Early development of podocyte injury independently of hyperglycemia and elevations in arterial pressure in nondiabetic obese Dahl SS leptin receptor mutant rats

Abstract

The current study examined the effect of obesity on the development of renal injury within the genetic background of the Dahl salt-sensitive rat with a dysfunctional leptin receptor derived from zinc-finger nucleases (SS^LepRmutant strain). At 6 wk of age, body weight was 35% higher in the SS^LepRmutant strain compared with SS_WT rats and remained elevated throughout the entire study. The SS^LepRmutant strain exhibited impaired glucose tolerance and increased plasma insulin levels at 6 wk of age, suggesting insulin resistance while SS_WT rats did not. However, blood glucose levels were normal throughout the course of the study. Systolic arterial pressure (SAP) was similar between the two strains from 6 to 10 wk of age. However, by 18 wk of age, the development of hypertension was more severe in the SS^LepRmutant strain compared with SS_WT rats (201 ± 10 vs. 155 ± 3 mmHg, respectively). Interestingly, proteinuria was substantially higher at 6 wk of age in the SS^LepRmutant strain vs. SS_WT rats (241 ± 27 vs. 24 ± 2 mg/day, respectively) and remained elevated until the end of the study. The kidneys from the SS^LepRmutant strain displayed significant glomerular injury, including podocyte foot process effacement and lipid droplets compared with SS_WT rats as early as 6 wk of age. By 18 wk of age, plasma creatinine levels were twofold higher in the SS^LepRmutant strain vs. SS_WT rats, suggesting the presence of chronic kidney disease (CKD). Overall, these results indicate that the SS^LepRmutant strain develops podocyte injury and proteinuria independently of hyperglycemia and elevated arterial pressure that later progresses to CKD.

chronic kidney disease (CKD) is a major growing health problem and is one of the leading causes of death in the United States (30). While the two primary causes of CKD are hypertension and diabetes, the prevalence of obesity has increased dramatically within the last decade and is now considered an independent risk factor for CKD (14, 32). Moreover, a recent study revealed that 15% of nondiabetic patients with CKD suffer from morbid obesity (58). Furthermore, a report by Ejerblad et al. (32) suggested that obesity may play a significant role in the development of renal injury in patients without a history of hypertension, diabetes, and/or preexisting renal disease. These studies indicate that functional changes in the kidney occur in response to obesity that contribute to CKD.

The mechanisms by which obesity stimulates the development of renal injury in the absence of hypertension and diabetes remain unclear. The early stages of renal disease associated with obesity are renal vasodilation, hypertrophy, and increased renal blood flow (RBF) and glomerular filtration rate (GFR) (hyperfiltration). There are several components of obesity, including hyperinsulinemia (92), hyperglycemia (12), and elevations in arterial pressure (102, 103, 105), that are known to promote renal hyperfiltration. However, obesity in the absence of hypertension and diabetes has also been shown to cause elevations in RBF and GFR (14, 33, 68, 101). These early functional changes in renal hemodynamics associated with obesity may lead to increased transmission of systemic pressure to the glomerulus, causing damage to the glomerular filtration barrier and leading to proteinuria. In fact, previous studies have demonstrated that the development of proteinuria in obese patients is due, in part, to alterations in renal hemodynamics since lowering arterial pressure and GFR attenuates proteinuria (11, 14, 75).

Previous studies have demonstrated that patients suffering from obesity have a more than twofold greater risk of developing albuminuria and CKD than lean subjects (5, 16, 55, 60, 69, 98). However, a major complication in studying the obesity-CKD association is that some of the cardiovascular and metabolic disorders (i.e., hypertension, diabetes, dyslipidemia, and insulin resistance-metabolic syndrome; MetS) that are the result of obesity also contribute to the development and progression of CKD (16, 60). The consequences of obesity as an independent risk factor for the development of glomerular injury and proteinuria has received less attention due to a lack of an appropriate animal model. Currently, there are several obese rodent models that develop characteristics of MetS (1, 9, 21, 35–37, 43, 45, 47, 65, 71, 72, 89, 108, 111). Most of these models have a disruption in leptin receptor signaling that results in hyperphagia and leads to the development of obesity. Although these obese rodent strains have also served as models of renal disease, the onset and severity of renal injury vary widely in these models. The obese Zucker fatty (OZF) rat, which spontaneously developed a missense mutation (fatty, fa) in the leptin receptor gene (LepR) (112, 113), exhibits mild hypertension and hyperglycemia (≤200 mg/dl) along with characteristics of CKD including progressive proteinuria and a decline in renal function (21). A substrain of the OZF rat, the Zucker diabetic fatty (ZDF) rat, was created by breeding OZF rats that develop hyperglycemia (≥300 mg/dl) (20). The ZDF strain displays glomerular injury and proteinuria after the development of robust diabetes in the absence of hypertension (48). In another obese rodent model, the Koletsky rat carries a nonsense mutation in the leptin receptor (56, 57) and displays glomerular injury and proteinuria in the presence of hypertension along with impaired renal function, suggesting the presence of CKD (34, 35, 110). Similarly, the spontaneously hypertensive heart failure strain containing the corpulent mutation in the LepR gene (SHHF/Mcc-cp), which results from several backcrosses between the Koletsky and spontaneously hypertensive (SHR) rats, develops severe hypertension and renal disease that includes progressive proteinuria with elevated plasma creatinine (P_cr) levels (108). The obese, diabetic ZSF1 rats, which were developed by a F1 hybrid cross between a ZDF rat and lean SHHF rat, suffer from hypertension, glomerulosclerosis, and proteinuria with a decline in renal function by 20 wk of age (9, 43, 74). The diabetic obese db/db mouse model contains a mutation in the leptin receptor and develops little to no renal injury unless hypertension is induced by decreasing nitric oxide (NO) availability within its genetic background (65, 111). Taken together, while these obese rodent models develop some form of renal disease, the onset of renal injury occurs after the development of hypertension, diabetes, and/or age-related mechanisms. Hence, these models have limited use when one is investigating the direct impact of obesity on the development of renal injury in the absence of hypertension and diabetes.

