Fig 2: Indirect immunofluorescence of IRBIT in rat kidney. (A) Merged overview of double staining of IRBIT (red) and α1 (green) in a rat kidney section. The original unmerged images of IRBIT and α1 were shown in Supplementary Figure S1. (B–D) High magnification view showing that IRBIT is abundantly expressed in mTAL. (E–G) Localization of IRBIT and NKCC2 in mTAL. (H–J) Localization of IRBIT (red) and α1 (green) in cortex. G, glomerulus; mTAL, medullary thick ascending limb; CX, cortex; OM, outer medulla; IM, inner medulla. Blue indicates the nuclei counter-stained with DAPI.

Fig 3: Interaction of IRBIT and L-IRBIT with NBCn1. (A) Alignment of the initial Nt of NBCn1 with the unique Nt of NBCe1 (variant B). (B) Characterization of structural elements of NBCn1 essential for binding of IRBIT by yeast-2-hybrid assay. (C) Summary for the effect of the auto-inhibition domain of NBCn1 (residues 1–115) on transport activity of NBCe1. (D) Summary for the transport activity of NBCe1 containing variants of AID of NBCn1. (E) Co-immunoprecipitation analysis showing the interaction of NBCn1 with L-IRBIT in rat kidney. In Panels (C,D), data are presented in mean ± S.D. in Panels (C,D). The numerals in the parentheses indicate the number of individual oocytes included in each bars. For statistical analysis, one-way ANOVA followed by Tukey’s comparison was performed. *p < 0.05; ****p < 0.001. ns: not significant.

Fig 4: Schematic diagram summarizing the localization of NBCn1, IRBIT, L-IRBIT, PP1, NKCC2 along the nephron. The expression localizations of IRBIT, L-IRBIT, and PP1 in the kidney were systematically characterized for the first time in the present study. The expression of NBCn1, NBCn2 and AE2 in the mTAL was established previously (Alper et al., 1997; Kim et al., 2003; Praetorius et al., 2004; Guo et al., 2017). The present study confirmed the localization of NBCn1 in the mTAL, but not in other segments of the nephron. The specific localization of NCC in DCT was established previously (Campean et al., 2001). NKCC2 is a specific marker for the mTAL and cTAL (Ecelbarger et al., 1996; Gimenez and Forbush, 2003). Calbindin is a specific marker for DCT, CNT, and CCD (Campean et al., 2001; Wetzel and Sweadner, 2001). G, glomerulus; PCT, proximal convoluted tubule; PST, proximal straight tubule; DTL, descending thin limb; ATL, ascending thin limb; mTAL, medullary thick ascending limb; cTAL, cortical thick ascending limb; MD, macula densa; DCT, distal convoluted tubule; CNT, cortical connecting tubule; CCD, cortical collecting duct; OMCD, outer medullary collecting duct.

Fig 5: Indirect immunofluorescence of L-IRBIT in rat kidney. (A) Merged overview of the double-staining of L-IRBIT (red) and α1 (green). The original unmerged images of L-IRBIT and α1 were shown in Supplementary Figure S4. (B–D) Localization of L-IRBIT and α1 of Na+ -K+ -ATPase in outer medulla. (E–G) Localization of L-IRBIT and NKCC2 in outer medulla. (H–J) Localization of L-IRBIT (red) and α1 (green) in cortex. CX, cortex; IM, inner medulla; OM, outer medulla; mTAL, medullary thick ascending limb; OMCD, outer medullary collecting duct. Blue indicates the nuclei counter-stained by DAPI.