Abstract
Recently, we reported that the early progression of renal injury in obese Dahl salt-sensitive leptin receptor mutant (SSLepRmutant) rats was associated with increased macrophage inflammatory protein 3-α (MIP3α) expression prior to puberty. Therefore, this study tested the hypothesis that MIP3α plays a role in recruiting immune cells, thereby triggering renal inflammation and early progressive renal injury in SSLepRmutant rats prior to puberty. Four-week-old Dahl salt-sensitive (SS) and SSLepRmutant rats either served as control (IgG; intraperitoneal, every other day) or received MIP3α-neutralizing antibody (MNA; 100 µg/kg) for 4 weeks. MNA reduced circulating and renal MIP3α levels and proinflammatory immune cells by 50%. Although MNA treatment did not affect blood glucose and plasma cholesterol levels, MNA markedly decreased insulin resistance and triglyceride levels in SSLepRmutant rats. We observed no differences in mean arterial pressure (MAP) between SS and SSLepRmutant rats, and MNA had no effect on MAP in either strain. Proteinuria was significantly increased in SSLepRmutant rats versus SS rats over the course of the study. Treatment with MNA markedly decreased proteinuria in SSLepRmutant rats while not affecting SS rats. Also, MNA decreased glomerular and tubular injury and renal fibrosis in SSLepRmutant rats while not affecting SS rats. Overall, these data indicate that MIP3α plays an important role in renal inflammation during the early progression of renal injury in obese SSLepRmutant rats prior to puberty. These data also suggest that MIP3α may be a novel therapeutic target to inhibit insulin resistance and prevent progressive proteinuria in obese children.
SIGNIFICANCE STATEMENT Childhood obesity is increasing at an alarming rate and is now being associated with renal disease. Although most studies have focused on the mechanisms of renal injury associated with adult obesity, few studies have examined the mechanisms of renal injury involved during childhood obesity. In the current study, we observed that the progression of renal injury in obese Dahl salt-sensitive leptin receptor mutant rats was associated with an increase in MIP3α, a chemokine, before puberty, and inhibition of MIP3α markedly reduced renal injury.
Introduction
In recent decades, obesity has become an epidemic globally and in the United States (Ogden et al., 2015; Ogden et al., 2016). The prevalence of obesity has risen not only in adults but also in children (Cattaneo et al., 2010). According to the World Health Organization, more obese and overweight children have died than underweight children due to breathing difficulties, elevated risk of fractures, hypertension, and early markers of cardiovascular disease and insulin resistance. Obese patients have an increased risk to develop diabetes and hypertension, the two leading causes of kidney disease (Kramer et al., 2006; Srivastava, 2006). Childhood obesity is positively correlated with markers of renal injury such as elevated serum creatinine and microalbuminuria (Ferris et al., 2007; Savino et al., 2010; Kaneko et al., 2011; Önerli Salman et al., 2019). Interestingly, renal injury in obese children and adolescents starts long before the development of hypertension or diabetes. Therefore, it is necessary to identify novel targets to prevent early progressive renal disease in this unique population. Recently, we reported that the Dahl salt-sensitive leptin receptor mutant (SSLepRmutant) rat develops renal injury without hyperglycemia and elevations in arterial pressure, prior to puberty, and is therefore a novel model to study mechanisms of childhood obesity-induced renal disease (McPherson et al., 2016; McPherson et al., 2019; McPherson et al., 2020; Poudel et al., 2020; Poudel et al., 2022).
An early hallmark characteristic of obesity-induced renal disease is elevations in glomerular filtration rate (renal hyperfiltration) (Hostetter et al., 1982; Kasiske and Napier, 1985). We hypothesized that renal hyperfiltration damages various renal cells such as podocytes and tubular cells, which are a source of proinflammatory cytokines (Rayego-Mateos et al., 2020). In support of our hypothesis, studies from our laboratory have demonstrated that early progressive proteinuria in obese SSLepRmutant rats was associated with renal hyperfiltration and inflammatory cytokines such as macrophage inflammatory protein 3-α (MIP3α) (McPherson et al., 2020; Brown et al., 2021). Moreover, prevention of renal hyperfiltration in SSLepRmutant rats prior to puberty slowed the progression of glomerular injury and decreased renal MIP3α levels (Brown et al., 2021). MIP3α is a low-molecular-weight chemotactic cytokine that recruits dendritic cells (DCs) and lymphocytes (Th17s, regulatory T cells, and B cells) (Wiede et al., 2013) and is secreted by podocytes and tubular cells, as well as immune cells such as macrophages (Nandi et al., 2014). However, the role of MIP3α in the progression of obesity-induced renal injury has not been studied. Therefore, the goal of the current study was to test the hypothesis that MIP3α plays a role in recruiting immune cells, thereby triggering renal inflammation and early progressive renal injury in SSLepRmutant rats prior to puberty.
