Chronic treatment with IL-25 increases renal M2 macrophages and reduces renal injury in obese Dahl salt-sensitive rats during the prepubescent stage

Abstract

Recently, we have reported that the early progression of proteinuria in the obese Dahl salt-sensitive (SS) leptin receptor mutant (SS^LepRmutant) strain was associated with increased renal macrophage infiltration before puberty. Macrophages can be divided into two distinct phenotypes: M1 (proinflammatory) and M2 (anti-inflammatory). Moreover, previous studies have demonstrated that interleukin (IL)-25 converts resting macrophages and M1 into M2. Therefore, the present study examined whether treatment with IL-25 would reduce the early progression of renal injury in SS^LepRmutant rats by increasing renal M2. We also investigated the impact of IL-25 on M2 subtypes: M2a (wound healing/anti-inflammatory), M2b (immune mediated/proinflammatory), M2c (regulatory/anti-inflammatory), and M2d (tumor associated/proangiogenic). Four-wk-old SS and SS^LepRmutant rats were treated with either control (IgG) or IL-25 (1 µg/day ip every other day) for 4 wk. The kidneys from SS^LepRmutant rats displayed progressive proteinuria and renal histopathology versus SS rats. IL-25 treatment had no effect on these parameters in SS rats. However, in the SS^LepRmutant strain, proteinuria was markedly reduced after IL-25 treatment. Chronic treatment with IL-25 significantly decreased glomerular and tubular injury and renal fibrosis in the SS^LepRmutant strain. Although the administration of IL-25 did not change total renal macrophage infiltration in both SS and SS^LepRmutant rats, IL-25 increased M2a by >50% and reduced M1 by 60% in the kidneys of SS^LepRmutant rats. Overall, these data indicate that IL-25 reduces the early progression of renal injury in SS^LepRmutant rats by inducing M2a and suppressing M1 and suggest that IL-25 may be a therapeutic target for renal disease associated with obesity.

NEW & NOTEWORTHY For the past few decades, immune cells and inflammatory cytokines have been demonstrated to play an important role in the development of renal disease. The present study provides strong evidence that interleukin-25 slows the early progression of renal injury in obese Dahl salt-sensitive rats before puberty by increasing systemic anti-inflammatory cytokines and renal M2a macrophages.

INTRODUCTION

Prepubertal childhood obesity (PPO) has emerged as an epidemic and major health problem in the United States (1–3). According to National Kidney Foundation reports, nearly $150 billion per year is spent treating obesity-related medical conditions (4). Recent studies have suggested that childhood obesity is associated with an increased risk of renal injury (5, 6). However, the underlying mechanisms of renal disease associated with PPO have received less attention due to a lack of an appropriate animal model of obesity that displays the progression of renal disease in this unique population. Recently, we observed that the obese Dahl salt-sensitive (SS) leptin receptor mutant (SS^LepRmutant) strain displays progressive renal injury before puberty in the absence of hyperglycemia and elevations in arterial pressure (7–11). Therefore, studying the SS^LepRmutant strain could be a valuable tool in identifying novel mechanisms involved in the early development of obesity-induced renal disease before puberty.

Over the past decade, immune cells have been recognized to play an important role in the development of various cardiovascular diseases including renal injury in humans and animals (12–15). We have recently reported that the early progression of proteinuria in SS^LepRmutant rats was associated with increased renal macrophage infiltration (7, 10), and depletion of renal macrophages using liposomal clodronate significantly reduced proteinuria, glomerular injury, and renal fibrosis (7). Macrophages can be divided into two distinct phenotypes: M1 macrophages (classical; proinflammatory) and M2 macrophages (alternative; anti-inflammatory) (16). M1 macrophages secrete various proinflammatory cytokines including interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ, which contribute to the development of renal inflammation and injury (14, 17). However, M2 macrophages produce various anti-inflammatory cytokines including IL-4, IL-5, IL-10, and IL-13, which reduce renal injury (15–17). Unlike M1 macrophages, M2 macrophages are divided into four major subtypes based on their function and key markers: M2a, M2b, M2c, and M2d (18). M2a macrophages, often referred to as M2 macrophages, are known as wound-healing and anti-inflammatory macrophages (19, 20). M2b macrophages are immune complex-mediated M2 macrophages, which are activated during glomerulonephritis (16). M2c macrophages are also known as regulatory macrophages, which are often generated in response to IL-10 and help to reduce renal inflammation and fibrosis (16, 19, 20). M2d macrophages, which are tumor associated, are proangiogenic and express vascular endothelial growth factor (VEGF) (19, 20). There are several approaches to raise M2 macrophages in vivo, including administration of IL-4, IL-10, or IL-25 or adoptive transfer of ex vivo programmed M2 macrophages (21–23). However, IL-25 induces a T lymphocyte 2 [T helper (Th)2] response characterized by the overproduction of IL-4, IL-5, and IL-13 and increases M2 macrophages along with reducing M1 macrophages (22–25). IL-25 (also known as IL-17E) is a member of the IL-17 cytokine gene family, which is produced by various cells such as T cells, eosinophils, basophils, mast cells, and epithelial cells (21). Moreover, previous studies have demonstrated that the decrease in renal injury in response to IL-25 administration was associated with elevated renal M2 macrophages (22, 23). Based on this evidence, we hypothesized that treatment with IL-25 would reduce renal injury in SS^LepRmutant rats before puberty by increasing renal M2 macrophages.

