Altered renal hemodynamics is associated with glomerular lipid accumulation in obese Dahl salt-sensitive leptin receptor mutant rats

Abstract

The present study examined whether development of renal injury in the nondiabetic obese Dahl salt-sensitive leptin receptor mutant (SS^LepRmutant) strain is associated with elevations in glomerular filtration rate and renal lipid accumulation. Baseline mean arterial pressure at 6 wk of age was similar between Dahl salt-sensitive wild-type (SS_WT) and SS^LepRmutant rats. However, by 18 wk of age, the SS^LepRmutant strain developed hypertension, while the elevation in mean arterial pressure was not as severe in SS_WT rats (192 ± 4 and 149 ± 6 mmHg, respectively). At baseline, proteinuria was fourfold higher in SS^LepRmutant than SS_WT rats and remained elevated throughout the study. The early development of progressive proteinuria was associated with renal hyperfiltration followed by a decline in renal function over the course of study in the SS^LepRmutant compared with SS_WT rats. Kidneys from the SS^LepRmutant strain displayed more glomerulosclerosis and glomerular lipid accumulation than SS_WT rats. Glomeruli were isolated from the renal cortex of both strains at 6 and 18 wk of age, and RNA sequencing was performed to identify genes and pathways driving glomerular injury. We observed significant increases in expression of the influx lipid transporters, chemokine (C-X-C motif) ligand 16 (Cxcl16) and scavenger receptor and fatty acid translocase (Cd36), respectively, and a significant decrease in expression of the efflux lipid transporter, ATP-binding cassette subfamily A member 2 (Abca2; cholesterol efflux regulatory protein 2), in SS^LepRmutant compared with SS_WT rats at 6 and 18 wk of age, which were validated by RT-PCR analysis. These data suggest an association between glomerular hyperfiltration and glomerular lipid accumulation during the early development of proteinuria associated with obesity.

INTRODUCTION

Obesity increases the risk for diabetes and hypertension, the two primary causes of chronic kidney disease (CKD), and generally has been reported as a secondary risk factor for CKD (14). However, recent evidence suggests that the increasing rate of obesity has paralleled the incidence of CKD and is now considered an independent risk factor for CKD (5, 30, 35). Moreover, with the onset of obesity at an early age in children and adolescents, it is extremely important to understand the mechanisms that are involved during the early development of renal injury associated with obesity that lead to CKD later in life. Some of the renal histopathological abnormalities associated with obesity include glomerulosclerosis, podocyte hypertrophy and foot process effacement, and glomerular lipid accumulation, often referred to as obesity-related glomerulopathy (ORG) (9, 11, 37, 38, 68, 76). Since not all obese individuals develop glomerular disease, it is possible that obesity alone is not the primary cause of ORG, and there may be additional factors that increase susceptibility of obese patients to glomerular injury. One of the hallmark characteristics of renal injury associated with obesity is glomerular hyperfiltration (8, 23, 26). Glomerular hyperfiltration is a result of the early changes in intrarenal hemodynamic function, including increased renal blood flow and glomerular capillary pressure, in response to various stimuli such as hyperglycemia, hypertension, and obesity (21, 25, 43). Recent studies have suggested that insulin resistance due to obesity, rather than traditional risk factors, including hypertension and hyperglycemia, is the most significant cause of glomerular hyperfiltration (42, 53, 59, 75). While the mechanisms whereby glomerular hyperfiltration or elevations in glomerular filtration rate (GFR) contribute to glomerular injury remain unclear, we know that hyperfiltration induces changes in podocyte morphology, which lead to podocyte apoptosis, progressive proteinuria, and severe glomerulosclerosis.

Abnormal lipid metabolism is one of the key features of ORG that contributes to increased triglyceride and cholesterol accumulation (64, 76, 88). Various cells in the glomerulus, including mesangial cells and podocytes, are sensitive to lipid accumulation, which contributes to the development of glomerular injury associated with obesity (12, 73). Lipid accumulation during ORG is the result of increased synthesis of cholesterol and triglycerides (11, 12), increased uptake via influx transporters [fatty acid translocase (Cd36) and scavenger receptor chemokine (C-X-C motif) ligand 16 (Cxcl16)] (19, 28, 40, 51, 89), decreased metabolism, and efflux [ATP-binding cassette subfamily A members 1 and 2 (Abca1 and Abca2, respectively)] of these lipids (28, 48, 58, 74). However, the factor(s) that initiates this response in the kidney or, more specifically, in the glomerulus during ORG remains unknown. Furthermore, studies examining whether there is an association between changes in GFR and lipid accumulation during development of renal disease associated with obesity alone are limited. Recently, we reported that the obese Dahl salt-sensitive leptin receptor mutant (SS^LepRmutant) strain develops glomerular injury, characterized by podocyte foot process effacement and lipid droplets and progressive proteinuria as early as 6 wk of age, independent of hyperglycemia and elevations in arterial pressure, that eventually progresses to CKD (47). However, GFR has not been measured in the SS^LepRmutant strain. Therefore, in the present study, we examined whether the development and progression of renal disease in the nondiabetic obese SS^LepRmutant strain are associated with temporal changes in GFR. In addition, we used RNA sequencing (RNA-Seq) analysis to identify genes and pathways driving glomerular injury. For the purpose of the present study, we focused on genes involved in lipid metabolism, including fatty acid transporters (i.e., Cxcl16, Cd36, Abca1, and Abca2), that could contribute to glomerular lipid accumulation from glomeruli isolated from the obese SS^LepRmutant strain and their lean littermates, SS_WT rats, during these same time points.

