PMC

Vasopressin and renin-angiotensin hormonal axes in the PCK rats after dietary treatment. (a,b) Shown are urinary copeptin (a) and circulating (plasma) Ang II (b) levels at the end of the 8 week long dietary treatment in the PCK rats fed a SD and a NS diet (for copeptin) or SD, NS and HS diets (for Ang II). Copeptin levels were undetectable in the urine of the HS diet fed PCK animals. (c) Aldosterone levels in the urine and plasma of the PCK rats fed a SD, NS and HS diets. Values were compared using a one-way ANOVA with a Tukey post-hoc test. In box plots, the box is SEM, whiskers represent SD, and line within the box shows median value.

Kidney injury in PCK rats fed diets with different sodium content. (a) Representative FITC-inulin clearance curves (left) and a summarizing graph showing GFR levels (right) obtained from PCK rats fed normal (0.4% NaCl, NS), high salt (4% NaCl, HS), and sodium-deficient (0.01% NaCl, SD) diets at the end of the experimental protocol. (b) Plasma BUN values in PCK rats fed a NS, HS and SD diets. N = 8 animals per group. Values were compared using a one-way ANOVA with a Tukey post-hoc test. In box plots, the box is SEM, the whiskers represent SD, and the line within the box shows median value.

ENaC activity is altered in the miR-9a-5 overexpressed experimental group. (a) Representative gap-free current traces from cell-attached patches made on mCCD cells of naïve, untransfected cells (upper), control (cont) miR with a non-specific targeting sequence (middle) and miR-9a-5 (lower) group. The closed (c) and open (o) states are denoted with dashed lines. Currents were recorded at the membrane voltage of −60 mV. # denotes p < .05 between control group and miR9a-5 group. (b) Current-voltage relationship and representative examples of ENaC single-channel current at different membrane potentials made on mCCD cells of miR group. (c,d,e) Summary graphs of NP_o (c), P_o (d), and N (e) for ENaC activity in naïve (white), control miR (dark grey) and miR-9a-5 (black) groups. *P < .05, **P < .01, ns – not statistically significant, miR9a-5 versus control-miR group by ANOVA. Values were compared using a one-way ANOVA with a Tukey post-hoc test. In box plots, the box is SEM, whiskers represent SD, and line within the box shows median value.

Study protocol and basic physiological parameters assessment throughout the study. (a) Experimental protocol showing the 3 groups of PCK rats studied on normal (0.4% NaCl, NS), high salt (4% NaCl, HS), and sodium-deficient (0.01% NaCl, SD) diets for 8 weeks starting at 6 weeks of age. Metabolic cage urine collections and GFR measurements were performed throughout the protocol. Values were compared using a repeated-measures ANOVA. (b,c) Daily urinary output (b) and food intake (c) of the experimental groups measured during the study. (d,e) Body weight (d) and two kidneys to body weight ratio (2 K/BW, (e)) data was obtained from the animals at the end of the experimental protocol. Values were compared using a repeated-measures ANOVA with Holm-Sidak post-hoc (d) and one-way ANOVA with a Tukey post-hoc test (e). In box plots, the box is SEM, whiskers represent SD, and the line within the box shows the median value. N = 8 animals per group.

Renal cyst development in PCK rats fed diets with different sodium content. (a) Upper panel shows representative images from H&E-stained kidney tissues of PCK rats fed a SD, NS and HS diets according to the experimental protocol. The scale bar (3 mm) is common for all top row images; the lower row of images demonstrates blown-up regions of cortical area from the upper images. (b) Summary graphs illustrating cystic index (percent of cysts relative to tissue slice area) in relation to whole kidney slices, and cystic index specifically in the cortical renal area of PCK rats fed a SD, NS and HS diets. Tissues from a minimum of 7 animals per group were analyzed. (c) Hepatic cyst development in PCK rats fed diets with different sodium content. Upper panel shows representative images from H&E-stained liver tissues of PCK rats fed a SD, NS and HS diets according to the experimental protocol. The scale bar shown is common for all top row images and is 2 mm; the lower row of images demonstrates liver regions expanded from the upper panel. (d) Summary graph illustrating cystic index (percent of cysts relative to tissue slice area) in the livers of PCK rats fed a SD, NS and HS diets. Tissues from at least 6 animals per group were analyzed. Values were compared using a one-way ANOVA with a Tukey post-hoc test. In box plots, the box is SEM, whiskers represent SD, and line within the box shows median value.

ENaC protein and mRNA expression in PCK rats fed diets with varying sodium content. (a) Western blotting showing expression levels of α-ENaC in the renal cortex of the PCK rats fed a SD, NS and HS diets, and a summary graph with densitometry values (normalized to β-actin expression for the same samples). Each lane on the blot is one animal. (b) mRNA expression for α-, β-, and γ-ENaC in the renal cortex of the PCK rats fed a SD, NS and HS diets. (c) Western blotting showing expression levels of AQP2 in the renal cortex of the PCK rats fed a SD, NS and HS diets, and a summary graph with densitometry values (normalized to β-actin expression for the same samples). (d) miRNAs found to be differentially expressed (p < .05) in the cortical tissues of the PCK rats fed a SD, NS and HS diets. *, p < .05, **p < .01. (e) Western blots demonstrating total ENaC subunit expression in cultured mCCD cells transfected with a control scrambled miR sequence or miR9a-5p, with summary densitometry graphs (normalized to β-Actin and relative to control mCCD expression). Both β- and γ-ENaC are significantly decreased in mCCD cells over expressing miR9a-5p. * p < .05. Values in (b) and (c) were compared using a one-way ANOVA with a Tukey post-hoc test. For (d), differential expression was determined by DESeq2 method []. False discovery rate within statistical analysis was controlled for using the Benjamini-Hochberg method, p value cutoff was 0.05. Graphs with whiskers show mean value in each group and SEM. In (e), following a Shapiro-Wilk normality test, control and experimental values were compared using an unpaired student t-test.