METHODS

General

Experiments were performed on 80 male and female wild-type Dahl SS and SS^LepRmutant rats at 4 wk of age. SS^LepRmutant rats were generated from our in-house colony of heterozygous SS^LepRmutant rats, which were originally derived from the Medical College of Wisconsin (Milwaukee, WI), as previously described (8). Genotyping was performed by the Molecular and Genomic Facility at the University of Mississippi Medical Center. Rats had free access to food and water throughout the study. Rats were fed a 1% NaCl diet (TD8640, Harlan Laboratories, Madison, WI). Rats were housed in the Laboratory Animal Facility at the University of Mississippi Medical Center, which has been approved by the American Association for the Accreditation of Laboratory Animal Care. All protocols were approved by the University of Mississippi Medical Center Institutional Animal Care and Use Committee.

Protocol

At 4 wk of age, SS and SS^LepRmutant rats were weighed and placed in metabolic cages for an overnight urine collection to determine protein concentration in the urine using the Bradford method (Bio-Rad Laboratories, Hercules, CA). A blood sample was collected from the tail vein for the measurement of blood glucose levels (glucometer from Bayer HealthCare, Mishawaka, IN). After the collection of baseline data, rats were randomly separated into the following four groups: 1) SS rats treated with control (IgG, ip), 2) SS^LepRmutant rats treated with control (IgG, ip), 3) SS rats treated with IL-25 (1 µg/kg ip every other day), and 4) SS^LepRmutant rats treated with IL-25 (1 µg/kg ip every other day) for 4 wk. Every 2 wk, rats were placed in metabolic cages, and proteinuria and blood glucose levels were measured at each time point. In addition, urine collected at the end of the protocol was used to assess tubular injury by measuring the excretion of kidney injury molecule (KIM)-1 (Rat KIM-1 ELISA, Abcam, Waltham, MA). At the end of the study, rats were placed under anesthesia, and a catheter was inserted in the carotid artery for the measurement of mean arterial pressure (MAP). After a 24-h recovery period, catheters were connected to pressure transducers coupled to a computerized PowerLab data-acquisition system (ADInstruments, Colorado Springs, CO), and MAP was recorded continuously for 30 min after a 30‐min equilibration period. In another cohort of 4-wk-old SS and SS^LepRmutant rats, telemetry transmitters (model HD-S10, Data Sciences, St. Paul, MN) were implanted into the femoral artery, and the unit was placed beneath the skin on the back to measure temporal changes in MAP. After a 3-day period of recovery, MAP was continuously recorded for three successive days to obtain baseline MAP data. After this, rats were randomly divided into four groups and treated with IgG or IL-25 as described above. MAP was then measured weekly for 4 wk to assess the effects of IL-25 on arterial pressure. After arterial pressure measurements, a final blood sample was taken from the abdominal aorta to measure plasma cholesterol (Cayman Chemical, Ann Arbor, MI), triglyceride (Cayman Chemical), and insulin (Mercodia Rat Insulin ELISA, Uppsala, Sweden) concentrations. Both kidneys were perfused with sterile saline, collected, and weighed. The left kidney was used to measure the infiltration of immune cells using flow cytometry analysis. The right kidney was cut in half, with one half placed in 10% buffered formalin solution for renal histopathology and the other half snap-frozen in liquid nitrogen and stored at −80°C. Plasma and renal cytokines were measured using the Bio-Plex Pro Rat Cytokine 6-Plex Assay Reagent Kit on a Bio-Rad Bioplex 200 System (for IL-4, IL-5, IL-10, IL-13, TNF-α, and IFN-γ, Bio-Rad Laboratories) and an IL-25 ELISA kit (MyBioSource, San Diego, CA), according to the manufacturers’ protocols.