Methods
General
Experiments were performed on a total of 59 female and male Dahl salt-sensitive (SS) and SSLepRmutant rats. Both rat strains were generated from our in-house colony of heterozygous SSLepRmutant rats, which were originally created at the Medical College of Wisconsin with the zinc finger nuclease technology (McPherson et al., 2016). Genotyping was done by the Molecular and Genomics Facility of the University at Mississippi Medical Center. The rats were given free access to food and water for the entire study. Rats were fed a 1% NaCl diet (Envigo, Madison, WI). MIP3α-neutralizing antibody (MNA) and IgG isotype control were purchased from R&D Systems (Minneapolis, MN). The rats were housed in the Laboratory Animal Facility of the University of Mississippi Medical Center, which is 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
These experiments were carried out on 4-week-old female and male SS and SSLepRmutant rats. At baseline, rats were weighed and placed in metabolic cages overnight to collect urine for the determination of proteinuria using the Bradford method (Bio-Rad Laboratories, Hercules, CA). Blood was collected from the tail vein to measure nonfasting blood glucose levels (glucometer from Bayer HealthCare; Mishwaka, IN). After the collection of baseline data, SS and SSLepRmutant rats were randomly separated into four groups as follows: 1) SS and 2) SSLepRmutant rats serving as control (IgG; 100 μg/kg i.p., every other day) and 3) SS and 4) SSLepRmutant rats receiving MNA 100 µg/kg i.p., every other day, for 4 weeks. The doses of MNA and IgG were selected from a previous study (Hu et al., 2016). Every 2 weeks, nonfasting blood glucose and proteinuria were measured in the rats. Urine collected at the end of the protocol were used to measure the excretion rates of kidney injury molecule 1 (KIM-1; Abcam, Waltham, MA), neutrophil gelatinase-associated lipocalin (NGAL; Abcam), albumin (Abcam), and nephrin (NPB2-76751, Novus Biologicals, Littleton, CO) via ELISA according to the manufacturer’s recommendations. Glomerular filtration rate was assessed via creatinine clearance (CrCl) (Bioassay Systems, Hayward, CA).
At the end of the study, rats were anesthetized, and a catheter was inserted into the carotid artery to measure mean arterial pressure (MAP). After a 24-hour recovery period, catheters were connected to pressure transducers (MLT0699, ADInstruments, Colorado Springs, CO) coupled to a computerized PowerLab data-acquisition system (ADInstruments). After a 30-minute equilibration period, MAP was recorded continuously for 30 minutes. Then, a final blood sample was drawn from the abdominal aorta for the measurement of plasma cholesterol (Cayman Chemical, Ann Arbor, MI), triglyceride (Cayman Chemical), insulin (Mercodia rat insulin ELISA, Uppsala, Sweden), and MIP3α (Bio-Rad Laboratories, Hercules, CA) concentrations. Next, the kidneys were perfused with saline until they appeared visibly pale. The kidneys were collected and weighed. The right kidney was cut into two equal halves; one half was fixed in 10% neutral buffered formalin solution for histologic analysis. The other half was snap-frozen in liquid nitrogen and stored at −80°C. Renal cytokines were measured using the Bio-Plex Pro Rat Cytokine 5-Plex Assay Reagent Kit on a Bio-Rad Bio-Plex 200 system as described by the manufacturer’s protocol (Bio-Rad Laboratories). The cytokines measured were MIP3α, interleukin (IL)-2, IL-4, IL-10, and IL-17. The left kidney was used to measure the renal infiltration of immune cells as described below.
