Dissociation of hypertension and renal damage after cessation of high salt diet in Dahl rats

Abstract

Background:

Every year, thousands of hypertensive patients reduce salt consumption in the efforts to control their blood pressure. However, hypertension has a self-sustaining character in a significant part of the population. We hypothesized that chronic hypertension leads to irreversible renal damage which remain after removing the trigger causing elevation of the initial blood pressure.

Methods:

Dahl salt sensitive (SS) rat model was used for chronic continuous observation of blood pressure. Rats were fed a high salt chow to induce hypertension, and then the diet was switched back to normal sodium content.

Results:

We found that a developed hypertension was irreversible by salt cessation: after a short period of reduction, blood pressure grew even higher than in the high salt phase. Notably, the self-sustaining phase of hypertension was sensitive to benzamil treatment due to sustaining ENaC hyperactivity, as shown with patch-clamp analysis. Glomerular damage and proteinuria were also irreversible. In contrast, some mechanisms, contributing to the development of salt-sensitive hypertension, normalized after salt restriction. Thus, flow cytometry demonstrated that dietary salt reduction in hypertensive animals decreased the number of total CD45⁺-, CD3⁺CD4⁺- and CD3⁺CD8⁺- cells in renal tissues. Also, we found tubular recovery and improvement of glomerular filtration rate in the post-salt period vs high salt diet.

Conclusion:

Based on earlier publications and current data, poor response to salt restriction is due to differential contribution of the factors, recognized in the developmental phase of hypertension. We suggest that proteinuria or electrolyte transport can be prioritized over therapeutic targets of inflammatory response.

INTRODUCTION

The World Health Organization estimates 1.4 billion people worldwide have high blood pressure ¹. Weinberger and colleagues reported that 26% of normotensive population was salt-sensitive; moreover, salt-sensitivity is a major contributor to cardiovascular diseases (CVDs) and mortality ². Association of high dietary salt with CVD rates and increased risk of stroke and fatal and other CVD events³. Hypertensive patients are routinely advised to reduce salt consumption (to the level of lower than 5g a day) to control their blood pressure ⁴. Although meta-analyses of the clinical trials confirm that salt restriction improves cardiovascular parameters ^5-7, there is a significant heterogeneity in the BP response ^8-10. Thus, Law and colleagues report that approximately one-third of the patients have an excellent BP response to a reduction of salt consumption, one-third have a modest response, and one-third have the little or no response ¹¹. This irreversibility of BP in this 1/3 of cases is associated with a plethora of mechanisms, underlying the self-sustaining nature of hypertension because the disorder causes multiple cardiorenal abnormalities which remain even after the trigger (dietary factors) is removed.

Origins of salt sensitivity (SS) are extensively studied in experimental settings. Thus, the Dahl SS rats are widely used as a model of hypertension developing as a response to high sodium intake. Elevation of blood pressure after switching to a high salt diet is driven by multiple pathological processes which include neural dysregulation, vascular incompliance, and renal dysfunction ^12-14. The latter process causes improper sodium and fluid retention in the body, increases cardiac output and builds up increased blood pressure. High renal perfusion pressure, in turn, leads to secondary renal damage, exacerbating the ability of the kidney to control blood pressure and complete the vicious cycle of systemic cardiorenal syndrome. Kidneys exhibit infiltration with immune cells, oxidative stress, and upregulation of epithelial sodium transporters ^15-18.

In 1961 L. Dahl reported that a third of salt-sensitive female rats demonstrated sustaining hypertension after cessation of the 8% NaCl challenge ¹⁹. Later incomplete ^20-24 or complete ²⁵ reversion of hypertension after weeks-long salt consumption was reported in Dahl SS rats, but little is known about factors maintaining a high BP in this period. Most studies collect data during high salt diet to characterize critical factors leading to the development of hypertension. In this phase, T-cell infiltration and oxidative stress are recognized as major contributors to renal injury and improper water-electrolyte balance. Immune cell infiltration in the hypertensive Dahl SS rat kidneys was found with histological analysis ^16,26 and flow cytometry^18,27. Using RNAseq approach, Abais-Battad and colleagues revealed that kidney-homing T-cells are dramatically modified in comparison to circulatory T cells of peripheral blood: 2/3 of genes in the transcriptome are differentially expressed ²⁸. T-cells are an important source of cytokines such as interleukin-6²⁹ which stimulates sodium transport in the distal nephron^30,31. Our earlier publications also show that increased activity of the Na-K-Cl cotransporter and epithelial sodium channel (ENaC) mediate abnormal sodium retention in the body ^32,33.

