Mild Hyperuricemia Attenuates Salt-Sensitive Hypertension and Kidney Damage

Abstract

BACKGROUND:

Asymptomatic hyperuricemia is associated with poor outcomes in kidney and cardiovascular diseases, but its causative role remains controversial. Clinical studies have shown a positive correlation between increased dietary sodium intake and urinary uric acid excretion, suggesting that hyperuricemia may influence salt-sensitive hypertension and associated kidney damage.

METHODS:

To study the effects of mild hyperuricemia on salt-sensitive hypertension, male and female Dahl SS rats were fed a diet containing a uricase inhibitor (2% oxonic acid) and 4% NaCl (high salt) or a high salt-only diet for 3 weeks. Analyses were conducted using radiotelemetry, immunohistochemistry, and RNA-Sequencing.

RESULTS:

Uricase inhibition resulted in a ≈3.5-fold increase in plasma uric acid levels in both sexes compared with their respective high salt controls. However, only the male high salt/oxonic acid group exhibited a significant increase in uric acid excretion. The mild hyperuricemia significantly attenuated the progression of hypertension in male but not female rats. The male high salt/oxonic acid group showed reduced kidney hypertrophy and protein cast formation, indicating mitigated kidney damage. Supplementation with oxonic acid reduced oxidative damage in the tubules, as evidenced by the decreased fluorescence intensity of 8-oxodG. RNA-Sequencing analyses of male kidneys revealed that oxonic acid administration was associated with increased expression of genes linked to the activation of various vasodilatory pathways.

CONCLUSIONS:

Our study demonstrates that in male, but not female, Dahl salt-sensitive rats, mild hyperuricemia accompanied by hyperuricosuria slowed the progression of salt-sensitive hypertension, potentially due to reduced kidney damage.

NOVELTY AND RELEVANCE.

What Is New?

• Mild hyperuricemia, accompanied by increased uricosuria, is associated with a significant attenuation in the progression of salt-sensitive hypertension and protects against kidney damage in Dahl SS rats.

What Is Relevant?

• The protective effects of elevated uric acid in male rats were not observed in female rats, highlighting a sex-specific response to hyperuricemia.

Clinical/Pathophysiological Implications?

• These data suggested that stratification by sex and salt sensitivity might be necessary for better therapeutic approaches in hyperuricemia.

Purines are key components of critical regulatory signaling pathways and energy metabolism. Uric acid (UA) is the final human purine oxidation product and can act as an antioxidant and a conditional pro-oxidant.¹ Unlike humans, rodents possess uricase, which breaks down UA to the more water-soluble allantoin. The evolutionary loss of the uricase gene caused humans to have higher UA levels than many other species.² While many epidemiological studies have shown a U-shaped relationship between serum UA levels and hypertension, as well as chronic kidney disease (CKD), the causal relationship is still unknown (reviewed in^3,4). Lowering UA has been shown to slow the progression of hypertension in humans with preserved kidney function.^5,6 Although hyperuricemia has been associated with worse outcomes in kidney damage, attempts to control UA levels in large-cohort clinical trials have not demonstrated significant benefits for CKD,^7,8 or diabetic kidney disease.⁹ Hyperuricemia is considered asymptomatic when the UA level is increased without gout or crystal deposition. A study conducted using an aristolochic acid-induced CKD mouse model suggested that only crystal-producing, but not asymptomatic, hyperuricemia promotes the progression of CKD.¹⁰

Salt-sensitive (SS) hypertension is a sexually dimorphic trait that results in kidney damage.^11,12 A few clinical studies have demonstrated that increased dietary sodium intake may lead to a decrease in serum UA levels.^13,14 The lack of uricase in humans is hypothesized to have been an adaptation to avoid lowering blood pressure (BP) under low-salt conditions.¹⁵ This hypothesis was built on studies where salt-resistant Sprague-Dawley rats became SS and hypertensive after a 7-week exposure to oxonic acid (OA).^15,16 Laakso et al¹⁷ showed that in Dahl SS, but not salt-resistant rats, the activity of the enzyme group producing UA, xanthine oxidoreductase increased dose-dependently with greater amounts of salt in the diet. However, inhibiting xanthine oxidoreductase was not consistently effective in Dahl SS rats to prevent hypertension, as some studies showed attenuation of SS hypertension while others were not able to confirm these findings.^18,19