The Dahl salt-sensitive (SS) rat is a well-established model of salt-dependent hypertension that is vastly susceptible to the development of renal injury (17, 26, 27, 70, 78, 81, 82, 90, 106). The kidneys of the SS rat are exposed to elevations in glomerular capillary hydrostatic pressure due to impaired renal autoregulation that ultimately leads to the development of glomerular and renal injury (104). However, the SS strain is not obese. In the current study, we created a 16-base pair frame-shift deletion in exon 11 of the leptin receptor gene (LepR) within the genetic background of the SS rat (SS^LepRmutant strain) using zinc-finger nucleases that develops obesity without overt diabetes. We hypothesize that the development of obesity in the SS^LepRmutant strain increases the early susceptibility to glomerular and renal injury in SS rats. In support of our hypothesis, SS rats fed a high-fat diet to induce a 15–25% increase in body weight display hypertension, proteinuria, and glomerular/tubular injury, suggesting that weight gain increases the susceptibility to renal disease in the SS strain (8, 52, 53, 67, 86). Similarly, Hattori and colleagues (46) observed progressive proteinuria, increased mesangial expansion, and reduced renal function in the obese DahlS.Z-Lepr^fa/Lepr^fa rat, a model created from a genetic cross between SS rats and heterozygous OZF rats, compared with their control counterparts. Moreover, these rats develop hypertension and hyperglycemia as early as 11 wk of age (46). However, the assessment of early changes in proteinuria and glomerular injury before 11 wk of age was not examined. Therefore, the aim of the current study was to assess the effects of obesity on the early temporal changes in metabolic and physiological parameters as well as the development of renal disease in our nondiabetic obese SS^LepRmutant strain compared with their lean wild-type littermates, the SS_WT strain.

METHODS

General.

Experiments were performed in 77 6–18 wk-old male SS_WT and SS^LepRmutant rats. SS_WT and SS^LepRmutant strains were obtained from our in-house colony of heterozygous SS^LepRmutant (SS_HET^LepRmutant) rats that were originally obtained from Drs. Aron Geurts and Howard Jacob from the Medical College of Wisconsin. The rats were housed in the Laboratory Animal Facility at the University of Mississippi Medical Center, which is approved by the American Association for the Accreditation of Laboratory Animal Care. The rats had free access to food and water throughout the study. Rats were fed a 1% NaCl diet (TD8640; Harlan Laboratories, Madison, WI) to minimize the development of hypertension. All protocols were approved by the Animal Care Committee of the University of Mississippi Medical Center.

Creation and genotyping of the SS^LepRmutant strain.