METHODS

General.

Eighty male SS_WT and SS^LepRmutant rats were used at 6, 12, or 18 wk of age. Genotyping was performed by the Molecular and Genomic Facility at the University of Mississippi Medical Center, as previously described, using tail snips collected from pups at day 12 (47). Rats were fed a 1% NaCl diet (TD58640, Harlan Laboratories, Madison, WI). Rats had free access to food and water except during the 2-h period of GFR measurement. Rat housing in the Laboratory Animal Facility at the University of Mississippi Medical Center is approved by the American Association for the Accreditation of Laboratory Animal Care, and all protocols were approved by the University of Mississippi Medical Center Institutional Animal Care and Use Committee.

Time course of changes in metabolic parameters in SS_WT and SS^LepRmutant rats.

Experiments were performed on 6-, 12-, or 18-wk-old SS_WT and SS^LepRmutant rats. At each time point, rats were weighed and placed in metabolic cages for an overnight urine collection to determine urinary total protein concentration using the Bradford method (Bio-Rad Laboratories, Hercules, CA). Blood samples were collected from the tail vein for measurement of blood glucose levels with a glucometer (Bayer HealthCare, Mishawaka, IN) and plasma insulin concentrations by ELISA (rat insulin ELISA, Mercodia, Uppsala, Sweden).

Temporal measurements of GFR in SS_WT and SS^LepRmutant rats.

At each time period, rats were anesthetized with isoflurane, and a catheter filled with heparinized saline for injection of FITC-sinistrin (catalog no. FTC-FS001, MediBeacon, Mannheim, Germany) was inserted into the jugular vein and exteriorized subcutaneously at the back of the neck. After a 24-h recovery period, rats were anesthetized briefly with isoflurane for assembly of the noninvasive clearance kidney device (MediBeacon) consisting of two light-emitting diodes that excite FITC-sinistrin at 480 nm, a photodiode that emits light at 531 nm, a microprocessor, and a battery. This device was attached to the back of the rat by a double-sided adhesive patch (MediBeacon) and secured with a rodent jacket to a region (~3 cm) on the back of the rat from which hair had been removed with a depilation cream. Rats were allowed to recover in separate cages for 15 min, and a baseline measurement was recorded. After baseline measurements, a bolus injection of FITC-sinistrin (5 mg/100 g body wt, prepared as 15 mg/mL in sterile isotonic saline) was administered via the jugular vein followed by a bolus injection of sterile saline. During a 2-h period after the bolus injection, excretion kinetics of FITC-sinistrin were measured transcutaneously at a sampling rate of 60 measurements/min with an excitation time of 10 ms/measurement and used to calculate the elimination half-life (t_1/2) of FITC-sinistrin using a one-compartment model with MDPLab evaluation software (MediBeacon), as previously described (55, 56, 66). GFR was determined from the t_1/2 of FITC-sinistrin with a validated empirically derived conversion factor, as previously described (66, 67, 90).

Measurement of mean arterial pressure in SS_WT and SS^LepRmutant rats.

At 2 days after conscious GFR measurements, rats were anesthetized, and a catheter was inserted into the femoral artery for the measurement of mean arterial pressure (MAP). After a 24-h recovery period, catheters were connected to pressure transducers (model MLT0699, ADInstruments, Colorado Springs, CO) coupled to a computerized data-acquisition system (PowerLab, ADInstruments), and MAP was recorded continuously for 30 min after a 30-min equilibration period. After arterial pressure measurements, a final blood sample was taken from the abdominal aorta for the measurement of plasma cholesterol and triglyceride concentrations (Cayman Chemical, Ann Arbor, MI), and kidneys were collected as previously described (47). Kidneys were weighed, cut in half, and fixed in a 10% buffered formalin solution.

Renal histopathology and oil red O staining.

Paraffin-embedded kidney sections were prepared from half of the kidneys collected from SS_WT and SS^LepRmutant rats at each time point. Kidney sections were cut into 3-µm sections and stained with periodic acid-Schiff (PAS) and Masson’s trichrome. Thirty glomeruli per PAS-stained section were scored in a blinded fashion to determine glomerular injury, and Masson’s trichrome-stained sections (10–15 representative fields/section) were analyzed to determine the degree of renal fibrosis, as previously described (47). Images were captured using a Nikon Eclipse 55i microscope equipped with a Nikon DS-Fil color camera (Nikon, Melville, NY). To determine renal lipid accumulation, the other half of the kidney was removed from the 10% buffered formalin solution and washed in 0.1% PBS for 5 h. The washed kidneys were placed in 30% sucrose overnight or until they sank to the bottom of the container. The kidneys were retrieved from the container, cut into 10-µm sections, and stored at −80°C overnight. On the next day, frozen sections were thawed, allowed to air dry for 20 min before fixation in 40% formalin, and washed with water. Thereafter, sections were stained in oil red O solution (EKI, Joliet, IL) for 10 min and washed with water. The washed sections were counterstained with Harris hematoxylin containing acetic acid (Stat Laboratory, McKinney, TX) for 1 min and washed again with water. Finally, sections were incubated in ammonia water to reveal the blue counterstain, washed again with water, and then mounted with aqueous mounting medium (Thermo Scientific, Waltham, MA).

