Fig 2: Effect of co-expression of MASP-2 and ENaC on current in Xenopus oocytes. (A) When co-injecting cRNAs for αβγENaC and MASP2, an increased amiloride-sensitive current was detected with a concentration of 8 and 12 ng MASP2. (B–D) In Xenopus oocytes injected with cRNAs encoding αβγENaC, no increased amiloride-sensitive current was detected when applying MASP-2 in the buffer, neither when adding the pattern recognition molecule collectin CL-LK (heteromer of collectin liver 1 and kidney 1), known to interact with MASP-2 (C), nor when decreasing the Na+ concentration of the buffer (D).

Fig 3: Changes in plasma concentration of MAp19 and MASP-2 in response to Na+ intake. (A,B) In a cohort of healthy young men (n = 10) ingesting a diet with high (250 mmol/day) and low (70 mmol/ day) Na+ content for five days, no difference was detected in plasma concentrations of MASP-2 and MAp19 in response to Na+ intake. Samples obtained on habitual diet were analyzed to allow comparison and increased power in correlation analysis (indicated as baseline). (C) The MAp19/MASP-2 ratio was significantly increased in conditions with high Na+ intake (p = 0.0009). (D,E) The ratio did not correlate with p-aldosterone (p = 0.16, R2 = 0.07), but the delta values of p-aldosterone in the individuals correlated significantly with the delta values of ratio MAp19/MASP-2 (p = 0.01, R2 = 0.55). (F) Ratio MAp19/MASP-2 correlated significantly with the fractional Na+ excretion (p = 0.03, R2 = 0.16). (G) No difference in urine MAp19/creatinine concentration ratio was detected with different Na + intake (p = 0.82). Intact MASP-2 was not detected in the urine. (H) In kidney cortex homogenate from patients receiving diuretics (Diu) before nephrectomy and therefore with predicted higher plasma aldosterone concentration than in patients not receiving medicine (Ctrl), no significant different in MASP-2 protein abundance was detected. Original uncropped blots are presented in Supplementary Fig. S2G. HCP = human kidney cortex pool, n = 4. p = plasma.

Fig 4: Detection of MASP-2 protein variants in liver, kidney and urine. (A) Three distinct murine monoclonal anti-human antibodies targeting MASP-2 were used, two reacting with epitopes within the SP-domain of MASP-2; an in-house (15-7-3) and a commercial (Origene), the third (13B7) reacting with an epitope in the N-terminal domain. The six domains of intact MASP-2 are illustrated, including the two CUB-domains, the epidermal growth factor (EGF)-like domain, the two complement control repeats (CCPs) and the serine protease (SP) domain. The alternatively spliced short variant MAp19 also shown. N- and C-terminals are outlined. (B) Predicted cleavage products detected by the two antibodies. Under non-reduced conditions, the antibody targeting the A-chain (13B7) would detect bands at ˜ 75 kDa (MASP-2) and ˜ 20 kDa (MAp19), and the antibody targeting the B-chain (15-7-3) would detect at ˜ 75 kDa. Under reduced conditions, 13B7 is expected to detect the 47 kDa A-chain if MASP-2 is activated, whereas 15-7-3 antibody is expected to detect the 27 kDa variant (the SP-domain) (Fig. 2B, lane 3 + 4). (C) When using the anti-SP-antibody (15-17-3), a band ˜ 70 kDa was detected in human kidney homogenate in both human pools of kidney cortex (HCP, n = 4), outer (HOM, n = 4) and inner medulla (HIM, n = 4) in reduced samples. (D). A similar band was detected in human liver homogenate, under reduced conditions, when using the anti-SP-antibody (15-17-3). HCP = human cortex pool (n = 4). (E) In human plasma, the anti-SP-antibody (15-17-3) detected a band migrating at ˜ 75 kDa. (F) Full length MASP-2 was not detected with protein from urinary extracellular vesicles (uEV) or in experimentally concentrated urine when using the 15-17-3 antibody. When testing the antibody targeting the A-chain (13B.7), no signal was detected in a kidney cortex pool (HCP, n = 4). In contrast, the alternative splice variant MAp19 was detected in uEVs and concentrated urine. (G) Under non-reduced condition, a band likely representing intact MASP-2 at 75 kDa was apparent in kidney cortex (HCP) and plasma with the in house 15-17-3 antibody whereas the commercial monoclonal antibody (OriGene #TA812533S) directed against the SP domain confirmed the presence of a 75 kDa band in non-reducing lanes. Both antibodies detected a band migrating slightly faster, at ˜ 70 kDa, in a human kidney cortex pool (HCP) and plasma. (H) Both the commercial antibody (OriGene #TA812533S) and the in-house monoclonal antibody (15-17-3) detected a band ˜ 75 kDa, when recombinant MASP-2 was applied to the membrane under non-reduced conditions. Original uncropped blots are presented in Supplementary Fig. S2A–F.

Fig 5: MASP-2 is localized in the intercalated cells of the collecting duct. (A,B) Using the antibody directed against the SP-domain of MASP-2 (15-17-3), immunohistochemical experiments revealed a positive signal in human liver sections that was associated with hepatocytes. (C) Negative control. (D–F) In human kidney sections, immunochemical signal from MASP-2 was detected in distinct tubular segments both in cortex and in medulla which were not proximal tubules. Along labeled segments, certain subsets of cells were not labeled (E,F). (G,H) MASP-2 co-localized with the intercalated cell marker ATP6V1B1. (I) MASP-2 did not co-localize with principal cell marker aquaporin-2 (AQP-2 turquoise, MASP-2 magenta, DAPI/nucleus = blue).