SS^LepRmutant rats were created using zinc-finger technology at the Medical College of Wisconsin (MCW) using the PhysGen Knockout (http://rgd.mcw.edu/) as previously described (41, 42). Zinc-finger nuclease (ZFN) constructs (Sigma-Aldrich, St. Louis, MO) targeting the following sequence: AGCATCGTACTGCCCacaatgGGACATGGTCACAAG on chromosome 5 in exon 11 were used to cause a 16-base pair frame-shift deletion in the leptin receptor (LepR) gene within the genetic background of the SS rat (SS-Lepr^em2Mcwi strain). The pronucleus of fertilized SS/McwiHsd rat embryos was injected with mRNAs encoding the LepR ZFNs (10 ng/μl) and transferred to pseudopregnant females. To identify SS^LepRmutant founders, genomic DNA extracted from ear punch tissues were screened with PCR and CEL-1 assay as previously described (64). The strain is designated as SS-Lepr^em2Mcwi (hereafter called SS^Lepmutant). Heterozygote animals (SS_HET^LepRmutant) from subsequent generations were used to generate homozygous mutants (SS^LepRmutant strain) and wild-type littermates (SS_WT strain). Genotyping and sequencing were performed by the Molecular and Genomic Facility at the University of Mississippi Medical Center. Briefly, genomic DNA collected from ear punch biopsies of pups at weaning was purified using the Wizard SV 96 Genomic DNA purification System (Promega, Madison, WI). For genotyping, the region of deletion targeted by the ZFNs within exon 11 of LepR was amplified via PCR using Platinum Taq DNA Polymerase High Fidelity Mix (Invitrogen, Grand Island, NY), dNTP mix (Invitrogen), and LepR-specific primers for the ZFN mutation site (forward 5′-CCACTGATGAAAAATGACTCAC-3′ and reverse 5′-ATGGGCCATGAGAACGTAAG-3′). The products were then loaded on a 3% agarose gel and visualized by ethidium bromide staining where the wild-type leptin receptor was 209 base pairs long and the mutated leptin receptor was 193 base pairs long. To validate the LepR disruption, PCR products were then purified using a PureLink Quick PCR Purification Kit (Invitrogen) to remove primers and excess reagents and prepared for Sanger sequencing using Dye Terminator Cycle Sequencing Mix (Beckman Coulter, Brea, CA) and the same primers used for genotyping described above. PCR products were then ethanol precipitated and resuspended in Sample Loading Solution (Beckman Coulter), then sequenced on the CEQ8000 Genetic Analysis System (Beckman Coulter). To confirm that the SS^LepRmutant strain contained dysfunctional leptin receptors, blood samples from the tail vein were collected to measure plasma leptin levels at 6 wk of age (Quantikine Mouse/Rat Leptin ELISA, R&D Systems, Minneapolis, MN).

Protocol 1: comparison of time course of changes in metabolic parameters in SS_WT rats and the SS^LepRmutant strain.

Experiments were performed in 6-wk-old SS_WT and SS^LepRmutant rats. Body weight and blood glucose levels were monitored every 4 wk until the rats reached 18 wk of age. Blood samples were collected from the tail vein for measurement of blood glucose levels (glucometer, Bayer HealthCare, Mishawaka, IN). The samples were collected with ad libitum feeding 3 h into the lights-on cycle between 10 and 11 AM. Baseline and terminal plasma cholesterol and triglyceride concentrations were determined by ELISA (Cayman Chemical, Ann Arbor, MI).

Measurement of glucose tolerance and plasma insulin concentration levels.

SS_WT and SS^LepRmutant rats were subjected to an intraperitoneal glucose tolerance test (IPGTT) at 6 and 18 wk of age. Three days before the IPGTT, a blood sample (300–500 μl) was collected from the tail vein to measure plasma insulin concentrations (Rat Insulin ELISA, Mercodia, Uppsala, Sweden). On the day of the IPGTT, after the determination of fasting (2 h) blood glucose levels, the rats received an injection of 2 g/kg of glucose solution (ip). Blood samples (5–10 μl) were collected from the tail vein at 15, 30, 60, 90, and 120 min after the glucose load.

Protocol 2: comparison of the time course of changes in systolic arterial pressure, proteinuria, and podocalyxin excretion in SS_WT rats and the obese SS^LepRmutant strain.

Experiments were performed in 6-wk-old SS_WT and SS^LepRmutant rats. Systolic blood pressure (SAP) was measured in conscious animals via the tail-cuff method (MC4000 BP Analysis System, Hatteras Instruments, Cary, NC) at 6, 10, 14, and 18 wk of age. Before baseline measurements, the rats were trained and acclimated to restraint for 20–30 min for 4 consecutive days, and SAP was measured at the same time of day. At each time period, the rats were placed in metabolic cages to collect an overnight urine sample to study time course changes in the excretion of protein and podocalyxin. Urinary total protein concentration was determined colorimetrically using the Bradford method (Bio-Rad, Hercules, CA), and urinary podocalyxin concentration, an indicator of podocyte injury, was determined by ELISA (Exocell, Philadelphia, PA). Baseline and terminal blood samples were collected to determine P_cr (Wako Chemicals, Richmond, VA) to assess renal function.

Renal histopathology.

Kidneys were collected, weighed, and fixed in a 10% buffered formalin solution at 6 and 18 wk of age. Paraffin sections (3 μm) were prepared and stained with periodic acid-Schiff and Masson's trichrome to assess the degree of glomerular injury and renal fibrosis, respectively, in ∼30 images/section. Thirty glomeruli per section were scored in a blinded fashion on a 0–4 scale with 0 representing a normal glomerulus, 1 representing a 25% of loss, 2 representing a 50% loss, 3 representing a 75% loss, and 4 representing >75% loss of capillaries in the tuft. Additional analysis was performed to determine the degree of renal fibrosis. Images were captured using a Nikon Eclipse 55i microscope equipped with a Nikon DS-Fi1 color camera (Nikon, Melville, NY) and analyzed for the percentage of the image stained blue (primarily collagen) in the Masson's trichrome-stained sections using NIS-Elements D 3.0 software. Ten to 15 representative fields were analyzed per section.