It remains unclear if elevated levels of UA in an SS subject can affect the progression of hypertension. In humans, the normal UA level is around 3.5 mg/dL, and hyperuricemia is defined as >6 mg/dL in women and >7 mg/dL in men. In the Dahl SS rats, the normal level of UA in plasma is ≈0.5 mg/dL.²⁰ Here, we attempted to mimic the fold difference of human UA increase to hyperuricemia. We deemed the elevation of UA as mild based on the previous studies.¹⁶ We hypothesized that mild asymptomatic hyperuricemia would not accelerate the progression of SS hypertension and the associated kidney damage. We tested this hypothesis using male and female Dahl SS rats, a model of SS hypertension.

Methods

Data Availability

The data from RNA-sequencing are available at: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE269511.

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

Animal Models and Experimental Design

All rats were housed under a 12:12-hour light-dark cycle and kept on a normal salt (NS) diet (0.4% NaCl [Dyet 113755]) and water ad libitum. All animal experiments adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

The University of South Florida Institutional Animal Care and Use Committee reviewed and approved each protocol that used rats (9421M and 9479R). Male and female rats from the Dahl SS (JrHsdMcwi) strain were bred in-facility. All custom diets for the rats were commercially made (Dyets, Inc, Bethlehem, PA). The uricase inhibitor added to the diet is potassium oxanate (OA; Sigma-Aldrich, St. Louis, MO).

The rats were cannulated with telemeters (DSI, St. Paul, MN) through the femoral artery at 8 to 9 weeks of age. After recovery, BP measurements were collected for 4 days on the NS diet and switched to either a high-salt (HS) diet (4% NaCl [Dyet 113756]), a HS + 2% OA diet (HS/OA Dyet 105030), or a NS+2% OA diet (NS/OA [Dyet 105203]) for 3 weeks. All BP measurements are the average mean arterial pressure from 9 am to 12 pm during the rat’s inactive day cycle. Before euthanasia under anesthesia (2% to 3% [vol/vol] isoflurane), the kidneys were perfused with PBS and harvested. The right kidney was fixed in 10% formalin for histology, and the left kidney was snap-frozen in liquid nitrogen for further analysis.

UA and Xanthine Oxidase Activity Measurements

UA measurements on rat plasma and urine samples were performed using a colorimetric assay (E-BC-K016-M; Elabscience, Houston, TX). Xanthine oxidase activity was measured in plasma using a fluorometric assay (10010895; Cayman Chemical, Ann Arbor, MI).

Histology and Immunohistochemistry

Histological preparations were done through the Children’s Research Institute Histology Core at the Medical College of Wisconsin (Milwaukee, WI) and AML Laboratories (St. Augustine, FL). Formalin-fixed kidneys and heart tissue were paraffin-embedded, sectioned, mounted, and stained for either Masson Trichrome, picrosirius red, or immune-stained with an antibody against kidney injury molecule 1 (Cat sc-518008, Santa Cruz Biotech, Dallas, TX). As a marker of oxidative DNA damage, 8-OHdG (sc-66036) was used and analyzed using a Leica SP8 confocal microscope with either paraffin-embedded or optimal cutting temperature-embedded samples.

A scoring investigator blinded to the sample identity assessed the glomerular damage (0–4, with each number representing a different level of damage: 0 for a healthy kidney, 1 for 1% to 25% mesangial expansion and sclerosis, 2 for 26% to 50% mild segmental hyalinosis, 3 for 51% to 75% diffuse sclerosis, and 4 for 76% to 100% mesangial expansion and extensive sclerosis).