Flow Cytometry

The left kidney from each rat was minced in RPMI-1640 containing 0.1% type II collagenase and 10 µg/mL DNAse 1 and was later grounded to make a homogenous solution using a tissue homogenizer. Next, the homogenized solution was filtered through a 100-µm filter and incubated for 30 min at 37°C. The kidney homogenate was then filtered through a 70-µm cell strainer, washed with FACS washing buffer (1× Dulbecco’s PBS without Ca²⁺ and Mg²⁺ with 2% FBS and 2 mM EDTA) at 4°C, and centrifuged at 4°C for 10 min at 300 g. The pellet was resuspended in 5 mL washing buffer and filtered through a 40-µm cell strainer. After centrifugation at 300 g for 5 min, the pellet was resuspended in 3 mL of 30% Percoll (Sigma, Life Science, prepared in RPMI-1640) at room temperature, layered over 3 mL of 70% Percoll (Sigma, Life Science, prepared in 1× PBS), and centrifuged at 25°C at 1,200 rpm with no breaks for 30 min. The mononuclear cell layer resting above the 70% Percoll was removed, washed with FACS washing buffer, and centrifuged at 4°C at 1,600 rpm for 10 min. The pellet was resuspended in 1 mL FACS buffer, and cells were counted on an Automated Cell Counter and Image Cytometer (Nexcelom Bioscience). The cell suspension was divided into two staining panels: 1) macrophage panel and 2) lymphocyte panel. In flow cytometry staining, each panel cell suspension (1 × 10⁶ cells) was first stained with Viobility 405/520 fixable dyes (Miltenyi Biotec, Auburn, CA) for 20 min at 4°C to exclude dead cells from live cells. The macrophage panel was established with the following antibodies: anti-rat CD68-APC (1:10 dilution, Miltenyi Biotec) and rabbit anti-rat inducible nitric oxide synthase (iNOS; 1:100 dilution, Abcam)/goat anti-rabbit Alexa Fluor 405 (1:100 dilution, Abcam) for M1 macrophages. M2 macrophage subtypes were defined with the following antibodies: 1) mouse anti-rat CD163-FITC (1:50 dilution, Bio-Rad) for M2a macrophages (wound healing anti-inflammatory), also known as M2 macrophages; 2) anti-rat CD86-PE (1:10 dilution, Miltenyi Biotec) and rabbit anti-rat iNOS for M2b macrophages (immune-activated proinflammatory); 3) mouse anti-rat CD163-FITC and rabbit anti-rat Toll-like receptor 1 (TLR1)-PerCP (1:50 dilution, Miltenyi Biotec) for M2c macrophages (regulatory anti-inflammatory); and 4) mouse anti-rat VEGF-APCCY7 (1:50 dilution, Miltenyi Biotec) for M2d macrophages (proangiogenic or tumor-associated macrophages). The lymphocyte panel was established with the following antibodies: anti-rat CD45-VioBlue (1:10 dilution, Miltenyi Biotec) for leukocytes; anti-rat CD45R-PE-Vio770 (1:10 dilution, Miltenyi Biotec) for B lymphocytes; anti-rat CD3-FITC (1:50 dilution, Miltenyi Biotec), anti-rat CD4-APC-Vio770 (1:10 dilution, Miltenyi Biotec), and anti-rat CD4-FITC (1:50 dilution, Miltenyi Biotec) for Th cells; mouse anti-rat GATA-3-Alexa Fluor 647 (1:10, BD Biosciences, Haryana, India) for Th2 cells; and anti-rat CD8a-PerCp-Vio700 (1:50 dilution, Miltenyi Biotec) for cytotoxic T cells. Flow cytometry was performed on the Miltenyi MACSQuant Analyzer 10 (Miltenyi Biotec), and data were analyzed using FlowLogic software (Miltenyi Biotec). The gating strategies for the macrophage and lymphocyte panels are shown in Fig. 1.

Figure 1.

Flow cytometry gating strategies for lymphocytes (A) and macrophages (B) in Dahl salt-sensitive (SS) and obese SS leptin receptor mutant (SS^LepRmutant) rats treated with either control or interleukin (IL)-25. A: after being gated for the mononuclear cell population using forward scatter (FSC) and side scatter (SSC), dead cells were excluded using Viobility staining, and doublets were excluded. CD45⁺ staining was used to gate for total lymphocytes. From this population, gates were placed for CD3⁻ cells and CD3⁺ T cells. CD45R⁺ B cells were identified in the CD3⁻ cell population, and CD4⁺ T helper (Th) and CD8⁺ cytotoxic T subsets were identified within the CD3⁺ T cell population. Within the CD4⁺ Th population, GATA-3⁺ Th2 were identified. B: similar to A, live, singlet CD45⁺/CD3⁻ cells were gated. Within this population, CD68⁺/inducible nitric oxide synthase (iNOS)⁺ M1 macrophages were identified. Additionally, M2 macrophage subtypes were identified: 1) CD68⁺/CD163⁺/Toll-like receptor (TLR)⁻ M2a macrophages, 2) CD68⁺/iNOS⁺/CD86⁺ M2b macrophages, 3) CD68⁺/CD163⁺/TLR+ M2c macrophages, and CD68⁺/vascular endothelial growth factor (VEGF)⁺ M2d macrophages.

Renal Histopathology

Paraffin kidney sections were prepared from the other half of the right kidney collected from both control and IL-25-treated SS and SS^LepRmutant rats after the termination of the study. Kidneys were cut into 5-µm sections and stained with periodic acid-Schiff and Masson’s trichrome. Thirty glomeruli per periodic acid-Schiff-stained section were scored to determine glomerular injury by scoring in a blinded fashion on a scale of 0–4 with 0 representing a normal glomerulus, 1 representing a 25% loss, 2 representing a 50% loss, 3 representing a 75% loss, and 4 representing a >75% loss of capillaries in the tuft (7). To determine the degree of renal fibrosis, 10 representative images per Masson’s trichrome-stained section from each animal were captured using a Nikon Eclipse 55i microscope equipped with a Nikon DS-Fi1 color camera (Nikon, Melville, NY). We analyzed the percentage of the image stained blue (primarily collagen) by identifying which animal had the most collagen and setting the threshold for blue staining in the Masson’s trichrome-stained sections using NIS-Elements D 3.0 software (7). Next, we used those same thresholding parameters for the blue staining for each image per rat in the study to measure renal fibrosis.

Statistical Analysis

Data are presented as means ± SE. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA). The significance of differences between the strains and the effect of IL-25 treatment values for a single time point were determined by two-way ANOVA followed by a Holm–Sidak’s multiple-comparisons test. The time course changes in proteinuria from baseline, between strains, and the effect from IL-25 treatment were determined using three-way ANOVA followed by a Tukey’s multiple-comparisons test. P values of <0.05 were considered significantly different.

