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. 2021 May 3;32(5):1131–1149. doi: 10.1681/ASN.2020081112

Figure 1.

Figure 1.

Male db/db but not female db/db mice display salt sensitivity. (A) Female and (B) male 30-week-old db/db and db/+ mice were exposed to an HS diet for 4 weeks. MAP was continuously monitored by radiotelemetry. Data are expressed as daily average. At the end of the experiment (week 34), mice were housed in metabolic cages for 24 hours. Mice treated with an MS diet were used as controls. *P<0.05, **P<0.01 versus db/+ mice by multiple t test. (C) Sodium and (D) potassium balance was calculated on the basis of urinary sodium and potassium concentration, urinary volume, and food intake (displayed in Supplemental Figure 1). Data are expressed as dot plots. Horizontal bars represent the mean±SD (n=6 per group). (E) Immunoblots for sodium-hydrogen exchanger isoform 3 (NHE3), NKCC2, NCC, NCC phosphorylated at serine 71 (NCCp), full-length (FL) and cleaved (CL) ENaC α subunit, βENaC, γENaC-FL and -CL, and SGK-1 were performed in kidney homogenates with a constant amount of protein per lane; β-actin was used as loading control. Relative abundance from each group is displayed below the corresponding blot. Female db/+ mice on MS was considered as 1.0. Blots from three representative samples are shown. Uncropped immunoblots and dot-plot analysis for all samples are shown in Supplemental Figures 3–9. *P<0.05, **P<0.01, ***P<0.001 versus the corresponding group treated with MS. † P<0.05, †† P<0.01, ††† P<0.001 versus male db/db HS by three-way ANOVA. (F) Summary of renal sodium-transporter profile expressed as the ratio between HS and MS values. Black arrows indicate those ENaC subunits not suppressed by the HS diet. (G) For the amiloride test, the difference (Δ) in urine sodium/creatinine concentration ([Na+]/[creat.]) between vehicle (0.9% saline) and amiloride injection (5 mg/g body wt in 100 ml of 0.9% NaCl) was calculated. Urine was collected for 4 hours. Data are expressed as dot plots. Horizontal bars represent the mean±SD (n=6 per group). *P<0.05, ***P<0.001 by three-way ANOVA. (H) Immunohistochemical analysis of αENaC in kidney samples. Black arrows indicate positive αENaC staining. K+, potassium; Na+, sodium.