Deficiency of Renal Cortical EGF Increases ENaC Activity and Contributes to Salt-Sensitive Hypertension

ENaC expression and activity are inappropriately upregulated in SS rats fed a HS diet. (A and B) Effects of a HS diet (4%, 3 weeks) on protein expression of ENaC subunits in the SS rats kidney cortex (n=7 in each group). The densitometry values are normalized to GAPDH. Equal loading control (GAPDH) is shown for analysis of α-ENaC expression. Similar equal loading control is done for analysis of β- and γ-ENaC subunits (not shown). (C and D) Western blots and summary graphs demonstrating expression of α-, β-, and γ-ENaC subunits in SS.13^BN rats fed a LS or HS diet (n=4 in each group), respectively (0.4 and 4%, 3 weeks). Equal loading control (β-actin) is shown for analysis of α-ENaC expression. Similar equal loading control is done for analysis of β- and γ-ENaC subunits (not shown). GAPDH, Glyceraldehyde 3-phosphate dehydrogenase. ***P<0.001 versus LS diet.

Expression of β-ENaC in SS and consomic SS.13^BN rats fed a HS diet. (A) Immunohistochemical staining and (B) summary graph of β-ENaC expression in the kidney cortex of SS and SS.13^BN rats fed a HS diet (8%, 1 week). ***P<0.001 versus SS rats fed a HS diet. Original magnification, ×40 in A.

SS rats demonstrate higher ENaC activity on a HS diet compared with a LS diet and SS.13^BN consomic rats. Representative single-channel traces (A) and summary graph (B) for ENaC from freshly isolated split opened CNTs/CCDs from SS and SS.13^BN rats fed a LS or HS diet, respectively. These patches were held at the test potential of V_h = −V_p = −60 mV. Inward Li⁺ currents are downward. Dashed lines indicate the respective current state, with “c” and “o_n” denoting the closed and open states. *P<0.05 versus other groups.

Chronically elevated RPP alters ENaC expression and glomerular injury. (A) Representative immunohistochemical staining for β-ENaC (shown in brown) in SS rat kidney cortical sections of servo-controlled and uncontrolled kidneys. Negative control tissue stained with secondary antibodies in the absence of primary antibodies is also shown. (B) Schematic diagram of servo-control experiments. (C) Densitometric analysis of relative β-ENaC expression in the cortex of servo-controlled and uncontrolled kidneys harvested from SS rats fed a HS diet. (D) Representative images of glomeruli in Trichrome-stained left and right kidneys. (E) Average glomerular injury score calculated in the kidneys of experimental animals (n=6). *P<0.05 and **P<0.01 versus servo-controlled kidneys (n=6). Original magnification, ×40 in A and D.

EGF concentration in the kidney cortex isolated from SS and SS.13^BN rats fed a LS or HS diet measured with ELISA assay. ***P<0.001 versus SS rats fed a LS diet and SS.13^BN rats on both diets.

EGF decreases ENaC activity in SS rats fed a HS diet. Effect of intravenous infusion (3 days; 10 µg/h per kilogram) of EGF on the ENaC activity in SS rats fed a HS (4%) diet. SS rats are fed with a HS diet for 3 days before and during EGF treatment. Representative single-channel traces (A) and summary graph (B) for ENaC activity from freshly isolated split opened CNTs/CCDs derived from SS rats after the treatment with EGF or vehicle are shown. ***P<0.001 versus SS rats treated with vehicle.

Chronic intravenous infusion of EGF prevents development of hypertension and kidney damage in SS rats fed a HS diet. Protocol (A) and effect of intravenous EGF infusion on the MAP (B) in the SS rats fed a HS diet as described in the corresponding protocol (n=3–4). (C) Light microscopy of Trichrome-stained sections of whole kidney (×1) and renal cortex (×60) of SS rats fed a HS diet for 10 days and intravenously infused with vehicle or EGF (2 μg/h). The averaged percentage of protein casts area in the renal outer medulla (D) and the glomerular injury score (E) are shown. *P<0.05, **P<0.01, and ***P<0.001 versus SS rats treated with vehicle.