RAAS Analysis

Renin-angiotensin-aldosterone system (RAAS) hormone levels in plasma (angiotensin I [Ang I] [1–10], Ang II [1–8], Ang III [2–8], Ang [1–7], Ang IV [3–8], and Ang [1–5]), were measured using ultra-pressure-liquid chromatography-tandem mass spectrometry and stable-isotope-labeled internal standards (Attoquant Diagnostics LLC, Vienna, Austria).

Patch-Clamp Analysis

Patch-clamp electrophysiology, in the cell-attached configuration (V_m=-V_pipette), was used to assess epithelial Na⁺ channel (ENaC) activity on the apical membrane of isolated, split-open cortical collecting ducts. Cortical collecting ducts were isolated, and single-channel data were acquired as described previously.²¹

RNA-Sequencing and Quantitative PCR

Total RNA was extracted from the kidney cortices or heart apex from snap-frozen tissue using Trizol reagent (Thermo Fisher Scientific, Waltham, MA). RNA-sequencing was commercially done through Novogene using the kidney total RNA (UC Davis, CA). Pathway analysis was performed using Qiagen Ingenuity Pathway Analysis (IPA) software. The figures were made using R and BioRender. The details of the quantitative PCR methodology on heart tissue, including primers for Nppa and Nppb, were published previously.²²

Western Blotting

Lysates from snap-frozen kidneys were prepared in Laemmli buffer (20 mg of tissue per mL) supplemented with protease and phosphatase inhibitors. The samples were briefly sonicated, then run on SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was first incubated with primary antibody for AKR1B10 (Cat# 18252-1-AP, Proteintech, Rosemont, IL) at 1:5000, then with a secondary antibody (Cat# 711-035-152, Peroxidase AffiniPure Donkey Anti-Rabbit IgG, Jackson ImmunoResearch Laboratories Inc., West Grove, PA) at 1:10000 in 2% BSA in Tris-buffered saline with Tween-20 (TBST). Detection was performed using enhanced chemiluminescence (Thermo Scientific, Rockford, IL). β- actin (Cat# HCA147, Bio-Rad, Hercules, CA) served as the loading control.

Statistical Analyses

Statistical analyses were done using GraphPad Prism software 10.2.0. Qiagen IPA software performed statistics for pathway analysis from transcriptomics. Details of specific tests used can be found in the figure legends. P<0.05 was considered significant. A statement of trend was only used when the P value was between 0.1 and 0.05.

For methods related to the supplemental tables and figures, please refer to the Supplemental Methods.

Results

Mild Hyperuricemia Was Associated With an Attenuated Progression of Hypertension in Male Rats

Male Dahl SS rats fed an HS/OA diet showed significantly reduced progression of hypertension compared with rats fed an HS diet alone (Figure 1A). When given NS/OA, their BP did not differ from those who were fed an NS/vehicle diet (Figure S1A). In contrast to males, female SS rats fed an HS/OA diet did not show differences in the progression of hypertension compared with HS-fed females (Figure 1B). Both sexes did not show differences in end point body weights compared with the vehicle-treated rats (Figure 1C; Figure S1B). Consistent with OA blocking uricase, male HS/OA, NS/OA and female HS/OA-fed rats had significant hyperuricemia compared with their respective vehicle groups (Figure 1D; Figure S1C). The excretion of UA (UA/creatinine) was significantly increased in male HS/OA but not in NS/OA and female HS/OA (Figure 1E and Figure S1D). No difference in plasma xanthine oxidase activity was observed between the different diet groups within each sex (Figure 1F). There was no differences observed in the circadian rhythm or heart rate variability associated with the OA treatment (Figure S2A and S2B).

Figure 1.

Hypertension progression and urate handling in Dahl SS rats. A, Mean arterial pressure (MAP) of male and (B) female Dahl SS rats fed a high-salt (HS) or high-salt/oxonic acid diet. C, Body weight (BW), (D) Plasma uric acid, (E) Urine uric acid/Creatinine, and (F) Plasma xanthine oxidase (XO) activity of male/female Dahl SS rats fed a HS or HS/oxonic acid diet at the end point. All rats were 8 to 9 weeks at the start of the experiment. ♂, male; ♀, female. Values are shown as mean±SEM. Two-way ANOVA was used separately for the MAP analyses for 0.4% and 4% NaCl conditions. An unpaired t test was used to measure the terminal blood pressure and all other parameters depicted. P value **<0.01, ***<0.001, P values not shown are not significant.