In the current study, we investigated how blood pressure and renal functions respond to switching of a high salt (HS) intake back to the normal salt (NS) diet. This approach determines what pathogenic factors, contributing to the development of high BP, are reversible or sustaining. The latter processes may present special interest in drug development for hypertension, resistant to dietary corrections.

METHODS

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Animals.

Dahl salt-sensitive rats were produced in the colony established in Henry Ford Health System (founders were obtained over 15 generations ago from Medical College of Wisconsin, Milwaukee, WI). All in vivo experiments were conducted according to the ARRIVE guidelines and approved by IACUC; the presented data were collected from males. Animals were bred and housed in a standard 12/12hr light/dark cycle with water and food (Envigo Teklad 8640, 0.4% NaCl) provided ad libitum. Blood pressure telemeters were implanted in the 9-week old rats similarly to earlier study ³⁴. Briefly, HD-S10 transmitters (Data Sciences International, Minneapolis, MN) were surgically installed in the femoral artery and affixed in a subcutaneous pocket in the loin area. After recovery period, starting at 9 weeks of age, blood pressure was recorded continuously via Ponemah software package. Blood pressure (BP) is represented daily as mean pressure (MAP) in 12hr-long inactive (light time) periods.

Protocol 1.

Rats with telemeters were challenged with a high salt diet (4% NaCl, #113756, Dyets, Bethlehem, PA) two times: for 3 days and, after a five-day-long washout period, for 3 weeks. Then high salt challenge was stopped, and BP was recorded for additional eight weeks (N=4). Sensitivity of the post-salt period to diuretics was tested a week after salt restriction with intraperitoneal administration of benzamil (5 mg/kg daily for three days). Age-matched control group (N=2) was kept on the normal salt diet.

Protocol 2.

Rats with telemeters were divided into two groups (N=4 in each group). The experimental group was challenged with a high salt diet for 4 weeks, and then switched back to a normal salt (NS, 0.4% NaCl) diet for one week. The control group stayed on the normal salt diet during the entire experiment. 24-hour urine samples were collected in both groups on a day before switching diet in the experimental group, and sodium and chloride excretion was measured with Carelyte analyzer (Diamond Diagnostics, Holliston, MA). In the end, their kidneys were collected for patch clamp analysis.

Protocol 3

used rats without telemeters. The control group was fed a normal salt diet between 9 and 16 weeks of age. A second aged–matched group was fed a 4% NaCl diet on weeks 12-16. Animals in the 3^rd group were fed a high salt diet for 4 weeks and then switched to the normal salt diet for an additional 3-week term (see protocol on Figure 3A), then the kidneys were collected for analysis by flow cytometry and histological assays (N=5-8 in different groups/assays).

Protocol 4.

Starting age of 4 weeks, rats were raised on a low salt diet (LS, 0.1% NaCl #113764, Dyets, Bethlehem, PA). Telemeters were implanted and, after recovery period, rats were fed the high salt diet for 4 weeks (8-12 weeks of age). Then diet was switched back to a low salt chow for eight weeks. Blood pressure was recorded continuously with periods of turning off to save battery life of the device (N=4).

Electrophysiology.

After Protocol 2, ENaC activity was assessed in freshly isolated non-dilated collecting ducts as previously described ^34-36. Briefly, cortical collecting ducts (CCDs) were isolated with forceps and split-open with sharp micropipettes. Cell-attached currents were acquired with an Axopatch 200B amplifier (Axon Instruments, CA) and Bessel filter LPF-8 at 200 kHz (Warner Instruments, Hamden, CT), interfaced via a Digidata 1550B to a PC running the pClamp 10.6 suite of software (Axon Instruments, CA). The bath solution contained (in mM): 150 NaCl, 1 CaCl2, 2 MgCl2, 10 HEPES (pH 7.4). The pipette solution contained (in mM): 140 LiCl, 2 MgCl2 and 10 HEPES (pH 7.4).