Transcriptome analysis by RNA-Seq.

Glomeruli were isolated from the whole kidney using a sieving technique, as previously described (17, 83–86, 91). Total RNA was isolated using TRIzol and Ambion kits, RNA quality was checked using a bioanalyzer (Bio-Rad Experion System), and concentrations were determined using the Qubit fluorometer. A RNA quality index score of ≥7.0 was required to proceed. RNA libraries were prepared using the TruSeq Stranded Total RNA with Ribo-Zero kit, set A (catalog no. FC-122-2501, Illumina) according to the manufacturer’s instructions. Each sample was prepared using 1 µg total RNA. The resulting cDNA libraries were quantified using the Qubit system (Invitrogen) and checked for quality and size using the Experion DNA 1K chip (Bio-Rad). The fragment size generated by the library was 200–500 bp with a peak at ~250 bp. A portion of each library was diluted to 10 nM, and all samples were pooled. The concentrations were verified again via Qubit and further diluted to 4 nM. A total of 5 µL of the 4 nM pooled libraries were diluted and denatured to a final concentration of 1.6 pM. The libraries were sequenced using the NextSeq500 High Output kit (300 cycles, 100 bp paired-end reads) on the Illumina NextSeq500 platform. Sequenced reads were assessed for quality using the Illumina BaseSpace cloud computing platform, and FASTQ sequence files were used to align reads to the rat reference genome (Rat Genome Sequencing Consortium 5.0/rn5) using the RNA-Seq alignment application (using STAR aligner). Differential expression was determined using Cufflinks Assembly & DE workflow (version 2.1.0). Gene expression differences are denoted as log₂ (ratio) and q > 0.05. Gene set enrichment analysis was performed using Enrichr (http://amp.pharm.mssm.edu/Enrichr/), which provides multiple tools for gene list enrichment analysis. All Supplemental Data are available at https://doi.org/10.6084/m9.figshare.11673582.v1.

Real-time PCR measurements.

Real-time PCR using SYBR green dye chemistry on the Bio-Rad real-time PCR platform was used to confirm key genes differentially expressed by RNA-Seq. RNA (the same samples used for RNA-Seq) was reverse transcribed to cDNA using the iScript cDNA synthesis kit. The gene primers were predesigned, validated primers from Bio-Rad PrimePCR assays followed by PCR using SsoFast EvaGreen supermix (Bio-Rad). Expression levels were normalized to GAPDH. Statistical analysis of real-time PCR data was performed using Bio-Rad Maestro software.

Statistical analysis.

Values are means ± SE. Statistical analysis was performed using SigmaPlot 12 software (Systat Software, San Jose, CA). The significance of difference in mean values for a single time point was determined by an unpaired t test. Temporal changes in renal and cardiovascular parameters were compared between and within strains using two-way ANOVA. P < 0.05 was considered significantly different.

RESULTS

Temporal changes in body weight and metabolic parameters.

Time-course changes in body weight and plasma concentrations of glucose, insulin, cholesterol, and triglycerides are provided in Supplemental Fig. S1. Body weight was 30% higher in 6-wk-old SS^LepRmutant than SS_WT rats and remained higher throughout the study (Supplemental Fig. S1A). Despite the development of obesity in the SS^LepRmutant strain, plasma glucose levels remained within the normal physiological nonfasting range (133 ± 15 mg/dL) during the study (Supplemental Fig. S1B). Insulin levels were markedly increased at 6 wk of age and remained elevated over the course of the study in SS^LepRmutant compared with SS_WT rats (Supplemental Fig. S1C). At 6 wk of age, plasma cholesterol levels were similar between SS^LepRmutant and SS_WT rats (94 ± 12 and 117 ± 18 mg/dL, respectively) but significantly increased to 277 ± 51 mg/dL in SS^LepRmutant rats, while they remained unchanged in SS_WT rats (118 ± 5 mg/dL) at 18 wk of age (Supplemental Fig. S1D). Plasma triglyceride levels were nearly sevenfold higher in 6-wk-old SS^LepRmutant rats than their SS_WT littermates (268 ± 45 vs. 41 ± 11 mg/dL) and rose to 877 ± 122 mg/dL in SS^LepRmutant rats and 139 ± 29 mg/dL in SS_WT rats by 18 wk of age (Supplemental Fig. S1E).

Temporal changes in arterial pressure and proteinuria.

