Early renal response to long-term salt loading: mitochondrial dysfunction, ER stress, and uromodulin accumulation in the kidney medulla

Abstract

Kidneys play a critical role in maintaining water and electrolyte balance, but prolonged salt loading can disrupt renal function by inducing osmotic and oxidative stress. Although high salt intake is well-known to contribute to hypertension and kidney damage, the early renal responses to mild, long-term salt intake, particularly in normotensive individuals, remain poorly understood. To address this knowledge gap, we investigated the effects of exposing normotensive Wistar Kyoto (WKY) rats to 1% NaCl over a 3-mo period, focusing on the medullary region and the adaptive cellular mechanisms in response to salt-induced stress. In addition, we examined the acute effects of 4 h of salt exposure on medullary tubules. The long-term salt intake did not significantly alter blood pressure or cause notable kidney damage but did lead to differential expression of proteins associated with mitochondrial dysfunction and endoplasmic reticulum (ER) stress in the renal medulla. Acute 4-h salt exposure triggered a rapid cellular response involving proteins linked to mitochondrial activity and oxidative stress responses. Both acute and chronic settings significantly reduced uromodulin (UMOD) excretion with altered trafficking indicating intracellular accumulation within medullary cells. This provides evidence that chronic salt loading disrupts normal protein handling without immediate renal injury, shedding light on adaptive mechanisms in the kidney to mitigate osmotic stress. These early adaptations provide insights into the mechanisms underlying salt-related renal pathologies and may inform therapeutic strategies for individuals susceptible to the effects of dietary salt.NEW & NOTEWORTHY This study reveals that even in normotensive Wistar Kyoto rats, mild long-term salt loading induces early renal stress without overt kidney damage or hypertension. Novel findings include reduced uromodulin (UMOD) excretion and altered intracellular trafficking in the renal medulla, alongside mitochondrial dysfunction and endoplasmic reticulum stress. These data highlight UMOD as a sensitive marker of salt-induced renal adaptation and provide insights into early cellular responses to salt before clinical disease onset.

Keywords: osmotic stress; renal physiology; sodium balance; uromodulin.

Graphical abstract

Figure 1. Long-term salt-loading increases urinary output and sodium excretion in WKY.

Systolic Blood Pressure (SBP) was assessed by tail -cuff measurements. (A) shows the difference in SBP in WKY from baseline measurements in control (n=6) and 3-month (n=6) salt-loaded groups. (B) Urinary output and (C) fluid intake of WKY rats were significantly increased during 3 months of salt-loading. Data represent mean ± S.E.M. **P<0.01, ***P<0.001, ****P<0.0001 (Mixed-effects analysis with Šídák's multiple comparisons test). (D) Urinary sodium excretion (u-sodium) was significantly increased in the salt-loaded group, represented as the delta change from baseline values for control and 3-month salt-loaded groups (n=6) (± S.E.M). ***P<0.001 (Unpaired t-test). Gene expression represented as fold change to control ± S.E.M of (E) kidney injury marker-1 (KIM-1) and (F) neutrophil gelatinase-associated lipocalin (NGAL), n=6 per group. mRNA levels were normalised to Ubiquitin C (Ubc) expression.

Figure 2. Long-term salt-loading changes total kidney proteome

(A) Schematic diagram showing the study. (B) Principal component analysis (PCA) displays distribution of the biological replicates from control (blue) and salt (pink) kidney proteome. (C) Volcano plot showing up- (red) and down- regulated (blue) proteins in long-term salt-loaded WKY. Top 20 differentially expressed proteins are labelled. (D) IPA-canonical pathway analysis of differentially expressed proteins shows mitochondrial dysfunction, glutathione-mediated detoxification, EIF2 signalling pathway and NRF2-mediated oxidative stress pathways.

Figure 3. High salt-loading causes acute change in medulla–

(A) Medullary tubules were extracted from WKY and ex-vivo incubated with 300mOsm NaCl and mannitol for 4 hours. The panel depicts the methodology for TMT-labelled proteomics. (B) Volcano plot showing distribution of significantly up-(red) and downregulated (blue) proteins in salt group. Top 20 differentially expressed proteins are labelled. (C) Gene ontology analysis showed key changes in medullary tubules after 4 hours of salt incubation. (D) Violin plot showing the differentially expressed proteins belonging to mitochondrial dysfunction. Data represent mean ± S.E.M. **P<0.01, ***P<0.001, ****P<0.0001. (E) Heatmap of various channels and transporters involved in ion homeostasis.

Figure 4. The medullary lysate was extracted from total kidneys of rats from the long-term salt study.

UMOD protein levels and representative Western blots for Binding Immunoglobulin Protein (BiP) and Protein Disulphide Isomerase (PDI) for total kidney lysates (A, B), and BiP and PDI for the medulla (C, D). Error bars represent mean ± S.E.M., *P<0.05 (Welch’s t test). n=6 kidney (per group) was evaluated.

Figure 5. Long-term salt-loading decreases urinary UMOD excretion in WKY.

