Mapping of Corticosteroids in Murine Kidneys Using Mass Spectrometry Imaging

Abstract

Renal sodium reabsorption is important for blood pressure homeostasis and is physiologically regulated by aldosterone; glucocorticoids may also contribute. Abnormal steroid hormone activity within the kidney contributes to hypertension but the mechanisms are not fully defined. Molecular profiling of receptors and metabolising enzymes indicates that steroid hormone action is compartmentalised within the kidney. Ambient steroid concentrations are a critical factor governing bioactivity at a cellular level, but this is largely unknown, and the kidney remains a “black box”. Mass spectrometry imaging (MSI) was applied recently to localise steroids in brain and testes, and here is applied to kidney. Image reconstruction permits characterisation and co-registration of kidney histological regions based on regional markers detectable by MSI. Our aim was to map and quantify glucocorticoids and aldosterone in different histological zones (cortex, medulla) of murine kidneys, using an optimised MSI method. This approach has the potential to map steroids within functional zones of the kidney, providing fundamental new information relevant to hormone action in health and in disease. Cryosections of male C57BL6 mouse kidneys (age 12 weeks, n=6) were subject to MSI following derivatisation using Girard T reagent and α-cyano-4-hydroxycinnamic acid matrix application. Images were reconstructed, and methods optimised to enhance signal and limit diffusion of analytes of interest. Matrix assisted laser desorption/ionisation (MALDI) was used as a sampling method, coupled to Fourier Transform Ion cyclotron mass spectrometry. Ions with m/z 458.3010, 460.3166 and 474.2957 were detected, using MALDI, in renal sections, close to the predicted masses of 458.3013 (Δppm=0.65), 460.3169 (Δppm=0.65), and 474.2962 (Δppm=1.05), for derivatives of 11-dehydrocorticosterone, corticosterone and aldosterone respectively. Untargeted evaluation of ions was conducted to find regional markers that would allow definition of kidney histological zones. The Heat maps generated indicated that corticosterone intensity was higher in the inner cortex area close to the corticomedullary junction than the rest of the kidney. In contrast 11-dehydrocorticosterone was detected mainly in medulla and aldosterone signal was equally strong in medulla and outer cortex. Thus, MSI can be used map the sites where glucocorticoid and mineralocorticoids are most active in regulating renal tubular function. Co-localisation of steroids of interest with zonal markers by MSI permits steroid mapping in functional renal zones of the kidney. This approach provides fundamental new insights into the physiological control of sodium transport by steroids and opens doors to understanding changes in disorders of blood pressure. The project was supported and funded by Kidney Research UK.