Flow cytometry

was used to assess infiltration of the kidneys with immune cells in the rats in Protocol 3. Under deep anesthesia, the kidneys were flushed and minced with a digestion solution containing 0.05% Collagenase A and 0.001% DNase I. Homogenate was sieved via 70μm mesh and separated with the use of Percoll gradient. The resulting heterogeneous cell preparation was washed, cells were counted and briefly (10 min) fixed at room temperature with 2% paraformaldehyde diluted in PBS. Then, the cells were stained for the following markers using the antibodies: CD45 APC-Cy7 (#202216), CD3 PerCP/Cy5.5 (#201418), CD4 PE/Cy4 (#201516), and CD86 PE (#200308) from Biolegend (San Diego, CA), CD8 BV510 (BD Biosciences, #740139), and CD68 ED1 (Novus NB600-985af488). Cells were acquired using a Fortessa flow cytometer (BD) and the results were analyzed using FACSDiva software (BD) v8.0.1. Instrument compensation was set with compensation beads. Cells were identified using forward vs side scatter light properties followed by the identification of singlets. Approximately 200,000 singlets were collected per sample. Singlets were characterized for CD45 expression. The CD45+ cells were characterized alone and for CD3 expression while CD45+/CD3+ cells were subsequently analyzed for CD4 or CD8. Analysis gates were positioned using unstained and single stained controls to identify positive and negative staining populations.

Glomerular filtration rate

(GFR) in conscious rats was studied in Protocol 3 to assess kidney function before high salt challenge (0.4% salt group), next day after high salt was stopped (4% salt group) and three weeks after high salt cessation (4% then 0.4% salt group). The method described by T. Rieg was adapted for the use in rats earlier ^34,37. 2% solution of FITC-labeled inulin in saline was injected in the tail vein (2 μL per 1 g of body weight), and then a series of blood samples (10 μL each) were collected from conscious animals over a 2-hour period of time. Plasma inulin clearance was measured using a NanoDrop 3300 Fluorospectrometer (Thermo Fisher Scientific) and analyzed in the two-compartment mathematical model.

Histopathology.

In Protocol 3, a portion of kidney was fixed with 10% neutral buffered formalin and stained with Masson’s trichrome to visualize abundance of protein casts as a marker of tissue damage and chronic kidney disease. Percentage area of protein casts to total slice is reported. Glomerular injury was assessed with semi-quantitative method described by Raij et al³⁸: ~200 glomeruli from each rat (N=5-10 rats in each group) were scored 0-4 based on abundance of fibrosis, Bowman space completeness, and capillary structure.

Statistical analysis

and graph plotting was performed in GraphPad Prism and Origin Pro software packages. All data are presented as mean±SEM. Independent groups were analyzed with ANOVA followed by correction for multiple comparisons. Difference in dependent events was confirmed with repeated measures ANOVA with Dunnett’s correction for multiple comparisons. Data in the groups were studied for normal distribution with Shapiro-Wilk test, log-transformation was applied if normality failed. Differences among group means were considered significant when p<0.05.

RESULTS

Partial reversibility of salt-sensitive hypertension in Dahl rats.

In Protocol 1 we challenged rats with a short period of high salt diet and then, after five days of salt restriction – with a second, 3 week-long high salt treatment. A short-term HS challenge reversibly increased mean arterial blood pressure (Figure 1). Then the diet was changed to 4% NaCl again for 3 weeks, and we observed development of hypertension reaching MAP ~160 mmHg in this period. Cessation of the salt challenge led to small but statistically significant drop of BP (Δ−6.2 mmHg). Further monitoring showed that BP stays high (~155 mmHg) with a trend to increase: eight weeks later, BP was even higher than on the last day of HS diet. Interestingly, the sustaining hypertension in the post-salt period was sensitive to benzamil, a diuretic drug blocking epithelial sodium channel. Intraperitoneal injections of benzamil (5 mg/kg) for three days reduced MAP to ~136 mmHg, but hypertension returned after benzamil treatment was stopped. Figure S1A reports systolic and diastolic blood pressure which show a trend similar to mean BP. Heart rate generally decreases with aging; however, benzamil treatment has a noticeable positive chronotropic effect (Figure S1B). BP monitoring in the control group demonstrated that BP elevates with aging in Dahl SS rats kept on normal salt diet, however it remains lower than salt-induced hypertension (Figure 1B).

Figure 1. Irreversibility of salt-induced hypertension.