Renal Immune Cell Isolation and Flow Cytometry
Immune cells from the left kidneys of control and MNA-treated SS and SSLepRmutant rats were isolated as previously described (Poudel et al., 2020). Briefly, the kidneys were minced in RPMI-1640 containing 0.1% collagenase and 10 µg/ml DNAse 1. These were homogenized, filtered through a 100-µm strainer, incubated at 37°C for 30 minutes, and subsequently filtered through 70-µm and 40-µm strainers. Mononuclear cells were separated by Percoll density gradient centrifugation at 1200 rpm for 30 minutes at 25°C. The pellet obtained was washed and resuspended in 1 mL fluorescence-activated cell sorter buffer, after which immune cells were counted using an Automated Cell Counter and Image Cytometer (Nexcelom Bioscience, Lawrence, MA.). Mononuclear cells were stained with viability (Viobility 405/520 for macrophage and dendritic cells, 1:50; 405/452 for lymphocytes, 1:50) fixable dyes (Miltenyi Biotec, Auburn, CA) for 20 minutes at 4°C to identify live cells. The macrophage panel consisted of the following antibodies: anti-rat CD68-PE-Vio770 (1:10; Miltenyi Biotec), anti-rat CD86-PE (1:50; Miltenyi Biotec), and mouse anti-rat CD163-FITC (1:50; Bio-Rad Laboratories). The DC panel consisted of the following antibodies: anti-rat CD103-APC (1:10; Miltenyi Biotec), anti-rat CD86-PE (1:50; Miltenyi Biotec), and mouse anti-rat CD80-BV421 (1:10; BD Biosciences, Haryana, India). The lymphocyte panel consisted of the following antibodies: anti-rat CD3-VioGreen (1:10; Miltenyi Biotec) for total T cells, anti-rat CD4-FITC (1:10; Miltenyi Biotec) for total T helper cells, anti-rat CD8a-APC-Vio770 (1:50; Miltenyi Biotec) for cytotoxic T cells, mouse anti-rat CD25 PE (1:50; BD Biosciences), mouse anti-rat RORγT PerCP (1:2; R and D systems, Minneapolis, MN) for Th17 cells, mouse anti-rat FOXP3-AlexaFluor-647 (1:2; R and D systems, Minneapolis, MN) for regulatory T cells, and anti-rat CD45R-PE-Vio770 (1:10; Miltenyi Biotec) for total B cells. Flow cytometry was carried out using the Miltenyi MACSQuant Analyzer 10 (Miltenyi Biotec), and data were analyzed using FlowLogic software (Miltenyi Biotec).
Renal Histology
Paraffin-embedded kidney sections were prepared from half of the right kidneys collected from SS and SSLepRmutant rats. Kidney sections were cut into 5-µm sections and stained with periodic acid-Schiff and Picrosirius red. Thirty glomeruli per periodic acid-Schiff section were scored blindly, on a scale of 0–4 to assess glomerular injury, where 0 represented a normal glomerulus, 1 represented a 25% loss, 2 represented a 50% loss, 3 represented a 75% loss, and 4 represented a greater than 75% loss of capillaries in the glomerular tuft (McPherson et al., 2016; Spires et al., 2018). To assess the extent of renal cortical and medullary fibrosis from Picrosirius red–stained sections, 10 representative images per kidney section per animal were taken with a SeBa microscope equipped with a colored camera (Laxco Inc., North Creek, WA). We analyzed for the percentage of each image stained red (collagen) by identifying the animal whose kidney had the most collagen. This was used to set a threshold for red staining in the sections using NIS-Elements D 3.0 software (McPherson et al., 2016; Spires et al., 2018). Next, those same thresholding parameters were used for the red staining on each kidney image per rat in the study to measure renal fibrosis.
Statistical Analysis
The data are presented as mean values ± S.D. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA). The significance of the difference in mean values for a single timepoint was determined by two-way ANOVA followed by Holm-Sidak’s multiple-comparisons test. Time-course changes in protein excretion were compared from baseline, between and within strains, using three-way ANOVA followed by Tukey’s multiple-comparisons test. P values of <0.05 were considered significantly different.
Results
Flow Cytometry
The flow cytometry gating strategy for immune cell infiltration is provided in Fig. 1. After gating for the mononuclear cell population using forward scatter and side scatter, dead cells were excluded using viability staining and doublet exclusion. From this population, CD3+ cells were gated as T lymphocytes, and CD4+ T Helper cells and CD8+ cytotoxic T cells were identified from the CD3+ cell population. From the CD4+ T Helper cell population, CD25+FOXP3+ cells were identified as regulatory T cells, whereas CD25-RORγT+ cells were identified as Th17s. CD45R+ B cells were identified from the CD3− cell population. Also, from the CD3− population, CD68+ and CD103+ cells were identified as macrophages and DCs, respectively. CD68+CD86+ and CD68+CD163+ cells were identified as M1 and M2 macrophages, respectively. CD103+CD80+ and CD103+CD86+ cells were identified as stimulatory DCs.