RESULTS

Measurement of Metabolic Parameters

The effects of chronic treatment with IL-25 on metabolic parameters including body weight, blood glucose, insulin, triglycerides, and cholesterol in SS and SS^LepRmutant rats are shown in Table 1. We observed a significant increase in body weight in SS^LepRmutant rats compared with SS rats, and chronic treatment with IL-25 had no effect on body weight in either strain. Insulin levels were significantly higher in SS^LepRmutant rats compared with SS rats (6.47 ± 0.94 vs. 0.94 ± 0.16 ng/mL, respectively), and treatment with IL-25 only reduced insulin levels in SS^LepRmutant rats (4.04 ± 0.41 ng/mL). Blood glucose levels were similar between SS and SS^LepRmutant rats with no effect from IL-25 treatment. Plasma triglyceride levels were more than sixfold higher in SS^LepRmutant rats compared with SS rats (389 ± 92 vs. 57 ± 8 mg/dL, respectively), and chronic treatment with IL-25 did not affect levels of triglycerides in either SS or SS^LepRmutant rats. Similar to triglycerides, total cholesterol levels were significantly higher in SS^LepRmutant rats compared with values measured in SS rats (178 ± 16 vs. 92 ± 8 mg/dL, respectively), and IL-25 treatment did not affect either strain.

Table 1.

Effects of IL-25 treatment on metabolic and cardiovascular parameters in SS and SS^LepRmutant rats

Metabolic Parameters	Vehicle Treatment		IL-25 Treatment
	SS Rats	SSLepRmutant Rats	SS Rats	SSLepRmutant Rats
Total animals	12	10	12	15
Numbers of female/male rats	7/5	4/6	7/5	7/8
Carotid mean arterial pressure, mmHg	135 ± 2	157 ± 5*	134 ± 3	155 ± 3*
Body weight, g	230 ± 17	293 ± 12*	220 ± 14	288 ± 9*
Glucose, mg/dL	113 ± 4	107 ± 5	113 ± 3	108 ± 2
Insulin, ng/mL	0.94 ± 0.16	6.47 ± 0.94*	1.05 ± 0.27	4.04 ± 0.41*†
Triglycerides, mg/dL	57 ± 8	389 ± 92*	67.5 ± 10.3	365 ± 61*
Total cholesterol, mg/dL	92 ± 8	178 ± 16*	97 ± 6	172 ± 14*

Values are means ± SE; n = 10–15 per group. IL-25, interleukin-25; SS rats, Dahl salt-sensitive rats; SS^LepRmutant rats, obese SS leptin receptor mutant rats. *P < 0.05 vs. SS rats within the same treatment; †P < 0.05 vs. vehicle-treated rats within the same strain.

Measurement of MAP and Temporal Changes in Proteinuria

The effects of IL-25 treatment on MAP and proteinuria in SS and SS^LepRmutant rats are shown in Fig. 2 and Table 1. Although we observed a tendency for elevations in MAP in SS^LepRmutant rats versus SS rats during the study via telemetry, it did not reach statistical significance. Treatment with IL-25 did not have a significant effect on MAP in either SS^LepRmutant or SS rats (Fig. 2A). At the end of the study, via chronic catheter in the carotid artery, we noticed a significant increase in MAP in SS^LepRmutant rats compared with SS rats (157 ± 5 vs. 135 ± 2 mmHg, respectively), and consistent with data obtained via telemetry, chronic treatment with IL-25 had no effect on MAP in either SS^LepRmutant or SS rats (155 ± 3 vs. 134 ± 3 mmHg, respectively; Table 1). At baseline, proteinuria was significantly higher in SS^LepRmutant rats compared with SS rats. Over the course of the study, proteinuria markedly increased in SS^LepRmutant rats compared with SS rats, and chronic treatment with IL-25 treatment significantly reduced proteinuria by 40% in SS^LepRmutant rats with no effect in SS rats (Fig. 2B). At the end of the study, KIM-1 excretion (a marker of tubular injury) was markedly increased in SS^LepRmutant rats versus SS rats. Treatment with IL-25 significantly reduced tubular injury in SS^LepRmutant rats (Fig. 2C).

Figure 2.

Effects of interleukin (IL)-25 treatment on temporal changes in mean arterial pressure (MAP; A), protein excretion (B), and tubular injury (C) in Dahl salt-sensitive (SS) rats and obese SS leptin receptor mutant (SS^LepRmutant) rats. Female rats and male rats in each group are represented by complete and partially filled symbols, respectively. The numbers of symbols in each bar dataset in C indicate the number of rats studied per group. Values are means ± SE. The significance of the difference in mean values for a single time point was determined by two-way ANOVA followed by a Holm–Sidak’s multiple-comparisons test. Temporal changes in protein excretion were compared between and within strains using three-way ANOVA followed by a Tukey’s multiple-comparisons test. P values of <0.05 were considered significantly different. *Significant difference from the corresponding value within the same strain at baseline; †significant difference from the corresponding value in SS rats within the same treatment; #significant difference from the corresponding value in vehicle-treated rats within the same strain. KIM-1, kidney injury molecule-1.