A – Mean arterial pressure (MAP) measured with telemetry in Dahl SS rats on a normal (NS) or high salt (HS) diet. Male rats (N=4, squares) were challenged with 4% NaCl diet for three days and then – for 3 weeks. Short-term HS diet reversibly increased blood pressure. But after cessation of long-term HS diet, the rats remained hypertensive. In the period of sustaining hypertension, BP was sensitive to ENaC-blocking diuretic benzamil. 8 weeks after switching back to a NS (0.4% NaCl) diet blood pressure stays high. *p<0.05 vs high salt. B – Extended scale of MAP after cessation of a HS det. Circles represent BP in control age-matched group kept on normal salt entire experiment (N=2). ^ p<0.05 vs before benzamil. #p<0.05 vs group challenged with HD diet.

Sustaining ENaC hyperactivity contributes to irreversibility of hypertension.

The hypotensive effect of benzamil shown on Figure 1 indicates that ENaC can be involved in maintaining high BP in the post-salt period. To test this hypothesis, we implanted telemeters in Dahl rats and placed them on normal or high salt diets. After 4 weeks of HS diet, 1/3 of the rats were euthanized, while others were switched to a NS diet for an additional week before they were euthanized. Collected kidneys were studied for ENaC activity in three conditions: 1) permanent NS diet, 2) HS diet and 3) after a week of salt restriction (Figure 2A, endpoints of these conditions are denoted with black, red and blue ovals, respectively).

Figure 2. ENaC contributes to self-sustaining hypertension.

Dahl SS rats were implanted with DSI telemeters, split into two groups (N=4 in each) and BP acquisition was performed continuously. The first group was fed 4 weeks with a HS diet and then switched back to a NS diet. The second group (control) was fed with a NS diet. A – Salt restriction did not cause return of BP to normotensive values. Black, red and blue ovals mark days of urine and organ collection for electrolyte analyses and patch clamp experiment in normal salt control, on high salt and in the post-salt period. B – Daily excretion of sodium and chloride before (black squares), during (red squares) and after (blue squares) HS diet, and in the control group on NS diet (black circles). C – Representative current traces of ENaC activity (holding potential is −60mV) recorded in cell attached configuration in split-open cortical collecting ducts. Summary graph illustrates that HS diet increases ENaC activity (NP_o) which stays high after cessation of the 4% NaCl diet. Each point represents a cell in distal nephron. ### p<0.01; * p<0.05 vs NS diet (2^nd group)

Telemetry confirmed that BP did not significantly reduce within a week after HS restriction and stayed significantly higher than in age-matched rats, fed a normal salt diet during entire experiment (Figure 2A). Urinary excretion of sodium chloride reflects changes in dietary NaCl consumption (Figure 2B) and is in accordance with our earlier data ¹⁷. Cortical collecting ducts, isolated from kidneys of animals on different diets, exhibited high ENaC activity in hypertensive rats fed 4% NaCl diet than in the group kept on a 0.4% NaCl chow. Interestingly, ENaC stayed hyperactive for 7 days after salt restriction. Representative current traces and summary graphs on Figure 2C reveal that the animals fed the normal salt diet had ENaC NP_o=0.72±0.17, whereas the channel activity in the CCDs, collected from the animals in the HS and post-salt periods was higher (1.61±0.34 and 1.48±0.3).

Reversibility of renal infiltration with T-cells in Dahl rats, fed a high salt diet.

Literature indicates that T-cell infiltration of kidneys during high salt diet amplifies BP elevation and renal damage in Dahl SS rats ³⁹. We hypothesized that the T-cell infiltration continues after cessation of a high salt diet and contributes to maintaining of hypertension. Immune cells were isolated from the kidneys and characterized with using flow cytometry in the samples from animals fed 1) normal diet, 2) four weeks of high salt diet, and 3) three weeks after salt restriction (Figure 3A). Figure 3B demonstrates representative analysis of flow cytometry detection of cells for the common leukocyte marker CD45 and T-cell marker CD3. Further characterization of CD45+ CD3+ cells was performed to identify helper T-cells (CD4+) or cytotoxic T-cells (CD8+). Similarly, to the previous reports ^16,18,26 Dahl SS rats exhibited a higher renal count of CD45+ cells at the end of high salt diet. Surprisingly, next three weeks of salt restriction reversed infiltration of kidneys with immune cells and reduced abundance of CD45⁺ cells from 276,450±35,791 to 54,218±9,187 cells per gram of kidney. Lymphocyte distribution followed the same trend: total population of lymphocytes, and particularly Tlymphocytes increased in hypertensive rats fed HS diet but reduced in the post-salt period but animals remained hypertensive (Figure 3C). Besides, total macrophages and their M1 sub-type in kidneys was counted but no statistically significant difference was detected between the groups (Table S1).