Electrolyte Handling in the Dahl SS Rats Fed an OA Diet

Plasma K⁺, Na⁺, Ca²⁺, Cl^-, creatinine, and blood urea nitrogen levels were not significantly different between HS/OA and HS/vehicle groups in either sex (Table S1). Urinary K⁺ excretion was higher, and Ca²⁺ excretion was lower in HS/OA groups in both sexes compared with the respective HS/vehicle groups (Table S1). No differences were observed in other urine electrolytes and creatinine excretion.

Hyperuricemia in Male Rats Was Linked to a Reno-Protective Phenotype

Histopathology (Masson Trichrome staining) demonstrated reduced tubular dilation and fewer medullary protein casts in HS/OA-fed male rats (Figure 2A). The kidney weight/body weight ratio was significantly lower in HS/OA-fed male rats consistent with smaller kidney size (Figure 2B). The percentage area of protein casts in males, but not fibrosis, was significantly lower in the HS/OA group compared with the HS group (Figure 2C and 2D). The glomerular damage score was significantly greater in the HS/vehicle group compared with the HS/OA group (Figure 2E). To further assess the potential kidney damage we analyzed the staining of kidney injury molecule 1, a commonly used proximal tubular injury marker. The precentage stained area was significantly lower in the HS/OA compared with the HS/vehicle group (Figure 2F). In contrast to males, female rats did not show differences in kidney damage, kidney weight/body weight ratio, the percentage area of protein casts, fibrosis, glomerular scoring or KIM staining following the hypertension progression (Figure 2A through 2F). We examined the frozen kidney tissue under polarized light microscopy which established that hyperuricemia did not result in crystal deposition of the kidney in either sex (Figure S3).

Figure 2.

Kidney damage assessment. A, Representative images of Masson Trichrome (MT) stained kidney (B) kidney weight (KW)/body weight (BW) ratio, (C) Percentage medullary protein cast quantification per area, (D) Percentage cortical fibrosis quantification per area, and (E) Glomerular damage score (0–4 with increasing damage) in male and female Dahl SS rats fed a high-salt (HS) or HS/oxonic acid diet. F, Representative images of kidney injury molecule 1 (KIM-1) staining in the kidney and Percentage quantification of KIM-1 staining in male Dahl SS rats fed a HS or HS/oxonic acid diet. ♂, male; ♀, female. Values are shown as mean±SEM. An unpaired t test was used for all parameters passing the normality test. P value *<0.05, **<0.01, P values not shown are not significant.

RAAS and ENaC Activity Were Not Affected by OA

Increased UA is known to be associated with worse outcomes for cardiovascular disease and changes in RAAS.^23,24 However, in our model of mild hyperuricemia, circulating angiotensin metabolites do not significantly change in treated male rats compared with the vehicle group (Figure 3A). Similarly, aldosterone levels, plasma renin activity (Ang I+Ang II), angiotensin-converting enzyme activity (Ang II/Ang I), and adrenal function (Aldo/Ang II) were not significantly different (Figure 3B). Consistent with the lack of change in RAAS activity, ENaC activity was not significantly different between groups (Figure 3C).

Figure 3.