Assessment of Renal Histopathology

Representative images of glomerular injury and renal fibrosis along with the corresponding analysis of renal histopathology in SS and SS^LepRmutant rats treated with IL-25 are shown in Fig. 3. The kidneys from SS^LepRmutant rats exhibited increased mesangial expansion and severe glomerulosclerosis compared with their SS counterparts (Fig. 3, A and C), and treatment with IL-25 significantly decreased glomerular injury in SS^LepRmutant rats. Similar to glomerular injury, renal fibrosis (percent blue staining) was markedly elevated in SS^LepRmutant rats versus their SS littermates (Fig. 3, B and D). Chronic treatment with IL-25 only decreased renal fibrosis in the SS^LepRmutant strain.

Figure 3.

Representative images and analyses of renal histopathology in Dahl salt-sensitive (SS) and obese SS leptin receptor mutant (SS^LepRmutant) rats treated with either control or interleukin (IL)-25. Female rats and male rats in each group are represented by complete and partially filled symbols, respectively. A: periodic acid-Schiff staining. B: Masson’s trichrome staining. C: glomerular injury score. D: renal fibrosis (percent blue staining). Values are means ± SE. The significance of the difference in mean values for a single time point was determined by two-way ANOVA followed by a Holm–Sidak’s multiple-comparisons test. P values of <0.05 were considered significantly different. †Significant difference from the corresponding value in SS rats within the same treatment; #significant difference from the corresponding value in control-treated rats within the same strain.

Flow Cytometry

The effects of chronic treatment of IL-25 on renal immune cell infiltration in SS and SS^LepRmutant strains are shown in Figs. 4 and 5. At the end of the study, kidney weights between lean SS and obese SS^LepRmutant rats were not significantly different (2.19 ± 0.13 and 2.57 ± 0.11 g, respectively). Therefore, kidney size did not have an impact on the infiltration of immune cells between SS and SS^LepRmutant rats. We observed a significant increase in renal infiltration of leukocytes in SS^LepRmutant rats compared with SS rats, with no effects observed with IL-25 treatment in either strain (Fig. 4A). Infiltration of total T cells and B cells was significantly elevated in the kidneys from SS^LepRmutant rats versus SS rats (Fig. 4, B and C), and treatment with IL-25 prevented the increase in total T cell and B cell infiltration in SS^LepRmutant rats. Similar results were noticed when measuring Th cells (Fig. 4D). Renal infiltration of Th cells was significantly higher in SS^LepRmutant rats compared with SS rats, and IL-25 treatment reduced Th cells by more than 40% with no effect in SS rats. We observed a significant increase in the renal infiltration of Th2 cells in SS^LepRmutant rats compared with SS rats, and chronic treatment with IL-25 resulted in an almost twofold increase in renal Th2 cells in SS^LepRmutant rats without affecting SS rats (Fig. 4F). Cytotoxic T cells were markedly elevated in the kidneys from SS^LepRmutant rats compared with values measured in SS rats, and chronic treatment with IL-25 had no effect in either strain (Fig. 4E).

Figure 4.

Comparison of leukocyte infiltration in Dahl salt-sensitive (SS) and obese SS leptin receptor mutant (SS^LepRmutant) rats treated with either control or interleukin (IL)-25. Female rats and male rats in each group are represented by complete and partially filled symbols, respectively. A: leukocytes. B: B lymphocytes. C: total T cells. D: T helper (Th) cells. E: cytotoxic T cells. F: Th2 cells. Values are means ± SE. The significance of the difference in mean values for a single time point was determined by two-way ANOVA followed by a Holm–Sidak’s multiple-comparisons test. P values of <0.05 were considered significantly different. †Significant difference from the corresponding value in SS rats within the same treatment; #significant difference from the corresponding value in control-treated rats within the same strain.

Figure 5.

Comparison of renal macrophage infiltration in Dahl salt-sensitive (SS) and obese SS leptin receptor mutant (SS^LepRmutant) rats treated with either control or interleukin (IL)-25. Female rats and male rats in each group are represented by complete and partially filled symbols, respectively. A: total macrophages. B: M1 macrophages. C: M2a macrophage subtype. D: M2b macrophage subtype. E: M2c macrophage subtype. F: M2d macrophage subtype. Values are means ± SE. The significance of the difference in mean values for a single time point was determined by two-way ANOVA followed by a Holm Sidak’s multiple-comparisons test. P values of <0.05 were considered significantly different. †Significant difference from the corresponding value in SS rats within the same treatment; #significant difference from the corresponding value in control-treated rats within the same strain.

Since IL-25 has been shown to regulate macrophage polarization, we examined the effects of IL-25 on total, M1, and M2 macrophages (Fig. 5). We observed a significant increase in renal total macrophages in SS^LepRmutant rats compared with SS rats, and IL-25 treatment did not have an effect on total macrophages in SS and SS^LepRmutant rats (Fig. 5A). M1 macrophages were significantly increased in the kidneys from SS^LepRmutant rats versus SS rats (Fig. 5B). Chronic treatment with IL-25 only significantly reduced renal M1 macrophage infiltration in SS^LepRmutant rats. Renal infiltration of M2a macrophages was similar in SS and SS^LepRmutant rats, but renal M2a macrophages were significantly elevated in IL-25-treated SS^LepRmutant rats but not SS rats (Fig. 5C). Surprisingly, we did not observe any differences in renal infiltration of M2b macrophages among the groups (Fig. 5D). Renal infiltration of M2c macrophages was significantly higher in SS^LepRmutant rats compared with values measured in SS rats, and treatment with IL-25 had no effect in both strains (Fig. 5E). Similar to M2b macrophages, there were no differences in renal M2d macrophages detected among the groups (Fig. 5F).

