Molecular Regulation of Renal Physiology and Pathophysiology by Naphthoquinones
Human Studies: In our study we initially looked to determine that the Tromsø chronic kidney disease (CKD) patient population mirrored the published reports on vitamin K deficiency. This required developing a new LC/MS/MS analytical method at UNN. This was established and is available for general clinical use. Healthy control subjects are to be drawn from the Tromsø Study. Another part of our submission to Helse Nord for support was to investigate the status of vitamin K catabolites in CKD patients. We began the development of vitamin K catabolite analysis method using LC/MS/MS, however, we encountered a problem in that the available standards at the time were of insufficient concentration to define the parameters of the catabolite daughter species. The value of the Tromsø Study samples meant that we could not risk getting a single-shot analyses only for vitamin K without the catabolite assay. Since completing the study year we have obtained external funding to continue these studies and the synthesis of both the vitamin K catabolite standards has been commissioned – together with their deuterated counterparts. This will allow completion of the analyses of all the CKD patient and Tromsø Study volunteer samples for total vitamin K and naphthoquinone status using LC/MS/MS in UNN. The standards and deuterated counterparts will be gifted to UNN. Cell-based Studies: At the same time that the analytical elements of the study were progressing a complimentary series of experiments were done in commercially obtained human primary kidney proximal tubule epithelial cells (RPTEC) and human mesangial cells. Human primary kidney proximal tubule epithelial cells were cultured according to the specifications of the cell supplier (Lonza) in the presence of defined growth factors. Near confluence cells were challenged with TGF-ß1. Cells were fixed with formaldehyde, washed and exposed to anti-human ZO-1 antibody, before 2ndary fluorescence tagged antibody exposure. The microscope slides were examined with a fluorescence microscope. The concentration and duration of exposure to TGF-ß1 was investigated with respect to the extent of expression of ZO-1 signal. These experiments suggested that previously employed concentrations of TGF-ß1 were potentially excessive, but a 48 hour exposure was adequate at lower concentrations. Adding vitamin K as the modulating factor on TGF-ß1 modulated (RPTEC) was found to exert a weak effect on the expression of ZO-1. However, the effects were limited. This caused us to readdress the nature of the naphthoquinone regulation as the molecular architecture to metabolise vitamin K is not present in the kidney. It is possible that the vitamin K resides in the membranes of cells and is limited at to acting on the cell membrane components, while catabolites of vitamin K may act intracellularly. Concluding remarks While not reaching all the initial aims, and finding unforeseen limitations, early promise from the Helse Nord funded project has been established. Some results could be published now, such as a vitamin K study based on domestic cats with and without CKD, in collaboration with colleagues at the Royal Veterinary College, London. We have decided, however, that further research will fundamentally improve these early findings making publication in higher ranking journals more feasible As mentioned above additional research funding has been obtained in order to continue the project initiated by the Helse Nord funding. The fact that we now have a range of naphthoquinones, cellular expression read-outs and intracellular signalling targets to widen the original study parameters, while setting any findings into getting a greater understanding of the vitamin K status of CKD patients our intention. With administrative support from UiT we are looking to appoint a post-doctoral fellow to continue the kidney studies working with Dr Ole-Martin Fuskevåg, Professor Lars Marius Ytrebø and Dr Hodges. The primary objectives of this work do not have immediate clinical impact on the delivery of health care.
Strong progress has been made in the areas of cell biology, analytical investigations, patient recruitment and sample collection. Furthermore, we have generated a substantial collaborative infrastructure to explore more fully human kidney disease. Project will run throughout May 2016 ann data compiled , analysed and reported by end 2016.Cellular Biology The human primary renal proximal tubule epithelial and mesangial cells have been expanded into a stock of cryopreserved viable cells of various passage age and remain a core asset of the study. These cells are slow growing and mature morphologically identifiable cells take some 7-9 days to reach 70-80% confluence before harvesting for storage of re-seeding for stock expansion. The renal proximal tubule epithelial cells have been used as the primary experimental template to examine the transformation of the cells from an epithelial phenotype to a mesenchymal phenotype (ETM). The primary cytokine driver used in these experiments was Transforming Growth Factor ß1 (TGF-ß1) and followed the protocol of Dudas et al. 2009. We have expanded our cell range by establishing strong links with Professor Moin Saleem (Specialist Consultant Nephrologist) at Bristol University Hospital, UK who has generously provided us with Sendai Virus transformed human podocytes. We have also engaged with Professor David Abraham (Specialist in Fibrosis) at The Royal Free Hospital, University College London, who has provided us with Sendai Virus transformed human myofibroblasts. These four cell types will continue to be interrogated individually and in co-culture into 2016. Measurement of vitamin K in human serum Using either ultra-performance convergence chromatography (UPC2) or ultra-performance liquid chromatography (UPLC) in combination with a sensitive tandem mass spectrometer we have established analytical methodology to measure vitamin K1 and menaquinone-4 (one of the forms of vitamin K2) and vitamin K1 epoxide in spiked human sera. Menaquinone-7 still presents some minor problems, but these issues are being actively addressed. Once the analytical measurement of this form of vitamin K2 has been established we will request QA standard materials from the KEQAS service in London for comparison to historical collective analysis data. Further comparative analyses will also be engaged in with London with the Tromsø Hospital laboratory joining prospectively with the KEQAS QA system operated out of St Thomas’ Hospital, London. Independent of Helse Nord, the University Hospital of Tromsø has also engaged with this project and has purchased deuterated standards in order to facilitate the accurate measurement of all four forms of vitamin K in human sera samples. Measurement of vitamin K catabolites in human serum and in urine The measurement of these molecules in human samples will be pursued in early 2016. While there are no available deuterated forms of these catabolites, indeed we are the only other laboratory outside Dr Harrington’s in St Thomas’ Hospital in London, that have these authentic compounds. Patient samples After ensuring full ethical approval for the collection of human blood and urine samples, we have begun the process of collecting and archiving samples for bulk analyses. Feline kidney disease In collaboration with Professors Pitsillides and Elliot at the Royal Veterinary College, London, cats who have previously presented clinically with Chronic Kidney Disease – and who have had blood and urine samples collected, have been identified in the freezer archives. Again once the analytical systems are defined we will analyses samples in bulk for the same vitamins and the vitamin K congeners and the vitamin K catabolites.