Measurements of circulating renin-angiotensin-aldosterone system (RAAS) and epithelial sodium channel (ENaC) activity in isolated cortical collecting duct (CCD) tubules. A, Ang I (1–10), Ang II (1–8), Ang III (2–8), Ang I (1–7), Ang (1–5) and Ang IV (3–8) in plasma in male Dahl SS rats fed a high-salt (HS) or HS/oxonic acid diet. Enzymes cleaving the angiotensin are shown inside arrows. B, Circulating RAAS activity: aldosterone, plasma renin activity (PRAs based on Ang I+Ang II levels), angiotensin-converting enzyme (ACE) activity (based on Ang II/Ang I), and Adrenal function (based on Aldo/Ang II) in male Dahl SS rats fed a HS or HS/oxonic acid diet. C, ENaC activity in isolated CCDs. Representative current traces cell-attached patches containing ENaC recorded from the apical membrane of split-open CCD cells of oxonic treated and vehicle Dahl SS rats on a HS diet. Image showing examples of split-open CCD dissected from the rat kidney. ENaC conductance, activity (NPo), and channel open probability (Po). n=4–6. An unpaired t test is used, P values not shown are not significant. AP indicates aminopeptidase; and NEP, neutral endopeptidase.

Transcriptomic Data Indicated Changes to Multiple Signaling Pathways That Use Calcium Signaling

To provide mechanistic insight into the protective phenotype seen in the male SS rats treated with OA, we performed RNA-Sequencing analysis of kidney cortices. Hierarchically clustering demonstrates the distinct transcriptomic signatures of the HS/Vehicle and HS/OA-treated group (Figure 4A). Considering a P<0.05, we observed 748 upregulated and 829 downregulated genes (Figure 4B).

Figure 4.

Mechanistic predictions by transcriptomics. A, Heatmap showing the hierarchical clustering of the gene expression. B, Volcano plot showing the top differentially expressed (DE) genes. C, The top (by significance [P<0.05]) canonical pathways in the oxonic treated vs vehicle comparison. The pathways were analyzed using IPA. D, Categories of signaling pathways. Categories were identified by manually studying the DE genes concerning each top canonical pathway. E, DE genes in oxonic treated vs vehicle comparison related to hypertension. Parts of the figure are created using BioRender.com.

The differentially expressed (DE) gene list (P<0.05 and log2[fold change] >|1|; DEG–84 upregulated and 60 downregulated) was used for further analyses using IPA. The top canonical pathways predictions included activation of IL-10 signaling; inhibition of white adipose tissue browning pathway, cytokine storm signaling, adrenergic receptor signaling, and calcium signaling by OA treatment (Figure 4C). Some pathways such as Gamma-Aminobutyric Acid (GABA) receptor signaling, glucocorticoid receptor signaling, and NRF-2-mediated oxidative stress response were enhanced but without directional predictions. Closely examining the DE genes associated with top canonical pathways (Table S2), we found that many enhanced pathways in the HS/OA group shared common signaling messengers such as cGMP, cAMP, and calcium signaling (Figure 4D).

Filtering the data set for DE genes associated with the term “hypertension” in the IPA knowledgebase (Figure 4E) identified that Fof1, Klk1, Mas1, Pcsk6, Snhg11, and Retn were upregulated; Cacnalb, Bglap, Tmem130, Ntan1, and Ptx3 were downregulated. Moreover, the gene expression relating to ion, urate, and water transport is provided in the Table S3.

OA Treatment Reduced Oxidative Stress in Renal Tubules

Since UA is known to be an antioxidant, especially in the hydrophilic environment of biological fluids, such as plasma^1,25 and the canonical pathways suggested some oxidative stress-related signaling, we further explored the expression of oxidative stress-related genes (Figure 5A). Eight DE genes related to “oxidative stress” and the “synthesis of reactive oxygen species” were identified. The genes Akr1b10, Mas1, Scd, and Retn were upregulated, and Echs1, Il1r2, Noxo1, and Il19 were downregulated in HS/OA-treated rats compared with the HS/vehicle rats. AKR1B10 is an aldo-keto reductase enzyme that plays a role in protecting cells from oxidative stress.²⁶ To confirm mRNA with protein and explore sex differences, we did Western blotting for AKR1B10 (Figure 5B). In male but not female HS/OA rat kidneys, AKR1B10 levels were significantly higher.

Figure 5.

