Jacek Klepacki

Endothelial dysfunction and oxidative stress in polycystic kidney disease.

Cardiovascular disease (CVD) is the leading cause of premature mortality in ADPKD patients. The aim was to identify potential serum biomarkers associated with the severity of ADPKD. Serum samples from a homogenous group of 61 HALT study A ADPKD patients [early disease group with estimated glomerular filtration rate (eGFR) >60 ml·min(-1)·1.73 m(-2)] were compared with samples from 49 patients from the HALT study B group with moderately advanced disease (eGFR 25-60 ml·min(-1)·1.73 m(-2)). Targeted tandem-mass spectrometry analysis of markers of endothelial dysfunction and oxidative stress was performed and correlated with eGFR and total kidney volume normalized to the body surface area (TKV/BSA). ADPKD patients with eGFR >60 ml·min(-1)·1.73 m(-2) showed higher levels of CVD risk markers asymmetric and symmetric dimethylarginine (ADMA and SDMA), homocysteine, and S-adenosylhomocysteine (SAH) compared with the healthy controls. Upon adjustments for age, sex, systolic blood pressure, and creatinine, SDMA, homocysteine, and SAH remained negatively correlated with eGFR. Resulting cellular methylation power [S-adenosylmethionine (SAM)/SAH ratio] correlated with the reduction of renal function and increase in TKV. Concentrations of prostaglandins (PGs), including oxidative stress marker 8-isoprostane, as well as PGF2α, PGD₂, and PGE₂, were markedly elevated in patients with ADPKD compared with healthy controls. Upon adjustments for age, sex, systolic blood pressure, and creatinine, increased PGD₂ and PGF₂α were associated with reduced eGFR, whereas 8-isoprostane and again PGF₂α were associated with an increase in TKV/BSA. Endothelial dysfunction and oxidative stress are evident early in ADPKD patients, even in those with preserved kidney function. The identified pathways may provide potential therapeutic targets for slowing down the disease progression.

A high-throughput U-HPLC-MS/MS assay for the quantification of mycophenolic acid and its major metabolites mycophenolic acid glucuronide and mycophenolic acid acyl-glucuronide in human plasma and urine.

Mycophenolic acid (MPA) is used as an immunosuppressant after organ transplantation and for the treatment of immune diseases. There is increasing evidence that therapeutic drug monitoring and plasma concentration-guided dose adjustments are beneficial for patients to maintain immunosuppressive efficacy and to avoid toxicity. The major MPA metabolite that can be found in high concentrations in plasma is MPA glucuronide (MPAG). A metabolite usually present at lower concentrations, MPA acyl-glucuronide (AcMPAG), has been implicated in some of the adverse effects of MPA. We developed and validated an automated high-throughput ultra-high performance chromatography-tandem mass spectrometry (U-HPLC-MS/MS) assay using liquid-handling robotic extraction for the quantification of MPA, MPAG, and AcMPAG in human EDTA plasma and urine. The ranges of reliable response were 0.097 (lower limit of quantitation) to 200 μg/mL for MPA and MPAG and 0.156-10 μg/mL for AcMPAG in human urine and plasma. The inter-day accuracies were 94.3-104.4%, 93.8-105.0% and 94.4-104.7% for MPA, MPAG and AcMPAG, respectively. Inter-day precisions were 0.7-7.8%, 0.9-6.9% and 1.6-8.6% for MPA, MPAG and AcMPAG. No matrix interferences, ion suppression/enhancement and carry-over were detected. The total assay run time was 2.3 min. The assay met all predefined acceptance criteria and the quantification of MPA was successfully cross-validated with an LC-MS/MS assay routinely used for clinical therapeutic drug monitoring. The assay has proven to be robust and reliable during the measurement of samples from several pharmacokinetics trials.

Proteomics and metabolomics in renal transplantation-quo vadis?

The improvement of long‐term transplant organ and patient survival remains a critical challenge following kidney transplantation. Proteomics and biochemical profiling (metabolomics) may allow for the detection of early changes in cell signal transduction regulation and biochemistry with high sensitivity and specificity. Hence, these analytical strategies hold the promise to detect and monitor disease processes and drug effects before histopathological and pathophysiological changes occur. In addition, they will identify enriched populations and enable individualized drug therapy. However, proteomics and metabolomics have not yet lived up to such high expectations. Renal transplant patients are highly complex, making it difficult to establish cause‐effect relationships between surrogate markers and disease processes. Appropriate study design, adequate sample handling, storage and processing, quality and reproducibility of bioanalytical multi‐analyte assays, data analysis and interpretation, mechanistic verification, and clinical qualification (=establishment of sensitivity and specificity in adequately powered prospective clinical trials) are important factors for the success of molecular marker discovery and development in renal transplantation. However, a newly developed and appropriately qualified molecular marker can only be successful if it is realistic that it can be implemented in a clinical setting. The development of combinatorial markers with supporting software tools is an attractive goal.