Electron microscopy.

For transmission electron microscopy, kidneys from SS_WT and SS^LepRmutant rats at 6 and 18 wk of age were perfused with 2% glutaraldehyde, then ∼1 mm of cortex was obtained and placed in 10% formaldehyde buffered at pH 7.4 (Carson-Millonig Formulation, Ricca Chemical, Arlington, TX). Next, cortical samples were postfixed for 1 h with OsO₄ followed by a graded series of chilled ethanol solutions (35–100%) ending with a final dehydration with 100% acetone. Samples were infiltrated overnight in Eponate 12 resin, and ultrathin sections (70–90 nm) were obtained and collected on copper support grids. Afterward, sections were poststained with uranyl acetate and lead citrate and examined using a JOEL 1400 plus transmission electron microscope (JOEL USA, Peabody, MA).

Statistical analysis.

Mean values ± SE are presented. The significance of difference in mean values for a single time point was determined by an unpaired t-test using SigmaPlot 12 software (Systat Software, San Jose, CA). The time course changes in metabolic and physiological parameters were compared between and within strains using a two-way repeated measures ANOVA followed by the Holm-Sidak test for preplanned comparisons with the same software mentioned above. A P value <0.05 was considered significantly different.

RESULTS

Validation of a dysfunctional leptin receptor in the SS^LepRmutant strain.

To determine whether the leptin receptor was dysfunctional in the SS^LepRmutant strain, we sequenced the targeted area and measured plasma leptin levels at 6 wk of age as well as temporal changes in body weight until the rats reached 18 wk age between SS_WT and SS^LepRmutant strains (Fig. 1). Sequencing revealed a 16-base pair frame-shift deletion in the LepR gene, resulting in a predicted stop codon 30 nonsense amino acids downstream (Fig. 1A). The genotypes of the SS_WT (209 bp), SS_HET^LepRmutant (209 and 193 base pairs), and SS^LepRmutant (193 base pairs) strains were verified using PCR as shown in Fig. 1B. The body weight at 6 wk of age was significantly higher in the SS^LepRmutant strain compared with SS_WT rats (238 ± 8 vs. 176 ± 6 g, respectively) and remained higher throughout the course of the study (528 ± 15 vs. 373 ± 4 g, respectively) (Fig. 1C). Moreover, plasma leptin levels were markedly elevated in the SS^LepRmutant strain vs. SS_WT rats (84.7 ± 4.5 vs. 2.1 ± 0.4 ng/ml, respectively) (Fig. 1D).

Fig. 1.

Sequencing (A), genotyping (B), and comparison of weight gain (C), and plasma leptin levels (D) of wild-type Dahl salt-sensitive (SS_WT) rats, SS heterozygous leptin receptor mutant rats (SS_HET^LepRmutant strain), and the nondiabetic obese SS leptin receptor mutant (SS^LepRmutant) strain. Numbers in parentheses indicate the number of rats studied per group. Values are means ± SE. *Significant difference from the corresponding value within the same strain at baseline (P < 0.05). †Significant difference from the corresponding value in SS_WT rats (P < 0.05).

Comparison of metabolic parameters in SS_WT and SS^LepRmutant rats.

The time course of changes in blood glucose, plasma insulin levels, glucose tolerance, and plasma lipid levels are presented in Fig. 2. At baseline, nonfasting blood glucose levels were normal (≤120 mg/dl) for both strains and remained within the normal physiological range throughout the course of the study (Fig. 2A). However, as early as 6 wk of age, the plasma concentration of insulin was markedly elevated in the SS^LepRmutant strain compared with the values measured in SS_WT rats (4.7 ± 0.1 vs. 0.3 ± 0.03 ng/ml, respectively) and continued to be significantly elevated until the end of the study (Fig. 2B). Glucose tolerance tests were performed at 6 and 18 wk of age (Fig. 2, C and D). We observed a 50% increase in peak blood glucose levels at 6 wk of age in the SS^LepRmutant strain vs. SS_WT rats. Blood glucose levels returned to baseline 90 min after administration of the glucose load in the SS^LepRmutant strain and 60 min in the SS_WT rats. By 18 wk of age, the impairment in glucose tolerance was significantly greater in the SS^LepRmutant strain compared with SS_WT rats. Plasma cholesterol (Fig. 2E) and triglyceride (Fig. 2F) levels were measured at 6 and 18 wk of age. Plasma cholesterol was significantly higher in the SS^LepRmutant strain vs. the SS_WT rats as early as 6 wk of age (178 ± 15 vs. 112 ± 5 mg/dl, respectively). Additionally, at this same time point, plasma triglycerides were higher in the SS^LepRmutant strain vs the SS_WT rats (180 ± 34 vs. 41 ± 6 mg/dl). At 18 wk of age, plasma cholesterol levels were threefold higher in the SS^LepRmutant strain vs. SS_WT rats. Similarly, serum triglycerides were significantly higher in the SS^LepRmutant strain compared with the values measured in their wild-type counterparts (1,550 ± 522 and 165 ± 27 mg/dl, respectively).