Oxidative stress in the kidney. A, Differentially expressed (DE) genes known to affect the synthesis of reactive oxygen species (ROS) and oxidative stress in male rats. B, Western blot for AKR1B10 expression in the kidney and its quantification in male and female rats. C, Representative images of paraffin-embedded 8-OxodG-stained kidney and the quantification of the fluorescence intensity of the staining in male age-matched Sprague-Dawley (SD) rats fed a normal diet (healthy control), Dahl SS rats fed a high-salt (HS) diet, or Dahl SS rats fed a HS/oxonic acid diet. D, Representative images of optimal cutting temperature (OCT)-embedded 8-OxodG-stained kidney of the female Dahl SS rats fed a HS diet, or Dahl SS rats fed a HS/oxonic acid diet. Red-8OxodG, blue- nuclei. Values are shown as mean±SEM. An unpaired t test was used. P value **<0.01, ****<0.0001. Parts of the figure are created using BioRender.com.

To confirm the transcriptomic data indicating lower oxidative stress in OA-treated rats, we used the marker 8-OxodG to assess oxidative DNA damage in the kidney. The male rats fed HS/OA had reduced amounts in proximal tubules compared with the HS/vehicle group (Figure 5C). Sprague-Dawley rats on a normal diet, serving as healthy controls, showed minimal staining compared with both HS-fed SS groups. Female rats fed either diet showed comparable levels of DNA damage, as indicated by 8-OxodG labeling (Figure 5D).

A previous study with mice fed OA diet suggests UA may prevent SOD inactivation by H₂O₂.²⁷ Therefore, we measured the CuZn SOD activity of the kidney cortices of all the groups (Figure S4A through S4C). There was no significant difference between groups, but a trend towards increased activity was observed in male rats fed the HS/OA diet compared with the HS-only group.

OA Treatment Had a Minimal Effect on the Cardiac Tissue

We assessed the hearts from the male HS/OA versus HS/vehicle groups to determine whether the protective phenotype extended to the cardiac tissue. There were no significant histopathologic differences between groups evident by picrosirius red, Masson Trichrome stainings, heart weight-to-body weight ratio, and quantification of fibrotic tissue in different areas (intraventricular septum, right and left ventricular walls; Figure S5A through S5D). Of note, there was a significant increase in the percentage of the fibrotic area when total area was taken into consideration (Figure S5D). Since we observed upregulation of the Pcsk6 gene in the kidney, we quantified the cardiac mRNA expression of Nppa (ANP) and Nppb (BNP), which are downstream targets of Pcks6,²⁸ and found no significant differences between groups (Figure S5E).

Discussion

Whether asymptomatic hyperuricemia should be managed to reduce cardiovascular and kidney disease risk is still a matter of debate.^29–31 The lack of outcome-based benefits of urate-lowering in clinical studies in patients with CKD and patients with CKD caused by diabetes raises questions about the role of UA in kidney damage in the absence of gout.^7,9 Our study demonstrates that mild hyperuricemia in the context of SS hypertension and kidney damage progression might be beneficial in a sex-specific manner, with males showing a benefit in contrast to females.

Previous studies have shown that Sprague-Dawley rats fed a salt-restricted diet (0.125% NaCl) with OA had increased UA associated with increased BP, increased juxtaglomerular renin expression and decreased macula densa NOS1 expression.¹⁶ We observed a significant attenuation in the progression of SS hypertension in male Dahl SS rats compared with the control groups despite a >3-fold increase in UA levels challenging the concept that elevated UA is universally harmful. Our findings also revealed that the increased UA did not activate circulating RAAS components in the Dahl SS model. Aldosterone is known to enhance the quantity and activity of ENaC, which subsequently influences BP. Previous studies have suggested that hyperuricemia might activate ENaC.³² We did not observe any changes in Na⁺ excretion or ENaC activity in hyperuricemic rats. Though plasma electrolytes were similar across the groups, an increase in K⁺ and a decrease in Ca²⁺ excretion was noted in OA-fed rats, regardless of sex or salt content in their diet. This increase is likely due to the OA, which is added to the diet as potassium oxonate. In a previously published study, we found not only that the K⁺ levels needed to attenuate BP in the same model were much higher than what is provided in this treatment (1.4% in the high K diet published versus 0.75% in the HS/OA and 0.4% in the HS diet in the current study) but also, it took 3 weeks of treatment to start showing any differences (as opposed to the 10 days in the current study).³³ Furthermore, a study by Manger et al³⁴ showed that 0.7% KCl was not enough to make changes to BP in SS rats. One can speculate that calcium may bind oxanate from OA in the kidney or gut. Further experiments are needed to confirm this hypothesis. It’s unlikely that changes in K⁺ and Ca²⁺ excretion influenced the hypertension phenotype, as these changes were observed across all groups regardless of their BP differences.