Measurement of Circulating and Renal MIP3α
The effects of chronic MNA administration on plasma and renal MIP3α are shown in Fig. 2. Plasma and renal MIP3α were markedly greater in control SSLepRmutant rats versus SS rats, and chronic MNA administration only decreased MIP3α in the plasma and kidneys of SSLepRmutant rats (Fig. 2, A and B).
Metabolic Endpoints
The effects of chronic MNA administration on metabolic endpoints are presented in Table 1. There was a marked increase in body weight in control SSLepRmutant rats in comparison with their SS counterparts, and MNA treatment did not affect body weight in either strain. We detected no differences in blood glucose levels in any of the groups. We observed an almost 10-fold increase in plasma insulin in control SSLepRmutant rats in comparison with SS rats, and the administration of MNA markedly reduced plasma insulin levels in SSLepRmutant rats by over 50% while not affecting SS rats. Plasma triglycerides were over 6 times higher in control SSLepRmutant rats compared with SS counterparts (467 ± 91 mg/dL versus 70 ± 28 mg/dL, respectively), and the administration of MNA decreased plasma triglycerides by over 50% (210 ± 30 mg/dL) while not affecting SS rats (71 ± 24 mg/dL). There was a marked increase in plasma total cholesterol in control SSLepRmutant rats in comparison with SS rats (252 ± 46 versus 143 ± 38 mg/dL, respectively), and the administration of MNA did not affect plasma total cholesterol in either strain.
Measurement of Mean Arterial Pressure and Markers of Renal Injury
The effects of MNA administration on MAP and markers of renal injury (proteinuria and albuminuria) are presented in Fig. 3. There were no marked differences in MAP among control and MNA-treated SS and SSLepRmutant rats (Fig. 3A). At baseline, we detected no differences in protein excretion between SS and SSLepRmutant rats. During the course of the study, proteinuria rose from 41 ± 7 mg/d to 426 ± 47 mg/d in control SSLepRmutant rats compared with control SS rats, where proteinuria only rose from 10 ± 3 mg/d to 51 ± 16 mg/d. Chronic MNA administration decreased proteinuria by over 50% in SSLepRmutant rats while not affecting SS rats (Fig. 3B). At the end of the study, we observed an over 30-fold increase in albumin excretion in control SSLepRmutant rats in comparison with SS rats, and chronic administration of MNA resulted in a near fivefold decrease in albumin excretion in SSLepRmutant rats while not affecting SS rats (Fig. 3C).
Renal Function Assessment and Markers of Glomerular and Tubular Injury
The effects of MNA on creatinine clearance (CrCl), as well as markers of glomerular and tubular injury, are presented in Fig. 4. There was a marked increase in CrCl in control SSLepRmutant rats in comparison with SS rats. Chronic MNA administration did not affect CrCl in SSLepRmutant rats and SS rats (Fig. 4A). We detected a fourfold increase in nephrin excretion in control SSLepRmutant rats in comparison with SS rats, and the administration of MNA decreased nephrin excretion by about 50% in SSLepRmutant rats while not affecting SS rats (Fig. 4B). There was a marked increase in excretion of urinary markers of tubular injury (KIM-1 and NGAL) in control SSLepRmutant rats in comparison with their SS counterparts (Fig. 4, C and D). The chronic administration of MNA markedly decreased KIM-1 and NGAL excretion in SSLepRmutant rats while not affecting the SS counterparts.
Renal Histology
Representative images and a corresponding analysis of renal histopathology in control and MNA-treated SS and SSLepRmutant rats are shown in Fig. 5. The kidneys from control SSLepRmutant rats showed increased mesangial expansion and glomerular injury in comparison with their SS counterparts (Fig. 5, A and D), and chronic MNA administration significantly reduced glomerular injury in SSLepRmutant rats. Greater fibrosis was seen in the renal cortex (Fig. 5, B and E) and renal medulla (Fig. 5, C and F) of SSLepRmutant rats in comparison with their SS counterparts, and chronic MNA administration reduced cortical and medullary interstitial fibrosis.