Fig. 2.

Comparison of time course measurements of blood glucose and plasma insulin levels (A and B), the progression of impaired glucose tolerance by IPGTT (C and D), and plasma cholesterol and triglyceride levels (E and F) in SS_WT rats and the SS^LepRmutant strain. Numbers in parentheses indicate the number of rats studied per group. Values are means ± SE. *Significant difference from the corresponding value within the same strain at baseline (P < 0.05). †Significant difference from the corresponding value in SS_WT rats (P < 0.05).

Time course of changes in SAP, protein excretion, and podocalyxin excretion in SS_WT rats and the SS^LepRmutant strain.

Comparisons of the development of hypertension, proteinuria, and podocyte injury between SS_WT rats and the SS^LepRmutant strain are presented in Fig. 3. While SAP was not significantly different at 6 and 10 wk of age in the SS_WT and SS^LepRmutant strains, SS^LepRmutant rats subsequently developed severe systolic hypertension compared with the values measured in SS_WT rats by the end of the study (201 ± 10 vs. 155 ± 3 mmHg, respectively) (Fig. 3A). Interestingly, protein excretion was significantly higher at 6 and 10 wk of age in the SS^LepRmutant strain vs. SS_WT rats before elevations in arterial pressure. Proteinuria increased to 569 ± 60 mg/day in the SS^LepRmutant strain while it only increased to 88 ± 5 mg/day in SS_WT rats by 18 wk of age (Fig. 3B). Similar to protein excretion, podocalyxin excretion was significantly elevated by more than threefold in the SS^LepRmutant strain vs. SS_WT rats by 6 wk of age (64 ± 14 vs. 17 ± 2 μg/day, respectively) without any differences in SAP and continued to increase to 247 ± 62 vs. 47 ± 7 μg/day, respectively, by 18 wk of age (Fig. 3C).

Fig. 3.

Time course measurements of systolic arterial pressure (SAP; A), protein excretion (B), and podocalyxin excretion (C), a marker of podocyte injury, in SS_WT rats and the SS^LepRmutant strain. Numbers in parentheses indicate the number of rats studied per group. Values are means ± SE. *Significant difference from the corresponding value within the same strain at baseline (P < 0.05). †Significant difference from the corresponding value in SS_WT rats (P < 0.05).

Renal pathology.

Images of renal histopathology in SS_WT rats and the SS^LepRmutant strain are represented in Fig. 4. Compared with SS_WT rats, the kidneys from the SS^LepRmutant strain exhibited severe glomerulosclerosis and mesangial expansion (PAS staining) as early as 6 wk of age (Fig. 4, A and B) and continued to progress until the end of the study (18 wk of age) (Fig. 4, C and D). At 6 wk of age, we did not observe any differences in renal fibrosis between SS_WT and SS^LepRmutant strains (Fig. 4, E and F). However, by 18 wk of age, kidneys from the SS^LepRmutant strain showed severe indications of interstitial fibrosis, protein casts, tubular necrosis, and dilated tubules while the SS_WT did not (Fig. 4, G and H).

Fig. 4.

Comparison of renal histology. Periodic acid-schiff (PAS) staining (A–D) and Masson's trichrome staining (E–H) in SS_WT rats and the SS^LepRmutant strain are shown.

Multianalyses of the degree of renal injury in SS_WT rats and the SS^LepRmutant strain are represented in Fig. 5. The glomerular injury score was significantly greater in the SS^LepRmutant strain at 6 wk of age and further increased by 18 wk of age compared with the values measured in SS_WT rats (Fig. 5A). While renal fibrosis (blue staining) in the SS^LepRmutant strain vs. SS_WT rats at 6 wk of age did not reach statistical significance (P = 0.053), there was a tendency for it to be elevated (Fig. 5B). However, renal fibrosis increased by threefold in the SS^LepRmutant strain compared with SS_WT rats by 18 wk of age. Additionally, we observed a significant increase in kidney weight (renal hypertrophy) in the SS^LepRmutant strain vs. the values seen in SS_WT rats (1.9 ± 0.089 vs. 1.41 ± 0.003 g, respectively). When evaluating renal function, we observed that P_cr levels were similar between both strains at 6 wk of age (Fig. 5C). However, P_cr levels more than doubled in the SS^LepRmutant strain vs. control SS_WT rats by 18 wk of age (1.31 ± 0.29 vs. 0.52 ± 0.03 mg/dl, respectively).

Fig. 5.

Comparison of glomerular injury score (A), renal fibrosis (B), and plasma creatinine (P_cr) levels (C) in SS_WT rats and the SS^LepRmutant strain. Numbers in parentheses indicate either the number of glomeruli/images and rats (A and B) or only the number of rats (C) studied per group. Values are means ± SE. *Significant difference from the corresponding value within the same strain at baseline (P < 0.05). †Significant difference from the corresponding value in SS_WT rats (P < 0.05).