Figure 3. Reversibility of T-cell renal infiltration in Dahl rats fed a high salt diet.

A – Protocol 3 (see Methods): 9-week old Dahl rats were fed NS, HS for 4 weeks, or HS (4 weeks) then NS (3 weeks) till 16 weeks age. At the end of treatment, the kidneys were collected for immunophenotyping of immune cells and histological characterization. B – Representative flow cytometry characterization of cells obtained from kidney tissue. Singlets were characterized for levels of CD45⁺ as well as for CD45+ CD3⁺. Cells that were CD45+ CD3+ were subsequently examined for levels of CD4⁺ and CD8⁺. C – Summary graphs of distribution of immune cells indicate that salt-sensitive hypertension is accompanied by an increase of lymphocytes and T lymphocyte populations, but subsequent salt restriction normalized their abundance in the kidney.

Effect of salt restriction on renal tissue damage and glomerular function.

End organ damage aggravates hypertension by forming of a vicious cycle where high BP produces renal injury, and the damaged kidneys, in turn, fail to control blood pressure. We studied whether glomerular and tubular damage contribute to the sustaining phase of hypertension. Similarly to humans, Dahl SS rats develop protein casts in the nephron, a hallmark of chronic kidney disease ⁴⁰. Using Protocol 3, we found that the increased abundance of protein casts in the animals fed the 4% NaCl diet was reversible. Figures 4A-C depicts kidney morphology stained with Masson’s trichrome in the three dietary regimens. The summary graph on Figure 4D reports that kidney casts significantly reduced from 7.5±1% to 3.4±0.7% of total slice area. In comparison, the intact age-matched rats fed NS diet had 3.1±0.4% cast area.

Figure 4. Effect of salt restriction on protein cast formation.

Morphological analysis with Masson’s trichrome staining detects tubular protein casts (arrows), a marker of chronic kidney disease, in the kidneys collected in Protocol 3. Protein casts are rare in intact rats kept on a NS diet (A), however, salt-induced hypertension leads to their massive development (B). Salt restriction leads to tissue recovery and reduction of protein casts abundance (C). D - Summary graph of protein casts percentage on the total slice area.

Proteinuria and associated glomerular damage are hallmarks of salt-sensitive hypertension in Dahl rats. We evaluated glomerular morphology and urinary protein level to characterize glomerular injury. High salt diet and hypertension caused glomerular damage and proteinuria (35±3 mg/day/100gBW). Both parameters remained high for 3 weeks after salt restriction, indicating irreversibility of glomerulopathy (Figure 5A-C). Filtration ability of the kidneys was assessed with plasma clearance after a bolus of intravenous FITC-inulin injection. Glomerular filtration rate was restored after 3 weeks of HS cessation (Figure 5D). Therefore, various aspects of kidney damage respond differently to salt restriction: tubular signs of chronic kidney disease are reversible but glomerulopathy remains causing proteinuria.

Figure 5. Effect of salt restriction on glomerular tissue and function.

A – Representative images of glomerular morphology stained with Masson’s trichrome. Bowman space, fibrosis (blue), structure of capillary tuft was evaluated and quantified and scored glomerular damage summarized (B). C – 24-hrs proteinuria measured with Bradford reaction. D –FITC-inulin clearance in conscious animals reveals an improvement of glomerular filtration rate after salt restriction.

Irreversibility of salt-sensitive hypertension on a lower salt diet.

Observations reported on Figures 1-2, generated a hypothesis that sodium chloride level of the 0.4% NaCl chow is still significant to maintain high blood pressure and, basically, has limited use as a “normal” salt diet. We tested blood pressure in Dahl SS rats raised on a 0.1% NaCl diet (referred “low” salt diet). Rats switched from the low salt diet to high salt for 4 weeks, developed MAP ~143 mmHg (Figure 6), similarly to the group in Protocol 2. Then animals were returned to the low salt diet that caused a -Δ18 mmHg drop in BP within 2 days, however later blood pressure increased and reached high values in 8 weeks. Figure S2 illustrates similar trend in systolic and diastolic arterial pressure during the experiment.

Figure 6. Self-sustaining hypertension after lowering salt consumption to 0.1% NaCl.