Measurement of Renal Immune Cell Infiltration
The effects of MNA on renal immune cell infiltration are shown in Figs. 6 and 7. There was a marked increase in renal CD103+ and CD103+/CD80+ DCs in control SSLepRmutant rats in comparison with SS rats, with no noteworthy differences detected in CD103+/CD86+ DCs. Chronic administration of MNA markedly reduced the renal infiltration of these DCs without affecting SS rats (Fig. 6, A and B). Renal total macrophages were significantly increased in control SSLepRmutant rats versus SS rats. Similar results were observed with M1 and M2 macrophages (Fig. 6, E and F). The administration of MNA significantly decreased the infiltration of total and M1 macrophages, but not M2, in SSLepRmutant rats (Fig. 6, D and E).
The effect of the administration of MNA on the renal infiltration of various lymphocytes is presented in Fig. 7. We detected a marked increase in the renal infiltration of total T cells, Th17s, and cytotoxic T cells in control SSLepRmutant rats in comparison with SS rats. The chronic administration of MNA markedly reduced the renal infiltration of Th17s and cytotoxic T cells, but not total T cells, in SSLepRmutant rats (Fig. 7, A, B, and E). No differences were detected in total T Helper cells or regulatory T cells across the groups (Fig. 7, D and F). Total B cells were markedly increased in the kidneys of control SSLepRmutant rats in comparison with SS rats. The administration of MNA only reduced the renal infiltration of B cells in SSLepRmutant rats (Fig. 7C).
Renal Cytokine Measurements
The effects of chronic MNA administration on other renal cytokines are presented in Fig. 8. There was a marked decrease in renal IL-10 in control SSLepRmutant rats in comparison with SS rats. Chronic MNA treatment did not affect renal IL-10 in SSLepRmutant rats (Fig. 8A). Similar to IL-10, we noticed a 50% reduction in renal IL-4 in control SSLepRmutant rats versus SS rats, and chronic MNA treatment did not affect renal IL-4 in SSLepRmutant rats (Fig. 8B). There were no differences in renal IL-2 or IL-17 expression for both control and MNA-treated SS and SSLepRmutant rats (Fig. 8, C and D).
Discussion
Obese adults and children are susceptible to renal disease independent of diabetes or hypertension (Kramer et al., 2006; Srivastava, 2006; Kovesdy et al., 2017). Although studies have focused on mechanisms of renal disease in adult obesity, little effort has been put into studying renal disease associated with childhood/prepubertal obesity. A major characteristic of obese patients is renal hyperfiltration (Ferris et al., 2007; Eirin et al., 2017; van Bommel et al., 2020). Studies have demonstrated that obesity-induced hyperfiltration leads to injury of specialized cells of the glomerulus, such as the podocytes, and tubular epithelial cells, which are a source of proinflammatory mediators (Brown et al., 2021). Recently, we reported that early progressive renal injury was associated with renal hyperfiltration, glomerular injury, renal macrophage infiltration, and increased renal MIP3α expression in obese SSLepRmutant rats before puberty (McPherson et al., 2016; Poudel et al., 2020; Brown et al., 2021; Poudel et al., 2022). Moreover, preventing renal hyperfiltration reduced MIP3α and renal disease during the prepubescent stage (Brown et al., 2021). Thus, the aim of the current study was to determine the role of MIP3α during the early progression of renal injury in SSLepRmutant rats prior to puberty. The administration of MNA decreased circulating and renal levels of MIP3α by over 50% in SSLepRmutant rats. Additionally, MNA administration reduced the renal infiltration of various immune cells (i.e., DCs, macrophages, Th17s, cytotoxic T cells, and B cells) in SSLepRmutant rats and significantly decreased renal injury in SSLepRmutant rats. In addition, the administration of MNA improved metabolic parameters in SSLepRmutant rats by decreasing insulin resistance and plasma triglyceride levels. Taken together, these results show that the neutralizing MIP3α ameliorates metabolic endpoints and decreases renal inflammation and renal injury in SSLepRmutant rats before puberty.