Electron microscopy.

Representative electron micrographs of the ultrastructure of glomerular capillaries in SS_WT and SS^LepRmutant rats are presented in Fig. 6. At 6 wk of age, SS_WT rats exhibited a normal appearance of the glomerular filtration barrier (Fig. 6A). However, glomerular capillaries from the SS^LepRmutant strain displayed minor podocyte foot process effacement (black filled arrows) and lipid droplets (black open arrows) at the same time period (Fig. 6B). By 18 wk of age, we observed severe foot process fusion and lipid accumulation in the SS^LepRmutant strain compared with SS_WT rats (Fig. 6, C and D).

Fig. 6.

Representative electron micrographs of the ultrastructure of the glomerular filtration barrier (A–D) in SS_WT and SS^LepRmutant strain. The filled black arrows represent podocyte foot process effacement, and the open black arrows represent lipid droplets.

DISCUSSION

Several rodent models of obesity have been developed. However, most of these models have limited use in the investigation of mechanisms responsible for the early development of renal injury associated with obesity due to the development of diabetes and hypertension. For example, the OZF rat develops mild hyperglycemia around 16 wk of age with proteinuria occurring at 40 wk of age, but P_cr levels remain within the normal range until it increases by more than twofold after 60 wk of age. Thus the slow progression of renal disease in the OZF rat cannot be separated from age-related mechanisms (21, 63). ZDF rats display severe hyperglycemia as early as 12 wk of age followed by the development of renal injury including severe glomerulosclerosis and albuminuria with a decline in GFR after 28 wk of age (48, 96). The obese Koletsky, SHHF/Mcc-cp, and ZSF1 strains, which contain the hypertensive genetic background of the SHR rat, present with hypertension as early as 6–8 wk of age but do not develop progressive renal injury until after 20 wk of age (9, 34, 35, 43, 74, 108, 110). Db/db mice only display albuminuria and glomerular abnormalities with a decline in GFR when mild to moderate hypertension is introduced by decreasing NO availability within its genetic background (48, 65). Therefore, it is difficult to identify any initial mechanisms specifically related to obesity in the absence of diabetes and hypertension. The current study determined the temporal changes in metabolic and cardiovascular parameters in a novel model of nondiabetic obesity which was created by mutating the leptin receptor in the SS rat genetic background using zinc-finger nuclease technology. The SS genetic background was used since SS rats exhibit impaired autoregulation of RBF and GFR and are more susceptible to the development of glomerular disease. The SS^LepRmutant strain developed obesity and insulin resistance in the absence of hyperglycemia. Proteinuria and podocalyxin excretion were markedly elevated before any significant increase in SAP in the SS^LepRmutant strain vs. SS_WT rats at 6 wk of age, suggesting early characteristics of glomerular injury. However, by the end of the study, the SS^LepRmutant strain developed severe elevated SAP and progressive proteinuria compared with their control counterparts. The kidneys from the SS^LepRmutant strain displayed renal abnormalities, including glomerulosclerosis, renal fibrosis, and tubular necrosis while kidneys from SS_WT rats did not. We also observed a twofold increase in P_cr levels in the SS^LepRmutant strain compared with SS_WT rats, suggesting the occurrence of CKD.

A large percentage of obese animal models exhibit type II diabetes. However, in the current study, the SS^LepRmutant strain developed obesity, insulin resistance, and hyperinsulinemia by 6 wk of age, which did not stimulate overt diabetes or hyperglycemia. Moreover, this nonhyperglycemic trend continued throughout the course of the study. Yet, we do not find this result quite surprising since the obese DahlS.Z-Lepr^fa/Lepr^fa rat only develops mild hyperglycemia with hyperinsulinemia by 11 wk of age (46). However, there are conflicting reports in the OZF rat model developing hyperglycemia (6, 13, 51). Rohner-Jeanrenaud et al. (79, 80) observed impaired glucose tolerance and elevated plasma insulin levels without hyperglycemia. In contrast, several studies have provided evidence that OZF rats display hyperglycemia after 16 wk of age (≥170 mg/dl) (21, 63). SHHF/Mcc-cp rats are hyperinsulinemic at an early age with hyperglycemia due to marked hyperplasia of pancreatic β cells (3, 97, 108). The ZDF strain exhibits hyperglycemia (≥300 mg/dl) between 10 and 12 wk age due to an autosomal recessive defect in the islet cells of the pancreas independently of the leptin receptor mutation (20, 23, 107). Plasma insulin levels increase significantly from 6 to 8 wk of age and fall dramatically after 10–15 wk of age, which contributes to the elevations in blood glucose levels in the ZDF rat (73, 95). The Koletsky and ZSF1 strains present with mild hyperglycemia and hyperinsulinemia that progresses to severe diabetes (≥300 mg/dl) after 20 wk of age (9, 34, 35, 43, 74, 110). Similarly, the db/db mouse develops severe hyperglycemia by 10 wk of age (≥400 mg/dl) (1, 45) with plasma insulin levels peaking at 12 wk of age and rapidly declining after 24 wk of age (7, 22, 45). These data suggest the varying differences in the levels of glucose in these obese rat and mouse strains may involve the degree of insulin sensitivity and resistance and/or when the decline of insulin secretion from the β cells of the pancreas occurs.