Measurement of Plasma and Renal Cytokines

The effects of IL-25 treatment on plasma and renal cytokine levels in SS and SS^LepRmutant rats are shown in Table 2. There was a significant decrease in renal expression of IL-25 in SS^LepRmutant rats versus SS rats, and chronic treatment with IL-25 resulted in a marked increase in renal IL-25 expression only in SS^LepRmutant rats. We observed a significant decrease in plasma IL-4 levels in SS^LepRmutant rats compared with values measured in SS rats. However, chronic treatment with IL-25 had no effect on plasma IL-4 levels in both SS and SS^LepRmutant rats. Plasma IL-10 levels were reduced by more than 45% in SS^LepRmutant rats compared with their SS counterparts, and treatment with IL-25 caused a significant increase in plasma IL-10 in SS^LepRmutant rats without affecting SS rats. We did not detect any differences in plasma levels of IL-5, IL-13, TNF-α, and IFN-γ in control and IL-25-treated SS and SS^LepRmutant rats. In addition, we did not observe any differences in the renal expression of cytokines among the groups.

Table 2.

Effects of IL-25 treatment on plasma and renal cytokine levels in SS and SS^LepRmutant rats

	Vehicle Treatment		IL-25 Treatment
	SS Rats	SSLepRmutant Rats	SS Rats	SSLepRmutant Rats
Plasma cytokines, pg/mL
IL-4	19 ± 4	9 ± 1*	17 ± 3	13 ± 2
IL-5	187 ± 15	187 ± 8	200 ± 10	184 ± 12
IL-10	105 ± 11	57 ± 4*	90 ± 7	93 ± 6†
IL-13	4 ± 1	4 ± 3	7 ± 2	6 ± 3
TNF-α	28 ± 4	19 ± 10	32 ± 18	36 ± 28
IFN-γ	39 ± 10	29 ± 5	37 ± 8	21 ± 4
Renal cytokines, pg/mg protein
IL-25	117 ± 5	72 ± 4*	104 ± 5	91 ± 3†
IL-4	21 ± 4	25 ± 2	26 ± 4	24 ± 2
IL-5	10 ± 1	11 ± 1	12 ± 1	11 ± 1
IL-10	225 ± 35	290 ± 26	278 ± 34	249 ± 17
IL-13	29 ± 6	32 ± 3	37 ± 5	33 ± 4
TNF-α	27 ± 6	28 ± 2	32 ± 5	26 ± 2
IFN-γ	31 ± 5	36 ± 3	38 ± 4	34 ± 2

Values are means ± SE; n = 10–15 per group in each cytokine. IFN, interferon; IL, interleukin; SS rats, Dahl salt-sensitive rats; SS^LepRmutant rats, obese SS leptin receptor mutant rats; TNF, tumor necrosis factor. *P < 0.05 vs. SS rats within the same treatment; †P < 0.05 vs. vehicle-treated rats within the same strain.

DISCUSSION

With the relationship between childhood obesity and renal disease on the rise, there is a growing need to identify mechanisms involved in the early development of obesity-induced renal injury before puberty. We have recently observed that obese SS^LepRmutant rats develop progressive renal injury before puberty (7–11) and that renal injury was associated with increased renal immune cell infiltration (i.e., macrophages and T cells) and proinflammatory cytokines (9, 10). Our laboratory demonstrated that depletion of renal macrophages using liposomal clodronate slowed but did not prevent the early progression of renal injury in SS^LepRmutant rats (7). Macrophages can be divided into two distinct phenotypes: M1 macrophages (proinflammatory) and M2 macrophages (anti-inflammatory) (16, 19, 26). In addition, M2 macrophages can be separated into subtypes: M2a (wound healing/anti-inflammatory), M2b (immune mediated/proinflammatory), M2c (regulatory/anti-inflammatory), and M2d (tumor associated/proangiogenic). We recently observed that renal injury in SS^LepRmutant rats was associated with increased renal M1 macrophages compared with their lean SS counterparts (10). Therefore, in the present study, we examined whether increasing M2 macrophages with IL-25 would prevent the early progression of renal disease in SS^LepRmutant rats. We also compared the composition of renal M2 macrophage subtypes between SS and SS^LepRmutant rats and tested the effects of IL-25 on these subtypes. The administration of IL-25 significantly increased renal IL-25 cytokine levels and M2a macrophages and decreased renal M1 macrophages in SS^LepRmutant rats. Treatment with IL-25 reduced plasma insulin levels, whereas triglycerides, total cholesterol, and blood glucose levels were found to be unaffected by IL-25 in SS^LepRmutant rats. Chronic treatment with IL-25 markedly decreased proteinuria by 40% and significantly reduced renal histopathology in SS^LepRmutant rats in the absence of lowering arterial pressure. In addition, IL-25 treatment significantly increased plasma levels of IL-10 in SS^LepRmutant rats while not having any effect on cytokine levels in the kidneys. When we examined the infiltration of various immune cells, chronic treatment with IL-25 significantly reduced total T cells, Th cells, and M1 macrophages while raising M2a macrophages. These results suggest that treatment with IL-25 may slow the early progression of renal injury in SS^LepRmutant rats before puberty by increasing plasma IL-10 and renal immune cells (M2a macrophages) and decreasing renal proinflammatory immune cells (T cells and M1 macrophages).

