47 ± 0.05 Effluent concentration to plasma concentration ratio of mAM 0.85 ± 0.07 Fig. 1 Lack of SHP099 clinical trial correlation between AM concentration in plasma and effluent Fig. 2 a A correlation between AM concentration in effluent and the D/P ratio of creatinine. b. A negative correlation between the mAM/AM ratio in effluent and the D/P ratio of creatinine AM, mAM

concentration, mAM/AM ratio and CA125 concentration in effluent AM and CA125 concentrations in effluent showed positive correlation (r = 0.51, p = 0.02) (Fig. 3a). However, mAM and CA125 concentrations in effluent showed no correlation (r = 0.33, p = 0.16) (Fig. 3b). Similarly, the mAM/AM ratio and CA125 concentration in effluent showed no correlation (r = −0.32, p = 0.17) (Fig. 3c). Fig. 3 a A positive correlation between AM concentration in effluent and EPZ5676 CA125 concentration in effluent. BI 2536 cell line b A lack of correlation between mAM concentration in effluent and CA125 concentration in effluent. c A lack of correlation between the mAM/AM ratio in effluent and CA125 concentration in effluent AM expression of PMCs in effluent Immunocytological study revealed that AM was diffusely expressed in the cytoplasm of PMCs. A representative example of PMCs producing AM is shown in Fig. 4. Rhodamine fluorescence, measured semi-quantitatively by confocal laser microscopy, was not detected in the

vimentin-negative cells. The fluorescence intensity using confocal laser microscopy for the anti-AM antibody on the cells identified as PMCs had a standard deviation 558 ± 142-fold next stronger signal than the cells which were vimentin-negative. The absence of AM indicated the cells were not PMCs. On the other hand, the vimentin-positive cells could be used to calculate the intensity of rhodamine. Fig. 4 A representative example of PMCs

showing diffuse expression of AM in the cytoplasm. Expression of AM was confirmed by double staining. Rhodamine showed expression of AM, and FITC-stained vimentin. The cytoplasmic portion with AM is shown in red. The overlap of AM and vimentin is shown in yellow Discussion AM was isolated from the adrenal medulla and is a potent vasodilative peptide [1]. mRNA of human AM is highly expressed in pheochromocytoma as well as in various tissues or cells, including normal adrenal medulla, kidney, lung, and heart [10]. AM levels in plasma of patients with poorly controlled diabetes were significantly higher than in healthy volunteers. This suggests that the elevated plasma levels of AM may originate from vascular AM exposed to hyperglycemia via protein kinase C-dependent pathway [5, 11]. Post-translational amidation turns AM into its active form, mAM [1], but precise mechanisms of amidation or an enzyme responsible for amidation has not been identified. In PD therapy, PMCs are exposed to high glucose by dialysate and they may express AM.