At 6 wk of age, MAP was similar between SS_WT and SS^LepRmutant rats (128 ± 2 and 126 ± 6 mmHg, respectively; Fig. 1A). However, by 18 wk of age, SS^LepRmutant rats developed severe hypertension (192 ± 4 mmHg) and SS_WT rats did not (149 ± 6 mmHg). While we observed no differences in arterial pressure at 6 wk of age, proteinuria was four times higher in SS^LepRmutant than SS_WT rats (111 ± 26 vs. 25 ± 5 mg/day; Fig. 1B). By 18 wk of age, SS^LepRmutant rats developed progressive proteinuria compared with SS_WT rats (778 ± 96 and 113 ± 10 mg/day, respectively).

Fig. 1.

Time-course measurements of mean arterial pressure (MAP; A) and proteinuria (B) in wild-type Dahl salt-sensitive (SS_WT) and obese Dahl salt-sensitive leptin receptor mutant (SS^LepRmutant) rats. Values are means ± SE; n = 8 of each strain per time point. *Significantly different from corresponding value within the same strain at baseline; †significantly different from corresponding value in SS_WT rats.

Measurement of GFR via clearance of FITC-sinistrin.

Time-course changes in GFR in both strains are shown in Fig. 2. The elimination t_1/2 of FITC-sinistrin was significantly shorter in 6-wk-old SS^LepRmutant than SS_WT rats (Fig. 2A). The elimination t_1/2 of FITC-sinistrin generally was unchanged in SS_WT rats throughout the course of the study. However, by 18 wk of age, the elimination t_1/2 of FITC-sinistrin was markedly elevated in SS^LepRmutant rats compared with baseline and age-matched SS_WT rats. In correlation with the elimination t_1/2, GFR normalized to body weight (mL·min⁻¹·100 g body wt⁻¹) was 30% higher in SS^LepRmutant than SS_WT rats at 6 wk of age (Fig. 2B). GFR normalized to body weight was significantly increased in both strains at 12 wk age compared with baseline values at 6 wk of age. After 12 wk of age, we observed a rapid decline in GFR in SS^LepRmutant versus SS_WT rats. Unnormalized GFR values (mL/min) were similar to GFR normalized to body weight (mL·min⁻¹·100 g body wt⁻¹) (Fig. 2C). When normalized to total kidney weight (mL·min⁻¹·g kidney wt⁻¹), GFR gradually declined in SS^LepRmutant compared with SS_WT rats (Fig. 2D).

Fig. 2.

Temporal changes in FITC-sinistrin elimination half-life (t½; A) and glomerular filtration rate (GFR) normalized to 100 g body weight (B), unnormalized (C), and normalized to total kidney weight (D) in wild-type Dahl salt-sensitive (SS_WT) and obese Dahl salt-sensitive leptin receptor mutant (SS^LepRmutant) rats. Values are means ± SE; n = 8 of each strain per time point. *Significantly different from corresponding values within the same strain at baseline; †significantly different from corresponding value in SS_WT rats.

Assessment of renal histopathology.

Representative images and corresponding analysis of renal pathology are provided in Supplemental Figs. S2 and S3 and are shown in Fig. 3. Glomerular injury increased over the course of the study in both strains, but the kidneys from SS^LepRmutant rats displayed increased mesangial expansion and severe glomerulosclerosis compared with their SS_WT littermates at each time point (Supplemental Fig. S2). Increased renal fibrosis was detected as early as 6 wk in SS^LepRmutant compared with SS_WT rats and remained elevated over the course of the study (Supplemental Fig. S3). While oil red O staining revealed small traces of lipid accumulation in the renal tubules of both strains, strong distinct punctuated lipid droplets were detected in glomeruli (Fig. 3). Moreover, lipid accumulation was markedly greater in glomeruli from SS^LepRmutant than SS_WT rats at 6 and 18 wk of age (Fig. 3, E–H).

Fig. 3.

Representative images of oil red O-stained kidneys from wild-type Dahl salt-sensitive (SS_WT) and obese Dahl salt-sensitive leptin receptor mutant (SS^LepRmutant) rats. Magnification: ×10 in A–D and ×40 in E–H.

Analysis of glomerular transcriptome by RNA-Seq.

RNA-Seq was performed on glomeruli isolated from SS_WT and SS^LepRmutant kidneys at 6 and 18 wk of age to provide an unbiased view of gene expression changes. At 6 wk of age, 1,255 genes were differentially expressed between SS_WT and SS^LepRmutant rats (>1.5 fold, q < 0.05): 554 genes were upregulated and 701 genes were downregulated in SS^LepRmutant rats compared with SS_WT rats (Supplemental Table S1). Several genes exhibited a large increase (>5-fold change) in expression in SS^LepRmutant rats: 5.3-fold increase in gremlin-2 (Grem2), 6.5-fold increase in leukotriene C₄ synthase (Ltc4s), and 7.5-fold increase in triggering receptor expressed on myeloid cells 2 (Trem2). Strikingly, 48 genes, many of which were associated with glomerular integrity, were downregulated more than fivefold (Supplemental Table S1). In total, upregulated genes were significantly enriched for pathways (reactome) linked to platelet activation, metabolism, and the innate immune system (Supplemental Fig. S4 and Supplemental Table S2), whereas the downregulated genes were enriched for pathways involving the extracellular matrix, nephrin interactions, and cell-to cell communication.