The SS rat is a commonly used animal model to study salt-sensitive hypertension-induced renal disease (17, 26, 27, 70, 78, 81, 82, 90, 106). Moreover, the development of renal injury in the SS rat is associated with a lack of autoregulation of RBF, which contributes to elevations in glomerular capillary pressure that eventually lead to glomerular injury independently of hypertension (104). Previous work from our laboratory recently demonstrated that inducing diabetes in the SS strain with streptozotocin is associated with renal hyperfiltration and progressive proteinuria without significant changes in arterial pressure (85). Similar results were observed in the current study in which proteinuria and podocalyxin excretion were significantly higher in the obese SS^LepRmutant strain compared with SS_WT rats at 6 wk of age in the absence of elevations in systolic arterial pressure and blood glucose levels. The results from the current study suggest that the SS rat is more susceptible to the development of renal injury associated with nondiabetic obesity. However, unlike the hypertension and diabetic studies in SS rats, the initial mechanism that triggers renal disease in the nondiabetic obese SS^LepRmutant strain remains to be determined but more than likely involves the lack of leptin signaling, causing obesity-related effects including changes in renal hemodynamics and insulin resistance. Obesity has been associated with increased albuminuria and renal injury independently of diabetes (11, 14, 25, 31, 76). Moreover, previous studies have demonstrated that the development of obesity-related proteinuria is due, in part, to alterations in renal hemodynamics since lowering arterial pressure and GFR attenuates proteinuria (11, 14, 75). Welsh et al. (100) observed that insulin resistance specifically at the podocyte level stimulates glomerular injury and albuminuria in the absence of hyperglycemia. These studies indicate that the early functional changes in the kidney that occur in response to obesity in the SS^LepRmutant strain may be due to elevations in GFR and insulin resistance.

Interestingly, we observed the severe development of hypertension in the SS^LepRmutant strain during the course of the study, which is not typical in similar models of obesity that are deficient in leptin receptor signaling. Db/db mice have similar or marked reductions in arterial pressure compared with their lean control littermates (10, 84, 109). The obese Zucker rat model develops moderate hypertension late in life (61) while their substrain counterpart, the Zucker diabetic fatty rat, does not (24, 29, 96). The lack of elevations in arterial pressure in these models is primarily due to the loss of leptin's mediated activation of the sympathetic nervous system. In contrast, there are a few models that develop hypertension in the wake of leptin receptor signaling. For instance, despite the LepR mutations on the SHR genetic background, the obese Koletsky, SHHF/Mcc-cp, and ZSF1 strains develop hypertension by 9 wk of age independently of leptin signaling (9, 34, 35, 43, 74, 108, 110). Similarly, deficiency of leptin receptor function in the DahlS.Z-Lepr^fa/Lepr^fa and SS^LepRmutant strains causes hyperphagia-induced increases in food/sodium intake that may lead to elevations in arterial pressure due to the genetic susceptibility to salt-sensitive hypertension. However, in the current study, baseline SAP measurements were in the hypertensive range (>140 mmHg) but were similar between both strains until 14 wk of age, suggesting the difference in sodium intake due to hyperphagia does not play a significant role in causing early elevations in arterial pressure in the SS^LepRmutant strain. We hypothesize that the severe development of hypertension after 10 wk of age observed in the SS^LepRmutant strain may be attributed to reduced renal function. Overall, these studies suggest that some rodent models of obesity that lack leptin receptor signaling and are genetically predisposed to hypertension may develop elevations in arterial pressure.

The major finding in the current study is that the renal disease in the nondiabetic obese SS^LepRmutant strain rapidly progresses to CKD by 18 wk of age. P_cr, a marker of renal function, was elevated by more than twofold in the SS^LepRmutant strain and averaged 1.3 mg/dl compared with the normal value of 0.5 mg/dl observed in the SS_WT strain, suggesting the presence of CKD. The reduction in renal function was associated with the severe development of SAP elevations and renal structural abnormalities, including podocyte foot process effacement, glomerulosclerosis, mesangial expansion, and renal fibrosis in the SS^LepRmutant strain. In contrast, development of renal disease in the other obese animal models with leptin receptor signaling deficiency progresses slowly or not all. Db/db mice and ZDF rats develop mild renal disease, which may be due to the lack of increased SAP (1, 2, 28, 48). While the Koletsky, SHHF-Mcc-cp, and ZSF1 strains develop severe hypertension as well as diabetes, these rats rarely present with any significant renal abnormalities before 20 wk of age (9, 34, 35, 43, 74, 108, 110) or unless fed a high-carbohydrate diet (93, 94). In OZF rats, the transition from renal injury to CKD occurs slowly and does not develop until these rats are >40 wk of age (21, 63). The DahlS.Z-Lepr^fa/Lepr^fa strain is very similar to our SS^LepRmutant strain, but the renal histological abnormalities are less severe. One possible reason for the progression of CKD in some of these models of obesity that lack leptin receptor signaling with insulin resistance and mild or no diabetes (i.e., DahlS.Z-Lepr^fa/Lepr^fa and OZF strains) is dyslipidemia (38, 46, 87). Dyslipidemia is a risk factor for the development of proteinuria and promotes the progression of CKD (62, 66, 83) by stimulating podocyte apoptosis and mesangial sclerosis (54). In the current study, the SS^LepRmutant strain exhibited significant hypertriglyceridemia at the end of the study that was associated with impaired renal function. However, further studies are needed to determine whether long-term lipid-lowering therapy (i.e., statins and fibrates) prevents the progression of renal injury in these unique rodent obese models that develop CKD.