The female SS rats did not respond favorably or negatively to the increase in UA. Interestingly, studies have shown that females are more likely to be SS than men because of differences in the RAAS.³⁵ In hyperuricemia, women tend to have more favorable outcomes, as premenopausal women have lower UA levels³⁶ which could be in part because of the uricosuric ability of estrogen.^36,37 Further evidence comes from studies in trans-sexual men treated with estrogens, where in the majority of treated patients, plasma UA levels decreased and urinary UA levels increased.³⁸ Although both sexes showed increased UA in plasma when given OA in their food, only male Dahl SS rats had increased UA excretion, the reason for these discrepancies remains to be determined. There is a lack of sex-specific data regarding the combination of salt-loading and hyperuricemia. Perhaps female rats may be closer to becoming symptomatic due to their lack of increased UA excretion. In either sex, these changes occurred without the presence of crystals in the kidney sections examined.

Utilizing IPA core analysis, we found evidence suggesting that IL-10 signaling was upregulated in the HS/OA group. This prediction was based on multiple downstream genes (Creb3l3, Hla-a, Il1r2, Map3k7, RT1-dma, RT1-dmb) that were differentially expressed rather than Il-10 itself. IL-10 is known to be antiinflammatory in kidney injury models.³⁹ Because asymptomatic hyperuricemia has been shown to promote recovery from ischemic organ injury,⁴⁰ it is possible that IL-10 signaling was one of the pathways that led to improved results in the current model. Most other canonical pathways noted by IPA were inhibitory and shared similar messengers typically involved in vascular resistance, such as calcium, cAMP, and cGMP. Upon examining DE genes related to “hypertension” in the literature, several genes known to activate vasodilatory pathways were upregulated. MAS-1 receptor activation through Ang 1 to 7 contributes to the alternative RAAS signaling and leads to vasodilation.⁴¹ Though Mas-1 mRNA increased, we did not observe an increase in Ang 1 to 7 in plasma. Intrarenal RAAS components have shown to be independent of circulating level and might provide better information.⁴² RAAS also works with the Kinin-Kallikrein system to change the vascular tone.⁴³ We observed an increase in Klk-1, which has been shown to contribute to the SS phenotype in Dahl rats.⁴⁴ PCSK6, another upregulated gene is essential in the cleavage of pro-corin to corin, which then activates the ANP pathway leading to vasorelaxation.²⁸ In Dahl SS rats, it was shown that the lack of ANP can lead to higher BP and infusion of ANP can decrease it.⁴⁵ Nevertheless, there was no increase in Nppa mRNA expression in the kidney or heart tissue of the current model. Overall, the transcriptomics from the kidney suggested a few possible vasorelaxation pathways that might mitigate the progression of hypertension in mildly hyperuricemic male rats.