One interesting finding from the present study was IL-25 reduced plasma insulin levels and improved insulin resistance in SS^LepRmutant rats. Although the mechanism by which IL-25 decreases insulin levels is unknown, one potential mechanism is its anti-inflammatory effects. In the present study, we observed that IL-25 increased plasma IL-10, which has been shown to increase insulin sensitivity (27–29). In addition, Hong et al. (28) demonstrated that muscle-specific overexpression of IL-10 prevented macrophage infiltration and improved insulin sensitivity in mice fed a high-fat diet. Overall, IL-25 may improve insulin resistance by increasing anti-inflammatory cytokines and inhibiting obesity-induced systemic inflammation.

One of the most intriguing findings from the present study was that IL-25 increased M2a macrophages and decreased M1 macrophages in the kidneys of SS^LepRmutant rats without altering total and other M2 subtype macrophages. Furthermore, treatment with IL-25 was associated with a significant increase in Th2 cells. Although IL-25-mediated Th2 responses have been shown to be involved in M2 macrophage polarization, studies have also demonstrated the ability of IL-25 to induce M2 macrophage polarization independent of Th2 effects (22, 30, 31). Therefore, we believe that both Th2-dependent and -independent effects of IL-25 may be involved in M2 macrophage polarization in the present study. In addition, previous studies have demonstrated that IL-25 suppressed proinflammatory macrophages and induced M2 macrophages in mice with adriamycin-induced nephropathy and ischemia-reperfusion injury (22, 23). In this study, we observed a significant decrease in plasma IL-10 in SS^LepRmutant rats, and administration of IL-25 was associated with an increase in plasma IL-10 in these rats. We hypothesize that the decrease in plasma IL-10 levels in SS^LepRmutant rats is due to the higher systemic inflammation in SS^LepRmutant rats versus SS rats. Moreover, the beneficial effects of anti-inflammatory IL-25 increased circulating IL-10 levels in SS^LepRmutant rats. Although not providing evidence of increasing M2 macrophages, other studies have reported that directly elevating IL-4 and/or IL-10 levels decreases total macrophages in a model of experimental glomerulonephritis (32–34). These data suggest that the increase in renal M2a macrophages in response to IL-25 may due to an increase in renal IL-25 expression, an increase in renal Th2 cells, and/or systemic anti-inflammatory effects of IL-10.

Several studies from Mattson and colleagues have demonstrated that the development of hypertension in SS rats fed a high-salt diet was associated with increased renal infiltration of T cells and macrophages, both M1 and M2 macrophages (12, 35). Moreover, a reduction of T cells has been shown to decrease arterial pressure in SS rats fed a high-salt diet (36–38), but depletion of total macrophages with liposome clodronate has not proven to reduce arterial pressure in SS rats in response to a high-salt diet (35). In the present study, we observed similar results, in which the significant elevation in arterial pressure in SS^LepRmutant rats was associated with increased renal M1 and M2c macrophages compared with their lean SS counterparts when measuring arterial pressure by chronic catheter via the carotid artery. When measuring arterial pressure via telemetry, we saw similar differences without statistical significance. However, reducing renal M1 macrophages and elevating M2a macrophages with IL-25 did not have an impact on arterial pressure in SS^LepRmutant rats. There is one potential reason of why we did not observe an effect of IL-25 on arterial pressure in SS^LepRmutant rats. SS^LepRmutant rats are on the SS genetic background and are susceptible to SS hypertension when fed a high-salt diet (12, 35, 39–41). Although SS^LepRmutant rats were not fed a high-salt diet in the present study, they consumed more food due to a dysfunctional leptin receptor, which contributes to their elevation in arterial pressure. These data suggest that elevations in arterial pressure in SS rats are associated with renal infiltration of T cells and macrophages, but only T cells seem to play a role in the development of hypertension in SS rats. However, further studies are needed to dissect the role of individual immune cells in the early development of elevations in arterial pressure associated with obesity.