Insulin is essential for the maintenance of physiologic levels of triglycerides as it promotes triglyceride storage while preventing its breakdown in adipose tissues (Czech et al., 2013). The SSLepRmutant rats displayed features of metabolic syndrome, and MNA administration decreased hyperinsulinemia/insulin resistance and dyslipidemia while not affecting their body weight or blood glucose levels. A plausible explanation for this is that the anti-inflammatory effects of MNA improved insulin resistance in SSLepRmutant rats. Hyperinsulinemia is a known indicator of systemic insulin resistance (Czech, 2017; Petersen and Shulman, 2018). The chemokine system contributes to insulin resistance during obesity by regulating immune cell recruitment, thereby triggering inflammation and impairing insulin sensitivity (Cancello et al., 2005; Huber et al., 2008; Neels et al., 2009; Kitade et al., 2012; Ota, 2013). This is supported by studies that show that inhibiting the chemokine signaling pathway, MCP1-CCR2, improved insulin sensitivity, whereas the overexpression of MCP1-CCR2 stimulated insulin resistance in mice (Kamei et al., 2006; Kanda et al., 2006; Weisberg et al., 2006). Although information on the role of MIP3α in metabolic syndrome is limited, a study reported that MIP3α is elevated in the serum of obese mice (Burke et al., 2015). In the current study, plasma MIP3α was elevated in SSLepRmutant rats, and the administration of MNA decreased plasma MIP3α and reduced hyperinsulinemia, suggesting a relationship between plasma MIP3α and obesity-related hyperinsulinemia and insulin resistance. Hyperinsulinemia and insulin resistance may, in part, contribute to dyslipidemia seen in SSLepRmutant rats, and the reduction of plasma triglycerides in MNA-treated SSLepRmutant rats may, in part, be due to decreased hyperinsulinemia and insulin resistance. These findings may be associated with the anti-inflammatory effects of MNA. Although various studies have demonstrated that improvements in insulin sensitivity are associated with weight loss (Ikeda et al., 1996; Clamp et al., 2017), chronic MNA administration did not lead to weight loss in SSLepRmutant rats despite improvements in insulin sensitivity. The lack of effect of MNA on body weight in SSLepRmutant rats may be due to the mutation in their leptin receptor that makes SSLepRmutant rats hyperphagic, leading to increased food intake and weight gain despite the effects of MNA on insulin resistance. These data suggest that MIP3α plays a role in insulin resistance and dyslipidemia during childhood obesity.
Despite overwhelming evidence to support the role of inflammation in the development of hypertension (Olsen, 1972; Kirabo et al., 2014; Mattson, 2019; Van Beusecum et al., 2019; Fehrenbach and Mattson, 2020), we did not observe a decrease in arterial pressure when inhibiting MIP3α in SS and SSLepRmutant rats. Furthermore, the inhibition of other chemokines has been shown to reduce arterial pressure by decreasing immune cell recruitment in various animal models of hypertension (Ruiz-Ortega et al., 2002; Chung and Lan, 2011; Alsheikh et al., 2020). There are three potential reasons that could explain our observation. 1) The first possible reason is the choice of diet used during the experiment. Mattson and colleagues have fed their SS rats a high-salt diet containing 4% NaCl or greater, which causes a rapid increase in arterial pressure within 3 weeks (Rudemiller et al., 2014). In the current study, both SS and SSLepRmutant strains are fed a diet containing 1% NaCl, which minimized the development of hypertension in the SS strain. 2) Another potential reason is the age of the rats used in our study. Previous studies have used SS rats older than 9 weeks of age (Mattson et al., 2006). Since the current study focused on progressive renal injury prior to puberty, it is reasonable not to expect differences in arterial pressure in response to various drugs that may typically lower arterial pressure in older animals. This is supported by recent studies from our laboratory that demonstrate that anti-inflammatory or arterial pressure–lowering drugs do not reduce arterial pressure in these rats during the prepubescent stage (Poudel et al., 2020; Brown et al., 2021). 3) The third reason is that the development of hypertension within SS rat genetic background is multifactorial, and specifically inhibiting MIP3α is not enough to reduce arterial in SSLepRmutant rats. These results suggest that the lack of arterial pressure reduction in SSLepRmutant rats’ response to MNA administration may due to the amount of NaCl content in the diet, age, and the genetic background of the SS rats.