Rats raised on a low salt diet were challenged with 4 weeks of HS diet and then returned to 0.1% NaCl chow. Blood pressure was continuously recorded with two breaks to save battery in the telemeters. *p<0.05 vs last day on high salt diet.

DISCUSSION

High salt diet leads to hypertension, and relationships between various involved mechanisms (ENaC activation, renal damage, immune cell infiltration) are still unknown, even described as “the chicken and egg problem”. We believe that the obtained comparative results uncover the sequence of cause-effect events in the pathogenesis of hypertension. Here we report a dissociation of a complex of pathogenic factors triggering hypertension and causing secondary BP increase. Although all studied parameters were previously shown to unidirectionally contribute to the development of hypertension in Dahl SS rats fed a HS diet, the cessation of NaCl does not reverse them all.

As previously summarized, most of the earlier reports indicate that hypertension can be sustained after cessation of a high salt diet. Van Vliet and colleagues demonstrated that reversibility is declining with longer progression of hypertension: experimenters switched HS and NS diets three times within 10 weeks, and found that the rats fed the HS diet for longer time were hypertensive, their blood pressure stayed higher in the periods of NS consumption ²¹. Authors concluded that there are two phases of salt sensitivity and in the second phase accumulation of pathogenic factors cannot be recovered by a normal diet. Results of our Protocol 1 confirmed and extended their data (Figure 1). In the Van Vliet et al article a 0.7% NaCl chow was used as “normal” salt diet and we used a 0.4% NaCl stock diet for most of the study. This aspect together with observation of growing pressure with aging in the rats kept on stock diet, raised a question: can 0.4% NaCl be used as normal salt diet? In a special experiment we demonstrated that reduction of salt content to 0.1% NaCl did not make salt-sensitive hypertension reversible (Figure 6).

The inability to engage water-electrolyte allostasis due to renal dysfunction is the central mechanism of blood pressure elevation in different types of hypertension. Limited reversibility of salt-sensitive hypertension in Dahl rats is likely maintained by excessive ENaC activity which can be detected even three days after HS diet start ¹⁷. The abnormal ENaC-dependent reabsorption is a hallmark of a salt-sensitive phenotype, because salt-resistant animals decrease ENaC activity on a salt load ³³. In earlier publications, we demonstrated that a HS diet abnormally increased ENaC activity and facilitated development of high blood pressure in Dahl rats ^17,33,41. In the current study, ENaC hyperactivity maintains high blood pressure in the post-salt period, according to benzamil-sensitivity of the latter.

Chronic kidney injury is associated with hypertension in Dahl rats. Thus, glomerulopathy, proteinuria and tubular lesions lead to impaired pressure-natriuresis relationship by low filtration, increased reabsorption and tubular congestion ¹⁵. Salt restriction have not reduced glomerular injury and proteinuria. Delivery of massive amount of protein to the distal nephron contributes to ENaC hyperactivity: many studies report that certain enzymes in the ultrafiltrate can cleave ENaC subunits and activate the channel⁴². Similarly to earlier findings⁴³, development of salt-sensitive hypertension reduced glomerular filtration rate but 3 weeks of feeding the normal salt diet restores GFR. Dissociation of GFR and proteinuria is in accordance with clinical data that proteinuria but not GFR has stronger association with systolic and diastolic blood pressure^44,45. Salt restriction also normalized tubular tissues as detected with quantification of protein casts.