At 18 wk of age, 1,572 genes were differentially expressed between SS_WT and SS^LepRmutant rats (>1.5-fold, q < 0.05): 945 genes were upregulated and 790 genes were downregulated in SS^LepRmutant rats compared with SS_WT rats (Supplemental Table S3). The top two most differentially expressed genes at 18 wk of age were proline-rich transmembrane protein 2 (Prrt2; −7.8, also the top gene at 6 wk of age) and serine palmitoyltransferase small subunit B (Sptssb; 8.9). Upregulated genes were enriched for pathways (reactome) linked to the immune system, the innate immune system, and extracellular matrix organization (Supplemental Fig. S5 and Supplemental Table S4), whereas the downregulated genes were enriched for pathways involving axon guidance, neural cell adhesion molecule 1 interactions, and extracellular matrix organization. As expected, given that transcriptome analysis was performed at an early (6 wk of age) and a later (18 wk of age) time point after significant renal injury was observed, the majority of genes that were only differentially expressed were unique to either 6 or 18 wk of age. However, almost a quarter (24%) of the genes (n = 540) were differentially expressed at both time points (Supplemental Fig. S6 and Supplemental Table S5).

Genes known to be involved in the maintenance of the glomerular filtration barrier and those that participate in fatty acid metabolism were queried from the RNA-Seq data sets (Fig. 4). The data are expressed as fold changes in SS^LepRmutant rats normalized to SS_WT rats at 6 and 18 wk of age. When we examined genes that contribute to the maintenance of the glomerular filtration barrier, we found that most were significantly decreased in SS^LepRmutant compared with SS_WT rats (Supplemental Tables S1 and S3 and Fig. 4A). However, transient receptor potential cation channel subfamily C member 6 (Trpc6) was the only gene that was markedly increased in SS^LepRmutant compared with SS_WT rats. When we investigated the influx and efflux transporter genes, we observed significant increases in Cxcl16 and Cd36 and significant decreases in Abca1 and Abca2 in SS^LepRmutant compared with lean SS_WT rats at 6 and 18 wk of age (Fig. 4B). Furthermore, the largest fold changes occurred in angiopoietin-like protein 2 (Angptl2) at 6 wk of age and peroxisome proliferator-activating receptor-γ (Pparg) at 6 and 18 wk of age.

Fig. 4.

Summary of transcriptome data of selected genes queried from RNA sequencing data obtained from isolated glomeruli from kidneys of wild-type Dahl salt-sensitive (SS_WT) and obese Dahl salt-sensitive leptin receptor mutant (SS^LepRmutant) rats. A and B: fold change (FC) in glomerular-specific genes that contribute to maintenance of the glomerular filtration barrier and genes that are involved in lipid metabolism at 6 and 18 wk of age. Data are expressed as log₂ fold change in SS^LepRmutant strain normalized to SS_WT rats at 6 and 18 wk of age; n = 8 of each strain per time point. Actn1, actinin-α₁; Actn2, actinin-α₂; Actn4, actinin-α₄; Cd2ap, CD2-associated protein; Col4a3, collagen type IV α₃-chain; Col4a4, collagen type IV α₄-chain; foxc1, forkhead box C1; Foxc2, forkhead box C2; Foxd1, forkhead box D1; Kirrel1, kirre-like nephrin family adhesion molecule 1; Kirrel2, kirre-like nephrin family adhesion molecule 2; Lmx1b, LIM homeobox transcription factor 1β; Nes, nestin; Nphs1, nephrin; Nphs2, podocin; Plce1, phospholipase C-ε₁; Podxl, podocalyxin-like; Robo1 and Robo2, roundabout guidance receptors 1 and 2; Synpo, synaptopodin; Trpc6, transient receptor potential cation channel subfamily C member 6; Wt1, Wilms tumor 1; Abca1 and Abca2, ATP-binding cassette subfamily A members 1 and 2; Abcg2 and Abcg3, ATP-binding cassette subfamily G members 2 and 3; Angptl2, angiopoietin-like protein 2; Cd36, fatty acid translocase; Cpt1a, carnitine palmitoyltransferase 1A; Cxcl16, chemokine (C-X-C motif) ligand 16; Fas, apoptosis antigen 1; Lpl, lipoprotein lipase; Pparg, peroxisome proliferator-activated receptor-γ; Scara3 and Scara5, scavenger receptor class A members 3 and 5; Scd2, stearoyl-CoA desaturase.

Validation of genes involved in lipid metabolism and maintenance of the glomerular filtration barrier by real-time PCR analysis.

Since we observed differences in a number genes by RNA-Seq analysis, we used real-time PCR analysis to validate a few of the genes involved in lipid metabolism and maintenance of the glomerular filtration barrier (Fig. 5). For the purposes of the present study, we focused on influx (Cd36 and Cxcl16) and efflux (Abca1 and Abca2) lipid transporters. The results from RNA-Seq and real-time PCR analyses were positively correlated (r = 0.81, P < 0.0001). At 6 wk of age, Cd36 and Cxcl16 were significantly increased in glomeruli from SS^LepRmutant compared with SS_WT rats (Fig. 5A). While we did not observe a significant decrease in Abca1, Abca2 was markedly reduced in SS^LepRmutant compared with SS_WT rats. Additionally, gene expression of the podocyte marker synaptopodin (Synpo) displayed a tendency to be decreased but did not reach statistical significance. We found similar results in these same genes at 18 wk of age (Fig. 5B). Cd36 and Cxcl16 were significantly elevated, and Abca2 was reduced, in glomeruli isolated from SS^LepRmutant compared with SS_WT rats. In contrast to 6 wk of age, Synpo was significantly decreased at 18 wk of age in SS^LepRmutant compared with SS_WT rats.

