Salt loading decreases urinary excretion and increases intracellular accumulation of uromodulin in stroke-prone spontaneously hypertensive rats

Abstract

Uromodulin (UMOD) is the most abundant renal protein secreted into urine by the thick ascending limb (TAL) epithelial cells of the loop of Henle. Genetic studies have demonstrated an association between UMOD risk variants and hypertension. We aimed to dissect the role of dietary salt in renal UMOD excretion in normotension and chronic hypertension. Normotensive Wistar-Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP) (n=8/sex/strain) were maintained on 1% NaCl for 3 weeks. A subset of salt-loaded SHRSP was treated with nifedipine. Salt-loading in SHRSP increased blood pressure (ΔSBP 35 ± 5 mmHg, P<0.0001) and kidney injury markers such as kidney injury marker-1 (KIM-1; fold change, FC 3.4; P=0.003), neutrophil gelatinase-associated lipocalin (NGAL; FC, 2.0; P=0.012) and proteinuria. After salt-loading there was a reduction in urinary UMOD excretion in WKY and SHRSP by 26 and 55% respectively, compared with baseline. Nifedipine treatment reduced blood pressure (BP) in SHRSP, however, did not prevent salt-induced reduction in urinary UMOD excretion. In all experiments, changes in urinary UMOD excretion were dissociated from kidney UMOD protein and mRNA levels. Colocalization and ex-vivo studies showed that salt-loading increased intracellular UMOD retention in both WKY and SHRSP. Our study provides novel insights into the interplay among salt, UMOD, and BP. The role of UMOD as a cardiovascular risk marker deserves mechanistic reappraisal and further investigations based on our findings.

Keywords: Salt; Tamm Horsfall protein; blood pressure; renal physiology; salt-sensitive hypertension.

Figure 1. Salt-loading increases BP in SHRSP

SBP was assessed by tail-cuff measurements. Each single dot represents an average of six to ten consecutive weekly SBP recordings. (A) shows the difference in SBP between 11-week-old WKY (n=32) and SHRSP (n=54). ****P<0.0001 (Welch’s t test). Sexual dimorphism in SBP was observed in SHRSP (female shaded gray). (B) shows differences in SBP (ΔSBP) between baseline (11-week-old) and after 3 weeks of salt loading. WKY were divided into two groups (n=16/group): normal salt (NS; no salt drinking water) and high salt (HS; 1% NaCl in drinking water). SHRSP were divided into three groups (n=16/group): NS, HS and high salt with nifedipine (HS-Nif). Nifedipine prevented the salt-induced increase in SBP in salt loaded SHRSP. **P<0.01, ***P<0.001 (Brown–Forsythe Welch ANOVA test). Symbols with gray fill indicate female. Bars indicate mean ± s.e.m.

Figure 2. Salt-loading induced kidney injury markers in SHRSP

(A) shows the significant difference in kidney weight among strains. Salt-loading significantly increased the kidney weight. **P<0.01, ***P<0.001, ****P<0.0001 (ANOVA with Bonferroni’s multiple comparison test). (B) shows the significant difference in ACR in SHRSP after salt-loading which is ameliorated with nifedipine treatment. *P<0.05 (Student’s t test). (C,D) represent mRNA expression of kidney injury marker-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL), respectively. *P<0.05, **P<0.01, ****P<0.0001 (Brown–Forsythe Welch ANOVA test). NS: normal salt, HS: high salt (1% NaCl) and HS-Nif: high salt with nifedipine, n=16 per group. Symbols with gray fill indicate female. Bars indicate mean ± s.e.m.

Figure 3. Urinary UMOD decreases with salt-loading in both WKY and SHRSP

Twenty-four hour urine samples were collected from rats caged individually once a week in metabolic cages. Urinary UMOD (uUMOD) concentration in urine samples were quantified by ELISA and normalised to 24-h urine output. (A) represents the difference in baseline (11-week-old rats) 24-h uUMOD between WKY (n=32) and SHRSP (n=54). *P<0.05 (Mann–Whitney test). Salt loading decreased uUMOD excretion in urine in both strains, and nifedipine treatment did not change the salt-induced reduction of uUMOD. (B) compares the 24-h uUMOD (ELISA) between groups at week 3 of salt-loading. Western blot represents pool of samples from each group. **P<0.01, ****P<0.0001 (Brown–Forsythe Welch ANOVA test). (C) represents the percentage change of uUMOD from baseline after 3 weeks of salt-loading in each group. Bars indicate mean ± s.e.m; NS: normal salt, HS: high salt (1% NaCl) and HS-Nif: high salt with nifedipine, n=16 per group. Symbols with gray fill indicate female.

Figure 4. uUMOD excretion does not reflect its mRNA and total kidney protein level

One-fourth of kidney (cut into sagittal plane and then into two halves) was used for RNA and total protein isolation (n=16 per group). (A) shows the significant difference in total kidney UMOD between strains and no significant difference with salt loading. Top: Graph represents results of UMOD ELISA normalised to total protein; Bottom: representative Western blot image of each group. (B) represents the comparison of δC_t of Umod normalised to β-actin within strains. (C,D) represents the ex-vivo experiment, wherein, medullary tubules were isolated from kidneys of WKY and SHRSP (12 ± 3 weeks old). The tubules were incubated either with 10 mM nifedipine (Nif) for 6 h at 37°C. The results are representation of two experiments performed each with WKY (n=4) and SHRSP (n=6) rats. (C) shows no significant change in UMOD secretion in media from both WKY and SHRSP tubules when incubated with nifedipine. (D) shows no change in UMOD in tubule protein lysate in both strains after incubation with nifedipine. Western blots at the bottom of panels (A,B) are representative images. Ctrl = control. Full image of Western blot and analysis can be found in Supplementary Figure S8. (E) shows the strains (n=9 per group) significant difference in hepsin expression in kidney, which is unaffected with salt. Western blot is representative image of each group. *P<0.05, **P<0.01, ****P<0.0001 (Brown–Forsythe Welch ANOVA test). Bars indicate mean ± s.e.m. NS: normal salt, HS: high salt (1% NaCl) and HS-Nif: high salt with nifedipine. Symbols with gray fill indicate female.

Figure 5. Salt loading effected the UMOD trafficking in WKY and SHRSP

(A,B) displays representative immunofluorescence analysis showing UMOD (red) and ER marker calnexin (green) in rat kidney sections. Co-localization of UMOD and calnexin is represented by yellow. Nuclei are stained in blue with DAPI. Scale bar represents 20 µm. Pearson’s correlation coefficients were calculated for four representative tubules (ROI, regions of interest) from eight samples per group. One-way ANOVA was performed to determine statistical significance. (C,D) represent changes in UMOD expression of WKY and SHRSP in cytosolic and membrane fractions of TAL tubule, respectively, when incubated ex-vivo with mannitol (Ctrl) and NaCl (Salt). Western blots are representative images for two experiments with n=4 WKY and n=4 SHRSP. Flotillin-1 was used as membrane marker. Filled black square and filled black star on blots represent the mature UMOD and immature/precursor UMOD. Blots were normalized to total protein intensity using Revert 700 stain. Ns: non-significant, *P<0.05, **P<0.01 and ***P<0.001 (Student’s t test). Bars indicate mean ± s.e.m.