Urine samples were collected over 24 hours over 4-time points in the study using metabolic cages, whereas blood was collected at the end of the study by cardiac puncture. Urinary and serum UMOD concentrations were quantified by ELISA. Urinary UMOD was normalised to 24-hour output for urine and represented as excretion rate (mg/h). (A) represents urinary UMOD excretion rate for the long-term salt study (n=6 control, n=6 salt). Data is shown as mean ± S.E.M. **P<0.01, ****P<0.0001 (Mixed-effects analysis). (B) shows plasma UMOD levels after 3 months in animals of the long-term salt study are unchanged (n=6 control, n=6 salt), suggesting salt is directly affecting urinary UMOD excretion. Data is shown as mean ± S.E.M. (C) represents urinary UMOD excretion rate for the intermittent salt study (n=4 control, n=6 salt). Data is shown as mean ± S.E.M. **P<0.01, ****P<0.0001 (Mixed-effects analysis).

Figure 6. Comparison of UMOD in long-term salt loading versus Intermittent salt loading.

(A) Total kidney (cortex and medulla) UMOD mRNA expression of long-term salt-loaded group n=6 control, n=6 salt. mRNA levels were normalised to Ubiquitin C (Ubc) expression. (B) Total kidney UMOD protein levels and representative Western blot of each group in long-term study (n=5 control, n=5 salt), showing increased UMOD and intracellular accumulation. Data represented as mean ± S.E.M. *P<0.05 (Welch’s t-test). (C) Total kidney UMOD mRNA expression of intermittent salt-loaded group n=4 control, n=6 salt. mRNA levels were normalised to Ubiquitin C (Ubc) expression. (D) Total kidney UMOD protein levels and representative Western blot of each group of the intermittent study (n=4 control, n=6 salt). (E) UMOD protein levels and representative Western blots from control (n=5) and salt-loaded groups (n=5) of the long-term study in the medullary fraction of their kidneys. (F) Total UMOD protein levels and representative Western blots from control (n=4) and salt-loaded groups (n=6) from the intermittent study in the medullary fraction of their kidneys. Data represented as mean ± S.E.M. The second lower molecular weight band in the Western blots for UMOD is non-specific.

Figure 7. Long-term salt-loading induces accumulation of immature UMOD in the ER of medullary TAL in normotensive WKY rats.

Kidneys of control and salt-loaded animals (n=6, per group) from the long-term study were isolated. (A) and cytosolic fractions (B) isolated from the medullary region of the control (n=6) and salt-loaded groups (n=6). Data represented as mean ± S.E.M. The plasma membrane (referred to as membrane fraction) was validated by the presence of membrane marker Flotillin-1. The cytoplasmic fraction contains the contents of all intracellular organelles (e.g. ER, Golgi apparatus, etc.) and the cytoplasm as confirmed by presence of ER chaperone calnexin, and Flotillin-2 as a marker of the cytosol. Vinculin was used as a loading control. *P<0.05, and **P<0.01 (Welch’s t test). (C) Representative immunofluorescence analysis showing UMOD (green) and ER marker calnexin (red) in from medulla of 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. The scatter plot on the right represents Pearson’s correlation coefficients which were calculated for four representative tubules (ROI, regions of interest) from 5 samples per group. Error bars represent mean ± S.E.M. **P<0.01 (Welch’s t test). Images were evaluated in a blinded fashion. (D) Endo H assay of the medulla samples from the control (n=6) and long-term salt-loaded (n=6) animals. Western blot shows representative bands of the endo H-resistant UMOD with complex glycans (mature, top band represented by “*”) and the ER form of UMOD (immature, bottom band). Error bars represent mean ± S.E.M. *P<0.05. (Welch’s t test). Western blots for UMOD from subcellular fractions of the medulla isolated from the intermittent salt-loading study, showing (E) membrane fraction and (F) the cytosolic fraction. Control n=4 and salt n=6. Data represented as mean ± S.E.M.

Figure 8. The proportion of UMOD at the membrane is decreased after salt incubation in MDCK cells.

Human UMOD-GFP expressing MDCK cells were incubated with DMEM media with additional 60mM salt or 120mM mannitol (osmotic control) for 18 hours. (A) Secreted UMOD protein levels in the media and representative Western blots. Media samples were normalised to total protein stain revert 700 (B) UMOD protein levels in the cell lysate and representative Western blots. UMOD was normalised to Vinculin. (C) mRNA expression shown as fold change relative to mean of control ± S.E.M and normalised to HPRT expression. (n=5 mannitol, n=5 salt). Data is shown as mean ± S.E.M. (D) Immunofluorescence analysis of UMOD in UMOD-GFP expressing MDCK cells after incubation with 60mM salt or 120mM mannitol as an osmotic control for 18 hours at 40X magnification. Representative images of non-permeabilized and permeabilized cells with UMOD (green) are shown and associated relative fluorescence units (RFU) (non-permeabilized (E) and permeabilized (F) calculated from the images displayed as scatter plots. White dashed boxes and arrows represent zoomed in regions of interest. Data is represented as mean ± S.E.M (n=6 images per group) **P<0.01(Welch’s t-test). Scale bar represents 20μm. DAPI used as a nuclear stain (blue). Images were evaluated in a blinded fashion