Chronic treatment with IL-25 markedly reduced proteinuria and renal injury in obese SS^LepRmutant rats independent of lowering arterial pressure. Our findings are consistent with previous studies demonstrating that exogenous IL-25 inhibited renal injury in proteinuric mice (22, 23). There are several potential reasons for the renoprotective effects of IL-25 in SS^LepRmutant rats. One potential reason is that IL-25 treatment increased plasma IL-10 in SS^LepRmutant rats. In obese and diabetic states, systemic metabolic stress and a proinflammatory environment characterized by low levels of IL-10 are associated with renal inflammation and kidney damage (42–47). Moreover, Mu et al. (48) demonstrated that increasing plasma IL-10 suppresses renal inflammation and fibrosis during the development of chronic kidney disease independent of changes in renal IL-10 expression. Another possible reason is IL-25 reduces total T cells and Th cells and increases Th2 cells in SS^LepRmutant rats. Although we did not measure other subsets of Th cells, we hypothesize that the anti-inflammatory actions of IL-25 would decrease proinflammatory Th cell subsets (i.e., Th17 and Th1). Previous studies have demonstrated the role of T cells in the development of renal injury in SS rats, and depletion of these immune cells or blockade of their pathways have prevented renal disease observed in various strains of SS rats (36–38). In addition, subsets of proinflammatory Th cells have been shown to be involved in the pathogenesis of chronic inflammation in acute kidney injury and glomerulonephritis (49–53). The exact mechanism by which these proinflammatory subsets of Th cells cause renal injury remains unclear but may involve promoting podocyte apoptosis (54–57). However, blockade of anti-inflammatory Th2 cells has been shown to lead to the development of renal injury (56, 57). Treatment of IL-25 reduced proinflammatory M1 macrophages while increasing anti-inflammatory M2a macrophages. In support of this finding, previous studies have demonstrated that chronic treatment with IL-25 raised M2 macrophages and decreased M1 macrophages, which led to a reduction of renal injury in mice with severe renal disease (22, 23). Mattson and colleagues (35) observed that macrophage depletion augmented the development of renal injury in SS rats fed a high-salt diet, which indicated that the observed increase in renal injury stimulated the elevations in arterial pressure. There are three potential reasons why we did not observe similar results in the present study in response to IL-25: 1) the animals in the present study were not fed a high-salt diet, 2) these strains of SS rats were younger (4–8 wk of age), and 3) SS^LepRmutant rats are obese. Taken together, these results indicate that the beneficial effects of IL-25 on renal injury in SS^LepRmutant rats may be due to its anti-inflammatory actions that preserve the glomerular permeability barrier and inhibit the development of renal fibrosis.

The overall goal of the present study was to examine whether treatment with IL-25 would reduce the early progression of renal injury in SS^LepRmutant rats by increasing renal M2 macrophages. Although the results support the main goal, there were some limitations in the study that should be noted. One limitation is we did not examine the influence of IL-25 on renal infiltration of other different subsets of Th cells (i.e., Th1, Th17, and regulatory T cells). Measuring these subsets of Th cells would have been beneficial, since they play a role in arterial pressure regulation and renal disease (58, 59). Another limitation is we did not perform either a glucose test or an insulin tolerance test to directly examine whether IL-25 treatment improved insulin sensitivity. An additional limitation is we did not evaluate the effect of IL-25 on glomerular filtration rate, since IL-25 reduced plasma insulin levels in SS^LepRmutant rats. Increased insulin levels have been shown to stimulate elevations in glomerular filtration rate (60–62). The lowering of proteinuria in response to IL-25 treatment may be due to reducing renal hyperfiltration, often observed in prepubertal SS^LepRmutant rats (9–11), via decreasing insulin levels. Future experiments from the laboratory will be designed to consider these limitations.

Perspectives

With the association of renal injury in obese children rising, there is an important need to identify novel targets to prevent the development of renal injury in this young population. Similar to our previous studies, we found that obese SS^LepRmutant rats develop renal disease before puberty that was associated with renal inflammation (7, 9–11). The present study demonstrated that reducing renal proinflammatory M1 macrophages and increasing plasma anti-inflammatory cytokines and renal Th2 cells and M2a macrophages with IL-25 was linked to decreased insulin resistance and renal injury in SS^LepRmutant rats. Overall, these results indicate drugs that induce anti-inflammatory pathways may be beneficial for obese children suffering from renal injury before puberty. In addition, targeting macrophage-related signaling pathways may serve as a novel therapeutic to slow early progression of renal disease associated with obesity.

DATA AVAILABILITY

Data will be made available upon reasonable request.

GRANTS

This work was financially supported by National Institutes of Health (NIH) Grants R01DK109133 and R01AG057842 (to J.M.W), NIH Grant R01HL151407 (to D.C.C), and American Heart Association Predoctoral Fellowship 23PRE1025798 (to U.S.E.). The work performed through the University of Mississippi Medical Center Molecular and Genomics Facility was supported, in part, by funds from NIH, including Mississippi INBRE (NIH Grant P20GM103476), Obesity, Cardiorenal and Metabolic Diseases-COBRE (NIH Grant P20GM104357), and Mississippi Center of Excellence in Perinatal Research (MS-CEPR)-COBRE (NIH Grant P20GM121334).

DISCLAIMERS

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

B.P., U.S.E, G.E.W., C.A.S., D.C.C., and J.M.W. conceived and designed research; B.P., U.S.E., G.E.W., C.A.S., D.C.C., and J.M.W. performed experiments; B.P., U.S.E., S.M., C.A.S., D.C.C., and J.M.W. analyzed data; B.P., U.S.E., S.M., C.A.S., D.C.C., and J.M.W. interpreted results of experiments; B.P., U.S.E., S.M., C.A.S., D.C.C., and J.M.W. prepared figures; B.P., U.S.E., S.M., G.E.W., C.A.S., D.C.C., and J.M.W. drafted manuscript; B.P., U.S.E., S.M., G.E.W., C.A.S., D.C.C., and J.M.W. edited and revised manuscript; B.P., U.S.E., S.M., G.E.W., C.A.S., D.C.C., and J.M.W. approved final version of manuscript.