Fig. 5.

Real-time PCR analysis of isolated glomeruli from wild-type Dahl salt-sensitive (SS_WT) and obese Dahl salt-sensitive leptin receptor mutant (SS^LepRmutant) rats at 6 and 18 wk of age. Values are means ± SE; n = 5 rats of each strain per time point. †Significantly different from corresponding value in SS_WT rats. Abca1 and Abca2, ATP-binding cassette subfamily A members 1 and 2; Cd36, fatty acid translocase; Cxcl16, chemokine (C-X-C motif) ligand 16; Synpo, synaptopodin.

DISCUSSION

While diabetes and hypertension are the two primary causes of CKD, recent evidence suggests that obesity alone is becoming an independent risk factor for CKD (5, 30, 35). Some of the common characteristics of renal disease associated with obesity include glomerulosclerosis, podocyte injury, and glomerular lipid accumulation, collectively known as ORG (9, 11, 37, 38, 68, 76). Recently, we reported that, as early as 6 wk of age, SS^LepRmutant rats develop podocyte injury and progressive proteinuria, independent of hyperglycemia and elevations in arterial pressure, that progress to CKD by 18 wk of age (47). The SS^LepRmutant strain, unlike other models of obesity, can be used to study mechanisms involved in the early development of renal injury associated with obesity without the complications of hyperglycemia and hypertension. In the present study, we examined whether development of glomerular injury in the SS^LepRmutant strain is associated with temporal changes in GFR and glomerular lipid accumulation. Similar to our previous report (47), rats of the SS^LepRmutant strain developed dyslipidemia and severe proteinuria in the absence of hyperglycemia and elevations in arterial pressure that were associated with glomerular hyperfiltration and increased glomerular lipid accumulation compared with SS_WT rats at 6 wk of age. Moreover, we observed significant increases in the expression of Cxcl16 and Cd36 (influx lipid transporters) and significant decreases in the expression of Abca1 and Abca2 (efflux lipid transporters) in SS^LepRmutant compared with lean SS_WT rats at 6 and 18 wk of age. These findings provide strong evidence of lipid accumulation at the glomerulus level. By 18 wk of age, kidneys from the SS^LepRmutant strain displayed more glomerulosclerosis, glomerular lipid accumulation, and renal fibrosis that correlated with a decrease in GFR. These data provide evidence of an association between glomerular hyperfiltration and glomerular lipid accumulation during the early development of proteinuria in the obese SS^LepRmutant strain. Further studies are needed to determine the direct impact of elevations in GFR on glomerular lipid accumulation.

One of the early characteristics of renal disease associated with obesity alone is glomerular hyperfiltration (8, 21, 25, 26, 43). In the present study, we found a 30% increase in GFR in SS^LepRmutant rats compared with their SS_WT littermates at 6 wk of age. Since SS^LepRmutant rats do not develop hyperglycemia and their arterial pressure is similar to that of SS_WT rats, the elevations in GFR may not be attributed to diabetes or hypertension. However, two factors may contribute to the early development of glomerular hyperfiltration in the obese SS^LepRmutant strain. One factor is the lack of autoregulation of the renal microcirculation in the SS genetic background. We previously observed that the development of renal injury in the SS rat is associated with a lack of autoregulation of the renal microcirculation, which contributes to elevations in glomerular capillary pressure that eventually lead to glomerular injury independent of hypertension (84, 85). Similar results were detected when diabetes was induced in the SS strain with streptozotocin; these rats developed glomerular hyperfiltration without significant changes in arterial pressure (69). However, additional experiments are needed to determine if the autoregulation of the renal microcirculation is further impaired in obese SS^LepRmutant rats. Moreover, even with a similar degree of impaired autoregulation, if the renal vasculature of mutant rats is more dilated, then a greater degree of arterial pressure transmission to the kidney would be expected. The other potential contributing factor is increased Na⁺ intake due to elevated food intake in the SS^LepRmutant strain. Recently, Cowley and colleagues demonstrated that feeding SS rats a high-salt (e.g., 4% NaCl) diet decreases GFR over time (10). However, in the present study, SS^LepRmutant rats were not fed an extremely high-salt diet but, rather, a 1% NaCl diet and exhibited elevations in GFR as early as 6 wk of age. Although obese SS^LepRmutant rats consumed more Na⁺ than lean SS_WT rats, this increase in Na⁺ intake may not be enough to raise arterial pressure. Therefore, the early development of glomerular hyperfiltration in the obese SS^LepRmutant strain could more than likely be a consequence of the increase in body weight, high-salt diet, and genetic lack of autoregulation of the renal microcirculation.