Like the other models of obesity that are of limited use in examining the direct impact of obesity on renal injury, the SS^LepRmutant strain has limitations as well. As early as 6 wk of age, the SS^LepRmutant strain displays characteristics of MetS, including insulin resistance, elevated SAP, and increased plasma triglyceride levels. Insulin resistance is associated with a higher prevalence of renal injury (15, 18). In the current study, the SS^LepRmutant strain displayed impaired glucose tolerance and hyperinsulinemia along with progressive proteinuria at 6 wk of age. To support the relationship between insulin resistance and renal disease, Welsh and colleagues (100) demonstrated that insulin resistance specifically at the podocyte level contributes to podocyte injury and albuminuria as early as 5 wk of age. While we did not observe any early differences in arterial pressure between the SS_WT and SS^LepRmutant strains, SAP was in the hypertensive range. We and others have previously demonstrated that the SS strain is genetically susceptible to the development of hypertension-induced glomerular injury due to impaired renal autoregulation (4, 39, 40, 88, 103, 104). However, the level of podocyte injury and proteinuria was more severe in the SS^LepRmutant strain compared with SS_WT rats, suggesting that hypertension alone does not play a significant role in the early development of renal injury in the SS^LepRmutant strain. Hypertriglyceridemia is a risk factor for developing proteinuria (91). Interestingly, we observed increases in plasma triglyceride levels and lipid droplets in the podocytes at 6 wk of age that was associated with glomerular injury and proteinuria in the SS^LepRmutant strain. Wang et al. (99) recently observed treatment with simvastatin had no effect on plasma triglyceride levels but prevented renal lipidosis and renal disease, suggesting that lipid accumulation contributes to renal injury. Overall, the early development of these MetS features may contribute to the early podocyte and renal abnormalities in the SS^LepRmutant strain. However, the SS^LepRmutant strain is the first reported obese rodent model that develops progressive renal disease at an early age in the absence of hyperglycemia and elevated arterial pressure compared with its lean littermates.

Recent studies suggest that patients with obesity in the absence of diabetes have an increased risk for the development of CKD, but the mechanisms remain unclear (19, 44, 49, 50, 98). Despite this major health concern, little is known about the pathogenesis of renal disease associated with nondiabetic obesity due to the lack of an appropriate animal model, which has inhibited our understanding of the causes and progression of disease symptoms that could lead to development of new therapeutic interventions. However, in the current study, we observed that the SS^LepRmutant strain develops many of the cardiovascular and metabolic characteristics of MetS along with CKD. Furthermore, the development of renal injury occurs at an early age independently of hyperglycemia and elevations in arterial pressure. Thus the SS^LepRmutant strain may be useful in investigating the potential mechanisms involved in both the early and late stages of development of CKD associated with nondiabetic obesity. Another area of research that may benefit from using the SS^LepRmutant strain is studying the effects of obesity on the progression of renal injury in a rodent strain that is susceptible to renal disease. This may be of use in high-risk minorities, including African Americans and Hispanics that are at greater risk for developing obesity and CKD than Caucasians (59, 77).

GRANTS

J. M. Williams is supported by funds from the National Institutes of General Medical Sciences of the National Institutes of Health (NIGMS/NIH) Obesity, Cardiorenal and Metabolic Diseases-COBRE (P20GM104357) and the American Heart Association (12SDG9440034). S. P. Didion is supported by the National Heart, Lung and Blood Institute of the National Institutes of Health (NHLBI/NIH) Grant HL107632. M. R. Garrett is supported by NHLBI/NIH Grant HL094446. The work performed through the UMMC Molecular and Genomics Facility is supported, in part, by funds from the NIGMS/NIH including Mississippi INBRE (P20GM103476), Center for Psychiatric Neuroscience (CPN)-COBRE (P30GM103328), and Obesity, Cardiorenal and Metabolic Diseases-COBRE (P20GM104357). The SS^LepRmutant strain was developed as part of funds from the NIH (DP2-OD008396 to A. M. Guerts).

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

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