A high-performance liquid chromatography – tandem mass spectrometry – based targeted metabolomics kidney dysfunction marker panel in human urine

BACKGROUND Previous studies have examined and documented fluctuations in urine metabolites in response to disease processes and drug toxicity affecting glomerular filtration, tubule cell metabolism, reabsorption, oxidative stress, purine degradation, active secretion and kidney amino acylase activity representative of diminished renal function. However, a high-throughput assay that incorporates metabolites that are surrogate markers for such changes into a kidney dysfunction panel has yet to be described. METHODS A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for the quantification of ten metabolites associated with the Krebs cycle, purine degradation, and oxidative stress in human urine was developed and validated. Normal values were assessed in healthy adult (n=120) and pediatric (n=36) individuals. In addition, 9 pediatric renal transplant recipients patients were evaluated before and after initial dosing of the immunosuppressant tacrolimus in a proof-of-concept study. RESULTS The assay met all predefined acceptance criteria. The lower limit of quantification ranged from 0.1 to 1000 μmol/l. Inter-day trueness and imprecisions ranged from 91.4-112.9% and 1.5-12.4%, respectively. The total assay run time was 5.5 minutes. Concentrations of glucose, sorbitol, and trimethylamine oxide (TMAO) were elevated in pediatric renal transplant patients (n=9) prior to transplantation as well as before and immediately after initial dosing of tacrolimus. One month post-transplant urine metabolite patterns matched those of healthy children (n=36). CONCLUSIONS The LC-MS/MS assay will provide the basis for further large-scale clinical studies to explore these analytes as molecular markers for the patients with renal insufficiency.

Development and validation of an LC-MS/MS assay for the quantification of the trans-methylation pathway intermediates S-adenosylmethionine and S-adenosylhomocysteine in human plasma.

BACKGROUND Although increased levels of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) have been implicated as markers for renal and vascular dysfunction, until now there have been no studies investigating their association with clinical post-transplant events such as organ rejection and immunosuppressant nephrotoxicity. METHODS A newly developed and validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the quantification of SAM and SAH in human EDTA plasma was used for a clinical proof-of-concept pilot study. Retrospective analysis was performed using samples from a longitudinal clinical study following de novo kidney transplant patients for the first year (n=16). RESULTS The ranges of reliable response were 8 to 1024 nmol/l for SAM and 16 to 1024 nmol/l for SAH. The inter-day accuracies were 96.7-103.9% and 97.9-99.3% for SAM and SAH, respectively. Inter-day imprecisions were 8.1-9.1% and 8.4-9.8%. The total assay run time was 5 min. SAM and SAH concentrations were significantly elevated in renal transplant patients preceding documented acute rejection and nephrotoxicity events when compared to healthy controls (n=8) as well as transplant patients void of allograft dysfunction (n=8). CONCLUSION The LC-MS/MS assay will provide the basis for further large-scale clinical studies to explore these thiol metabolites as molecular markers for the management of renal transplant patients.

Regulation of Kynurenine Metabolism by a Ketogenic Diet

Ketogenic diets (KDs) are increasingly utilized as treatments for epilepsy, other neurological diseases, and cancer. Despite their long history in suppressing seizures, the distinct molecular mechanisms of action of KDs are still largely unknown. The goal of this study was to identify key metabolites and pathways altered in the hippocampus and plasma of rats fed a KD versus control diet (CD) either ad libitum or calorically restricted to 90% of the recommended intake. This was accomplished using a combination of targeted methods and untargeted MS-based metabolomics analyses. Various metabolites of and related to the tryptophan (TRP) degradation pathway, such as kynurenine (KYN), kynurenic acid as well as enzyme cofactors, showed significant changes between groups fed different diets and/or calorie amounts in plasma and/or the hippocampus. KYN was significantly downregulated in both matrices in animals of the CD-calorically restricted, KD-ad libitum, and KD-calorically restricted groups compared with the CD-ad libitum group. Our data suggest that the TRP degradation pathway is a key target of the KD.

Chapter Four – The Role of Proteomics in the Study of Kidney Diseases and in the Development of Diagnostic Tools

Proteins are the functional output of genes. The proteome is defined as the expressed protein and peptide complement of a cell, organ, or organism, including all isoforms and posttranslational variants. While an organism possesses a single genome, it possesses multiple proteomes depending on the cell compartment, the type of cell, type of tissue, and organ which undergo constant temporal changes. The term proteomics summarizes all of the procedures required for analysis of a proteome. Clinical proteomics in nephrology aims at providing the clinician with tools to accurately diagnose and monitor patients as well as predict treatment effects, thus, enabling individualized patient management. Protein-marker based strategies hold the promise of being sensitive, specific, predictive, and able to outperform the currently established clinical diagnostic tests overall. Proteomics research in nephrology has primarily focused on kidney tissues and urine. Urine is attractive since it is considered a “proximal” matrix, meaning it is a biofluid that is in direct contact with the site of disease. Proximal matrices are local sinks for metabolites, proteins, or peptides secreted, shed or leaked from the tissue of interest. Thus urine protein patterns often reflect disease processes of and drug effects on the kidney. Proteomics commonly involves sample fractionation, separation, mass spectrometry, and biostatistical analysis. The technologies required to enable implementation of truly nonbiased proteome analysis in clinical practice are not available yet. Proteomics is a hypothesis-generating technology that plays an important role in nephrology research today regarding molecular marker discovery and qualification of molecular markers during their development into potential clinical diagnostic tools. As evidenced by the regulatory approval of protein kidney injury markers in rats for preclinical drug toxicity studies, proteins in urine as diagnostic markers are starting to have an impact in clinical medicine. It is reasonable, therefore, to expect that proteins and the analysis of combinatorial protein patterns, especially in urine, will play an increasingly important role as clinical diagnostic tools in nephrology in the near future.