Glomerular hyperfiltration or an elevation in GFR is a suggested mechanism for the early development of renal injury in several clinical conditions, including obesity, and may even be considered an early marker of renal disease. The functional changes in renal hemodynamics in response to obesity may lead to increased transmission of systemic pressure to the glomerulus, which could initiate or damage the glomerular filtration barrier, leading to proteinuria. Previous studies have demonstrated that development of obesity-related proteinuria is due, in part, to alterations in renal hemodynamics, since lowering arterial pressure and GFR attenuates proteinuria (5, 8, 57). In the present study, we observed that the early glomerular hyperfiltration was associated with progressive proteinuria in the SS^LepRmutant strain that leads to the development of severe glomerulosclerosis, renal fibrosis, and CKD. In addition, we found a number of genes, including podocin (Nphs2), Synpo, and nephrin (Nphs1), that participate in the maintenance of the glomerular filtration barrier to be significantly decreased in obese SS^LepRmutant compared with lean SS_WT rats. Dysfunction or reduction of these genes has been shown to contribute to progressive albuminuria and renal injury (7, 52, 54, 63, 77–79, 91). The only gene that we found to be markedly increased was Trpc6; a gain in function of Trpc6 has been associated with focal segmental glomerulosclerosis (20, 29, 61, 87, 92). These data suggest that the early functional changes in renal hemodynamics in response to obesity stimulate glomerular hyperfiltration, which damages the glomerular filtration barrier and causes progressive proteinuria. Over time, this can lead to glomerulosclerosis, renal fibrosis, and, eventually, CKD.

During ORG, abnormal fatty acid metabolism stimulates increased lipid (i.e., cholesterol and triglycerides) accumulation in the kidney (4, 15, 44, 59). Animal and human studies have implied that dyslipidemia contributes to the progression of CKD by stimulating inflammation and oxidative stress. We detected dyslipidemia and markedly elevated glomerular lipid accumulation in the SS^LepRmutant strain as early as 6 wk of age that was further exacerbated by 18 wk of age. Elevated triglyceride accumulation can result from increased fatty acid production, increased uptake via Cd36, or decreased fatty acid oxidation (11). Likewise, cholesterol accumulation can occur from increased synthesis and uptake via Cd36 and Cxcl16 (19, 28, 40, 51, 89) or reduced cholesterol efflux via Abca1 and Abca2 (28, 48, 58, 74). In the present study, using RNA-Seq or real-time PCR analysis, we observed increases in Cd36, Cxcl16, and ATP-binding cassette superfamily G member 2 (Abcg2) expression and significant decreases in Abca1 and Abca2 expression in glomeruli isolated from the kidneys of SS^LepRmutant compared with lean SS_WT rats at 6 and 18 wk of age. All five transporters have been linked to the development of renal disease (19, 28, 40, 48, 51, 58, 74, 89). Stearoyl-CoA desaturase (Scd2) and carnitine palmitoyltransferase 1A (Cpt1a) were significantly decreased in the SS^LepRmutant strain, and both have been proven to be beneficial in preventing renal injury upon activation (32, 34). The largest fold change at 6 wk of age occurred in Angptl2, a proinflammatory circulating protein that has been demonstrated to play a significant role in the development of renal injury (33, 49, 50). However, Angptl2 expression was markedly decreased in SS^LepRmutant compared with SS_WT rats. Interestingly, Pparg was significantly increased in glomeruli from SS^LepRmutant rats during the progression of renal disease, which is contradictory to previous reports demonstrating a link between podocyte injury and decreased Pparg activation (27, 70). These data suggest that changes in these genes involved in lipid metabolism may stimulate glomerular lipid accumulation and play major roles in the early development of renal injury, proteinuria, and CKD associated with ORG.

The most intriguing findings from the present study are glomerular lipid accumulation and alterations in lipid metabolism at an early age in the obese SS^LepRmutant strain. However, the initiating factor(s) that causes these changes in the lipid transporters (i.e., Cxcl16, Cd36, Abca1, Abca2, Abcg2, and Abcg3) to stimulate glomerular lipid accumulation remains to be determined. Two factors could contribute to glomerular lipid accumulation: 1) hyperinsulinemia/insulin resistance and 2) glomerular hyperfiltration. Steneberg et al. (72) recently provided evidence that hyperinsulinemia directly increases the expression of Cd36 and lipid accumulation in the liver, not in the kidney, of mice fed a sucrose-enriched diet. Perhaps similar hyperinsulinemia-mediated effects occur in the kidney during the development of CKD. In obese patients with insulin resistance, such as diabetic nephropathy, efflux lipid transporters have also been implicated in the development of renal injury (28, 48, 58, 74). To our knowledge, the present study is the first to demonstrate a possible relationship between glomerular hyperfiltration and glomerular lipid accumulation during the development of renal injury in the absence of hyperglycemia and elevations in arterial pressure. In support of these findings, Li et al. (45) observed that increased GFR is associated with renal adiposity during the early stages of metabolic syndrome. However, future studies are needed to investigate the potential effects of hyperinsulinemia/insulin resistance and/or glomerular hyperfiltration on the expression and localization of these lipid transporters on cells in the glomerulus during the development of renal disease associated with obesity.

