Search

Renal sodium balance is the main regulator of blood pressure homeostasis. Sodium balance is regulated by aldosterone (ALDO) and, according to recent studies, also by glucocorticoids. Abnormal hormonal regulation of sodium equilibrium results in kidney disease and hypertension. In previous studies ALDO and the glucocorticoid corticosterone (CORT) and its inactive metabolite 11-dehydrocorticosterone (11DHC) have been quantified in plasma and urine samples but their sites of actions within the kidney remain unknown. Glucocorticoids were recently localised in brain and adrenal tissue using mass spectrometry imaging (MSI). MSI has the potential, therefore, to map CORT, 11DHC and ALDO to defined regions of the kidney. The hypothesis of this study was that the proportions of active and inactive corticosteroid change along the different kidney histological zones. The major aim was to optimise MSI technology for application to the kidney and use this approach to assess the distribution of steroids under control, high and low salt diets. In the second chapter corticosteroid amounts were assessed in kidney subregions using liquid chromatography tandem mass spectrometry (LC-MS/MS). Kidneys were physically separated into cortex and medulla with CORT levels being higher compared to 11DHC in both histological areas. Western blots were used to validate cortex-medulla separation indicating that following manual dissection, cortical contamination remained in medullary samples and vice versa. Therefore, MSI was explored as an alternative approach. Optimisation of a MSI method to assess renal steroids distribution is described in Chapter 3, adapting a method previously reported to assess brain steroids. Detection of corticosteroids derivatised with Girard-T (GirT) was achieved in kidney using matrix assisted laser desorption/ionisation (MALDI), which proved more successful than desorption electrospray ionisation. Subsequent experiments utilised MALDI coupled to a 12T FT-ICR instrument. Several different parameters such as cutting temperature, humidity level, matrix density and spatial resolution were optimised and the final approach validated. In the following chapter, the validated method was applied to kidneys from healthy male mice (age 10 weeks) to develop an approach for co-registration of MSI maps with histology images. MSI maps of corticosteroids were generated superimposed and used to define steroid localisation along the kidney sections. CORT was localised along papilla, medulla and inner cortex, while 11DHC was highly concentrated only in medulla. High levels of ALDO were found in medulla and outer cortex. Molecular markers of histology within the MSI datasets were screened and putatively identified. Data imputation was also implemented for later comparison of steroid signal intensity between experimental groups. In the last data chapter the effect of the varying dietary salt intake on the amounts and distribution of CORT, 11DHC, and ALDO in murine kidney was explored; low (0.03%), normal (0.3%), and the high (3%) salt diets were compared. The pattern of distribution of active and inactive glucocorticoids as well as ALDO remained the same, as described above, across the different salt diets. Quantitation of signal intensity showed that both CORT and 11DHC were an order of magnitude lower than ALDO and in all three diets ALDO remained the same. To conclude, the outcome of this thesis was to successfully develop approaches to allow MSI of corticosteroids within the mouse kidney. The major findings were: 1) Active and inactive corticosteroids were localised in different areas in mice that underwent normal salt diet, 2) Molecular markers were found in MSI datasets and used for definition of kidney areas and 3) Quantitation of corticosteroids in plasma and tissue samples both in normal and abnormal salt intake conditions showed that CORT is higher than ALDO in plasma but ALDO is higher in kidney regardless of the type of diet. MSI holds great promise to dissect corticosteroid signalling in functional zones of the kidney and thus will be a new tool to help uncover the mechanisms whereby the kidney contributes to high blood pressure and why this differs between individuals.