupropion was used as a probe substrate for hepatic CYP2B function using rat liver microsomes. The resulting metabolite, hydroxy-bupropion, and bupropion were quantified by ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry. Level of mRNA and protein were determined by RT-PCR and Western blot, respectively. The results of our study demonstrate that CYP2B1 is downregulated in a rat model of CKD. CYP2B1 mRNA level was significantly decreased (88%, P < 0.001) in CKD relative to control. Similarly, maximal enzymatic velocity (Vmax) for CYP2B was decreased by 46% in CKD relative to control (P < 0.0001). Previous studies involving patients with CKD demonstrated altered bupropion pharmacokinetics compared to control. Hence, our results suggest that these alterations may be mediated by attenuated CYP2B hepatic metabolism. This finding may partially explain the alterations in pharmacokinetics and nonrenal drug clearance frequently observed in patients with CKD.