Analysis of the glomerular transcriptome RNA-Seq data showed a few pathways that were associated with the development and progression of renal injury in the SS^LepRmutant strain. A number of the genes that were upregulated belong to pathways involved in the innate immune system and platelet activation. Over the last decade, the innate immune system and inflammation have been extensively examined in the development of renal injury. Fehrenbach and colleagues (18) recently reported that increases in immune cells from the innate immune system are associated with renal disease in the SS rat. While platelets play a crucial role in hemostasis, accumulating evidence demonstrates that platelets are heavily involved in the pathology of acute kidney injury (31, 36). However, the involvement of platelet activation during the development of renal injury associated with obesity is unknown. Not surprisingly, there were genes that were downregulated and linked to pathways involved in extracellular matrix and nephrin interactions, which supports the hypothesis that hyperfiltration alters the extracellular matrix and damages the filtration barrier, contributing to progressive proteinuria. In addition to pathways, we found a few genes that were markedly elevated in the SS^LepRmutant strain (Grem2, Ltc4s, and Trem2). Wen et al. (82) recently reported that increased protein expression of gremlin-2 was associated with albuminuria in a mouse model of obesity and that overexpression of gremlin-2 stimulates podocyte apoptosis under hyperglycemic conditions. Ltc4s is responsible for synthesizing LTD₄, which has been implicated in the reduction of GFR during acute glomerular injury (2, 3, 60) and has been observed to stimulate proteinuria by increasing glomerular capillary pressure (39). Although Trem2 has not been directly associated with renal disease, it is involved in the migration and maturation of dendritic cells (6, 24), which have been shown to contribute to renal injury (41, 62). The most differentially expressed genes, Prrt2 and Sptssb, have not been examined in the pathology of glomerular injury, but both belong to pathways that affect renal function: Prrt2 in regulation of glutamate release (13, 81) and podocyte injury and Sptssb in biosynthesis of sphingolipids (1, 16, 46, 65). Overall, the roles and contributions of these genes and pathways require further investigation in the development and progression of renal disease associated with obesity.

The main goal of the present study was to examine whether the development of renal injury in the obese SS^LepRmutant strain was associated with elevations in GFR and renal lipid accumulation via alterations in influx and efflux lipid transporters. While the results support the main goal, some limitations in the study should be noted. One limitation is measurement of arterial pressure by the chronic carotid catheter method, which involves stress on the animals, rather than by telemetry. We chose not to use telemetry, because the catheter could not be placed in the femoral artery of the animals, especially the lean SS_WT rats, which were too small at 6 wk of age. However, to confirm the current arterial pressure results, using the tail-cuff method, we previously reported no differences in arterial pressure between SS_WT and SS^LepRmutant rats at 6 wk age (47). Another major limitation of the present study was that we did not perform genetic analysis of extrarenal vascular tissue to serve as a control to determine whether the differences in gene expression were specific to the glomerulus. However, we have been using the same standard protocol to isolate glomeruli from the renal cortex of various rat strains for a number of years, and it has always yielded a very high percentage of glomeruli, rather than other sections of the nephron.

Clinical translational perspective.

Data from the present study suggest that the early development of renal disease associated with obesity in the absence of hyperglycemia and elevations in arterial pressure involves glomerular hyperfiltration. We also hypothesize that the hyperfiltration may act synergistically with dyslipidemia to stimulate glomerular injury via lipid accumulation, resulting in progressive proteinuria and renal injury that eventually leads to CKD. This process occurs as early as 6 wk of age and possibly before these animals reach puberty, which is clinically relevant. Over the past few decades, prepubertal or childhood obesity has emerged as a major health problem (22, 71, 80). Furthermore, obese children have an increased risk for development of renal injury. With prepubertal childhood obesity on the rise, there is an urgent need to understand the underlying mechanisms that contribute to future risk of CKD. The obese SS^LepRmutant strain, unlike most models of obesity, offers the advantage that the early mechanisms that may be involved in the development of renal injury associated with insulin resistance and hyperinsulinemia can be studied without the complications of hyperglycemia and hypertension. In conclusion, the SS^LepRmutant strain may be useful to study signaling pathways and mechanisms involved in the development of renal disease associated with prepubertal childhood obesity.

GRANTS

This work was financially supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 1-F31-DK-109571 (to K. C. McPherson), National Heart, Lung, and Blood Institute Grant HL-130456 and National Institute of General Medical Sciences Obesity, Cardiorenal, and Metabolic Diseases (COBRE) Grant P20-GM-104357 (to D. C. Cornelius), National Heart, Lung, and Blood Institute Grant HL-137673 (to M. R. Garrett), and National Institute of General Medical Sciences Grant P20-GM-104357 and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-109133 (to J. M. Williams). Work performed through the University of Mississippi Medical Center Molecular and Genomics Facility is supported, in part, by funds from the National Institute of General Medical Sciences, including Mississippi IDeA Networks for Biomedical Research Excellence Grant P20-GM-103476, COBRE Grant P20-GM-104357, and Mississippi Center of Excellence in Perinatal Research (MS-CEPR) COBRE Grant P20-GM-121334.

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

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