In SS hypertension, oxidative stress plays a role in BP control, kidney damage, and hemodynamics.^46–48 Studies show that antioxidants such as vitamin C, E, or catalase help improve hypertension in Dahl SS rats.^49–51 Soluble UA has been shown to protect against neurological diseases and cancer by scavenging peroxynitrite.^25,52,53 Furthermore, a study using aorta from ApoE−/− mice fed OA showed that another possible benificial effect of UA is through preventing the inactivation of SODs by H₂O₂ and regulating vascular redox states.²⁷ More recent studies suggest that the distinction between soluble and crystalline UA can determine whether it will be harmful to the kidney.^10,40 Our data imply that the increase in soluble UA potentially acts as an antioxidant, while reduced UA breakdown into allantoin may decrease H₂O₂ production. We observed a reno-protective effect in male but not female rats fed HS/OA, which was accompanied by reduced DNA damage and suggested lower oxidative stress. In 5 of 6 nephrectomized Sprague-Dawley rats, it was shown that OA-induced hyperuricemia reduces oxidative stress and improves NO-mediated vasorelaxation.⁵⁴ In contrast, other rodent models showed increased oxidative stress with increased UA or xanthine oxidoreductase.^55,56 The protective effect of UA as an antioxidant may depend on preexisting renal and vascular damage in different models.

The current study has some limitations. First, human and rodent purine metabolism differ, which may affect the translation. However, we addressed this by inhibiting uricase in our model. Although differences in salt sensitivity of BP exist in rodents and humans, the Dahl SS rat is the best-characterized noninvasive rodent model of this pathology. We focused on the renal effect of the OA treatment; the insights from transcriptomics are steering our attention toward a vascular phenotype, which requires further studies. It is also hard to distinguish if the reno-protective effect is causative for the decreased hypertension or consequential. Finally, we induced hyperuricemia simultaneously with the progression of SS hypertension. The effects of mild hyperuricemia, if OA is given after the rats have become hypertensive (ie, therapeutical approach), remain unclear.

Perspectives

In summary, we showed that mild asymptomatic hyperuricemia can attenuate the progression of SS hypertension and hypertensive kidney damage in male rats associated with lower renal oxidative damage. Transcriptomic analysis of renal tissue suggested activation of vasodilatory and antiinflammatory pathways. Further research is necessary to better understand the determinants of the beneficial effects as well as of the observed sexual dimorphism. Nevertheless, current data challenges the notion that asymptomatic hyperuricemia is inherently harmful.

ARTICLE INFORMATION

Acknowledgments

The authors gratefully acknowledge Dr Anna Manis for editing the article for language and readability; the Medical College of Wisconsin Children’s Research Institute Histology Core for processing tissue for immunohistochemistry; Dr Byeong Cha from the Lisa Muma Weitz Laboratory for Advanced Microscopy & Cell Imaging core at the University of South Florida for the assistance with imaging; Dr Christopher Banek from the University of Minnesota for the helpful suggestions; and the developers of the open-source programs R and QuPath.

Author Contributions

Conceptualization: L.V. Dissanayake, O. Palygin, I.S. Daehn, A. El-Meanawy, and A. Staruschenko; data curation, formal analysis, and investigation: L.V. Dissanayake, A. Zietara, R. Bohovyk, C.A. Klemens, O. Kravtsova, V. Levchenko, M. Lowe, A. Shapiro, V. Pasmanik, and T. Rieg; software, validation, and visualization: L.V. Dissanayake; funding acquisition: L.V. Dissanayake, T. Rieg, and A. Staruschenko; project administration, supervision, and resources: A. Staruschenko; writing – original draft: L.V. Dissanayake; writing – review and editing: All authors.

Sources of Funding

Research in the author’s laboratory was supported by the National Institutes of Health grants R01 DK135644 and R01 DK129227 (to A. Staruschenko) and T32 HL160529 (to R. Bohovyk), US Department of Veteran Affairs grants I01 BX004024 (to A. Staruschenko) and I01 BX004968A (to T. Rieg), Ben J. Lipps Research Fellowship Program from the American Society of Nephrology (to L.V. Dissanayake), American Heart Association Postdoctoral Fellowship 25POST1375066 (to L.V. Dissanayake) and USF Hypertension and Kidney Research Center Early Stage Investigator Pilot Award (to L.V. Dissanayake) and Multi-PI Award (to A. Staruschenko and T. Rieg). The contents do not represent the views of the Department of Veterans Affairs or the US Government.

Disclosures

None.

Supplemental Material

Supplemental Methods

Tables S1–S3

Figures S1–S5

Major Resources Table