A highly increased number of T-cells in the kidney in response to HS load and high BP is well documented in Dahl SS rats ¹⁸ and other models of hypertension ⁴⁶. Histopathology analysis of renal specimens show that T-cells and macrophages are abundant around glomeruli, blood vessels and intratubular space. Infiltrated immune cells exhibit high abundance of NADPH subunits and are recognized to participate in oxidative stress, which leads to renal tissue damage and dysfunction ¹⁶. In the current study, flow cytometry reveals reversibility of T-cells infiltration occurring on a high salt diet. This indicates that the salt rather than blood pressure elevation drives immune cell infiltration. Also, our data indirectly indicate that development of tubular injury is mediated by immune cell infiltration. However, additional experiments are required to provide direct evidence of mechanistic relationships between these factors, especially in the light of other studies. A significantly increased number of T-cells in the kidneys exposed to perfusion pressure was shown in Dahl SS rats fed a HS diet. If the contralateral kidney was simultaneously protected from hypertension by a servo-controlled cuff on the renal artery, immune cell infiltration into this kidney was significantly lower. This approach allows separation of systemic effects of high salt diet and intrarenal factors driven by high perfusion pressure. The authors found that a seven-day reversal of renal perfusion pressure did not return the T-cell infiltration of kidneys to control levels ¹⁸. We believe that our findings, in accordance with Evans et al, confirm a role of dietary salt as a positive regulator in the immune cell mobilization in the kidneys. Moreover, both their data and the earlier publication by Mori et al. ⁴⁷ report that servo-controlled kidneys are protected from formation of protein casts and glomerulopathy. Therefore, although high renal perfusion pressure leads to renal damage (which, in turn, aggravates hypertension), the tubular injury is likely driven by immune cell infiltration, and both factors are reversible by salt restriction. Because our current results provide evidence that reducing a dietary salt intake is an efficient way to limit renal inflammation and damage even in resistant hypertension, supporting the rationale for dietary salt control in pathogenic therapy of chronic and resistant hypertensive diseases.. Potentially, no specific pharmacological therapy directly targeting immune cells is needed to protect the kidney in the patients who lower their salt consumption.

Genetically modified Dahl rats with a lack of functional T- and/or B-cells reveal their involvement in pathogenesis of salt-sensitive hypertension. In Rag1^−/− Dahl rats, Wade and colleagues found a better reversal of hypertension as well as lower nephropathy due to a lack of mature T- and B-lymphocytes ⁴⁸. Differential contribution of T- or B-cells in Dahl rats remain controversial: genetic deficiency of CD247 receptor, which is required for T-cell maturation reduces proteinuria and renal damage, but both beneficial and low effects on blood pressure were reported ^49,50. Interestingly, no effect on macrophage infiltration were found in either current study (Table S1) or earlier investigation¹⁸, focused on hypertensive period after reversal of renal perfusion pressure or dietary salt.

Study limitations.

Salt-sensitive hypertension in Dahl rats is a one of many models of blood pressure studies. Here we have not investigated reversibility of hypertension in females, although sex difference of BP control is an important but poorly studied factor of cardiovascular diseases. It remains unknown whether reversibility typical for high fat induced hypertension in Dahl rats, DOCA-salt or high fructose+salt model in Sprague-Dawley rats. Reversibility of hypertension in AngII-induced hypertension is illustrated by a study where infusion of angiotensin II was used as a pressor agent ⁴⁸. Authors also found an incomplete recovery of tissue damage (protein casts and glomerular score) and proteinuria, while the renal immune cell count was lower in the reversal period than during administration of AngII. We assume that mechanisms of NaCl vs AngII as triggers of hypertension are similar in relation to adaptive immune response. Other limitations of our study include a lack of information about extrarenal mechanisms of hypertension in post-salt period such as vascular compliance and neural control of blood pressure. Except for ENaC analysis, this project focuses on systemic effects and does not provide molecular and intracellular mechanisms maintaining high blood pressure in the salt-restricted groups.

Perspectives:

We believe that a reversal of pressor stimuli is an affordable maneuver improving mechanistic insight of experiments and should be implemented in a wide spectrum of studies. Technical advances of the last decades such as telemetry allow longer in vivo observations with an opportunity to reverse dietary challenges (high salt, fat, fructose, and other components of Western diet). This is important because the most of population pays low attention to preventive measures. As a result, many hypertensive patients start seeking treatment, and changing dietary habits after hypertension is formed. Currently, there is a significant research gap in this field. In this study, we show that proteinuria and sodium reabsorption can be prioritized over immune targets in the treatment of hypertension resistant to salt restriction.

NOVELTY AND RELEVANCE.

What Is New:

After salt cessation, Dahl SS rats remain hypertensive due to improper water-salt balance and kidney damage, but not T-cell infiltration.

What Is Relevant:

Dahl SS rats may serve as a model of human hypertension resistant to salt restriction.

Clinical/Pathophysiological Implications:

Targeting T-cells has low priority in the choice of therapy in hypertensive individuals with low salt consumption.

Abbreviations:

BP

blood pressure

CD

cluster of differentiation

CCDs

cortical collecting duct

ENaC

epithelial sodium channel

FITC

fluorescein isothiocyanate

GFR

glomerular filtration rate

HS

high salt

LS

low salt

NS

normal salt

SS

salt-sensitive