A significant finding from this study was that the chronic administration of MNA markedly reduced early progressive renal injury in SSLepRmutant rats before puberty. Clinical and experimental studies have shown that the progressive renal injury in certain severe forms of renal disease is associated with increased renal MIP3α expression (Turner et al., 2010; Lu et al., 2017; González-Guerrero et al., 2018). MIP3α is a strong chemotactic for immune cells such as DCs, T cells, and B cells (Woltman et al., 2005; Wiede et al., 2013; Nandi et al., 2014). During renal injury, stimulatory DCs activate T cells via CD80/86 upregulation (Banchereau and Steinman, 1998; Kurts et al., 2020; Zhang et al., 2020), which elicits a proinflammatory response and macrophage recruitment, leading to renal inflammation and progressive renal injury (Kurts et al., 2020; Zhang et al., 2020). This is supported by the results of this study and preliminary reports from our laboratory demonstrating that inhibition of T-cell activation slows progressive renal injury in SSLepRmutant rats before puberty (Ekperikpe et al., 2021). Therefore, we believe that chronic MIP3α neutralization with MNA reduces progressive renal injury in SSLepRmutant rats by decreasing the recruitment of DCs and macrophages, thereby preventing T-cell activation. Furthermore, chronic MNA administration produces an overall anti-inflammatory effect by reducing renal MIP3α expression. Although treatment with MNA decreased renal Th17 infiltration, there were no noticeable differences in renal IL-17 expression. A plausible explanation for this observation is that there are multiple sources of IL-17 other than Th17s (Keijsers et al., 2014). Overall, these data reveal that MIP3α contributes to progressive renal injury in SSLepRmutant rats by mediating proinflammatory response during prepubertal obesity.
In conclusion, the data from this study suggest that during the early stages of renal injury in SSLepRmutant rats, renal hyperfiltration damages glomerular and tubular epithelial cells. These cells secrete MIP3α, leading to the recruitment of DCs, which activate T cells, causing a proinflammatory response and macrophage recruitment, stimulating renal inflammation and progressive renal injury associated with obesity prior to puberty. Moreover, chronic MIP3α inhibition ameliorated metabolic syndrome and renal injury in obese SSLepRmutant rats by decreasing renal inflammation. Although the current study specifically focused on the effects of MIP3α blockade on renal injury in young obese SSLepRmutant rats, we would speculate that similar results would be observed in older SSLepRmutant rats. Inflammation is known to contribute to renal injury in various adult animal models of obesity (Fernández-Sánchez et al., 2011; Wang et al., 2015; Lindfors et al., 2021). To the best of our knowledge, this is the first study to demonstrate a role for MIP3α in the early development of renal injury associated with obesity. Future studies from our laboratory will focus on downstream events after MIP3α-signaling stimulation during the early progression of obesity-induced renal injury such as T cell activation. These data indicate that MIP3α may be a pharmacological target for managing renal injury and metabolic disease associated with childhood obesity.
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Acknowledgments
The authors are grateful to Tyler D. Johnson and Sarah M. Safir for maintaining the animal colony.
Authorship Contributions
Participated in research design: Ekperikpe, Cornelius, Williams.
Conducted experiments: Ekperikpe, Poudel, Shields, Cornelius, Williams.
Performed data analysis: Ekperikpe, Cornelius, Williams.
Wrote or contributed to the writing of the manuscript: Ekperikpe, Mandal, Cornelius, Williams.
Footnotes
- Received August 8, 2022.
- Accepted November 23, 2022.
This work was financially supported by National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant R01-DK109133] (to J.M.W.) and the National Heart, Lung, and Blood Institute [Grant R01-HL151407] (to D.C.C.). The work performed through the UMMC Molecular and Genomics Facility is supported, in part, by funds from National Institutes of Health National Institute of General Medical Sciences, including Mississippi INBRE [Grant P20-GM103476], Obesity, Cardiorenal and Metabolic Diseases-COBRE [Grant P20-GM104357], and Mississippi Center of Excellence in Perinatal Research (MS-CEPR)-COBRE [Grant P20-GM121334].
No author has an actual or perceived conflict of interest with the contents of this article
Abbreviations
- CrCl
- creatinine clearance
- DC
- dendritic cell
- IL
- interleukin
- KIM-1
- kidney injury molecule-1
- MAP
- mean arterial pressure
- MIP3α
- macrophage inflammatory protein 3-α
- MNA
- MIP3α-neutralizing antibody
- NGAL
- neutrophil gelatinase-associated lipocalin
- SS
- Dahl salt-sensitive
- SSLepRmutant
- Dahl salt-sensitive leptin receptor mutant
- Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics