RENAL FUNCTION AND STRUCTURE IN DIABETIC, HYPERTENSIVE, OBESE ZDFxSHHF-HYBRID RATS

Abstract

The obese ZDFxSHHF-fa/fa^cp model was developed by crossing lean female Zucker Diabetic Fatty (ZDF +/fa) and lean male Spontaneously Hypertensive Heart Failure (SHHF/Mcc-fa^cp, +/fa) rats. The purpose of the present study was to determine renal function and morphology, hemodynamics, and metabolic status in ZDFxSHHF rats. Two sets of experiments were conducted. First, we evaluated heart and kidney function and metabolic status in aged (46 weeks old) male obese ZDFxSHHF and age matched obese SHHF rats, lean Spontaneously Hypertensive (SHR) and lean normotensive Wistar Kyoto (WKY) rats. In the second set of experiments, renal function and structure as well as metabolic and lipid status were determined in lean (LN) and obese (OB) adult (29-weeks of age) ZDFxSHHF rats. At 46 weeks of age ZDFxSHHF rats are hypertensive expressing marked cardiac hypertrophy associated with diastolic dysfunction and preserved contractile function. Fasted hyperglycemia and hyperinsulinemia are accompanied by moderate hypercholesterolemia and hypertriglyceridemia. Obese aged ZDFxSHHF have marked renal hypertrophy, a 3–8 fold decrease in creatinine clearance (compared with SHHF, SHR and WKY), a high percent of segmental + global glomerulosclerosis (59.8% ± 10.8), and severe tubulointerstitial and vascular changes. Obese ZDFxSHHF rats die at an early age (∼12 months) from end-stage renal failure. Studies conducted in 29-week animals showed that, although both LN and OB 29-week old animals are hypertensive, OB animals have more severely compromised renal function and structure as compared with lean littermates (kidney weight: 2.56 ± 0.16 vs. 1.61 ± 0.12 g; creatinine clearance: 0.42 ± 0.04 vs. 1.24 ± 0.13 L/g kid/day; renal vascular resistance 12.39 ± 1.4 vs. 6.14 ± 0.42 mmHg/mL/min/g kid; protein excretion: 556 ± 16 vs. 159 ± 9 mg/day/g kid, p < 0.05, OB vs. LN, respectively). Obesity is also associated with hyperglycemia (424 ± 37 vs. 115 ± 11 mg/dL), hyperinsulinemia (117.2 ± 8.8 vs. 42.3 ± 3.5 μU/mL), hypertriglyceridemia (5200 ± 702 vs. 194 ± 23 mg/dL), hypercholesterolemia (632 ± 39 vs. 109 ± 4 mg/dL), and presence of segmental + global glomerulosclerosis (20.1 ± 3.2% vs. 0.1 ± 0.1%) with prominent tubular and interstitial changes (p < 0.05, OB vs. LN, respectively). In summary, the present study indicates that the crossing of rat strains of nephropathy produces hybrids that carry a high risk for severe renal dysfunction. The ZDFxSHHF rats express insulin resistance, hypertension, dislipidemia and obesity and develop severe renal dysfunction. In addition, the hybrids do not develop some of the complications (hydronephrosis or congestive heart failure) common for the parental strains that may compromise studies of renal function and structure. Therefore, the ZDFxSHHF rat may be a useful model fore valuating risk factors and pharmacological interventions in chronic renal failure.

• Animal model
• Rats
• Hypertension
• Metabolic syndrome
• Chronic renal failure

INTRODUCTION

The triad of insulin resistance, hypertension and obesity is frequently observed in humans and carries with it an increased risk for renal disease [1-2]. In addition, the significance of hyperlipidemia in the progression of renal failure has been recently recognized [3-4] and hyperlipidemia has been identified as an independent risk factor for the development and progression of human renal disease [[5]]. Accordingly, development of experimental animal models that express those risk factors may provide useful tools for studying the effects of these risk factors, as well as the effects of different pharmacological intervention, in chronic renal failure.

Over the last three decades two significant mutations (i.e. fa and cp) that produce obesity have been identified in rats. The fa mutation, originally described by Zuckers in 1961([[6]]; Zucker fatty rat model) has been backcrossed into several strains [[7]]. The cp mutation described originally by Koletsky [[8]] has been also backcrossed into several strains, including SHHF/Mcc-fa^cprats (infra vide; [[9]]). All obese strains that carry those mutations have been shown to express renal dysfunction. Therefore, it may be expected that crossing of animals with those mutations would result in animal models that carry a high risk for renal disease.

The ZDFxSHHF-(fa-fa^cp)-hybrid rat model described below was developed by Genetic Models Inc, by crossing lean female Zucker Diabetic Fatty (ZDF, +/fa) and lean male Spontaneously Hypertensive Heart Failure (SHHF/Mcc, +/fa^cp) rats. The Zucker Diabetic Fatty Rat (ZDF/Gmi), one of the parental strains, is a relatively new partially inbred model that was derived from few Zucker rats with high blood glucose [[7]], [[10]]. This strain has been selectively inbred for more than 20 generations, and has metabolic abnormalities similar to human non-insulin-dependent diabetes mellitus (NIDDM). Both lean and obese ZDF have been shown to be normotensive [[11]]. Recently, longitudinal studies were performed to fully characterize renal function and structure and explore whether obese ZDF can be used as a experimental model of diabetic nephropathy [[11]]. The presence of a severe non-diabetic lesion (i.e. hydronephrosis) observed in both lean and obese animals makes the ZDF rats inappropriate for studying of renal diabetic disease.

The other parental strain, the SHHF rat, is a relatively new genetic model of spontaneous heart failure, associated with hypertension and renal dysfunction. This model was derived from obese Spontaneously Hypertensive Rat (SHR) carrying the corpulent cp gene (SHR/N-cp; [[12]]), and is genetically similar to SHR and normotensive Wistar Kyoto (WKY) rats. In SHHF rats, obesity is expressed as an autosomal recessive (cp/cp) trait and hypertension is multifactorial. Both obesity and gender make significant contributions to the onset and expression of heart failure, renal failure, insulin resistance and to the natural lifespan of the rats. Obese and male animals develop heart failure earlier, have more severe renal dysfunction, and die at an earlier age as compared with lean and female animals, respectively [[9]], [[13]]. Lean male SHHF animals, at 9 months of age, have significantly lower creatinine clearance and excrete significant amounts of protein as compared with age-matched SHR and WKY rats [[14]]. In lean males renal lesions occur spontaneously at the age of 5–6 months and are characterized by worsening proteinuria and glomerulosclerosis that progresses from focal and segmental to global by the time the animals die from overt congestive heart failure [[9]], [[13]]. Since both parental strains express renal dysfunction, we hypothesized that crossing of those two strains will produce hybrids with severe renal failure.

The first objective of this study was to determine how the mating of lean (heterozygote) ZDF and SHHF rats affects the cardiac and renal function in their obese hybrid (ZDFxSHHF, fa/fa^cp) offspring. More specifically we intended to determine whether aged obese male animals develop heart failure and hydronephrosis (i.e., complications observed in parental strains that may make the evaluation of renal function and structure difficult). Inasmuch as the ZDF rats are known to develop severe hydronephrosis, we used only the SHHF as a parental control strain. We also compared ZDFxSHHF hybrid to genetically similar hypertensive and normotensive control strains (i.e., spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats, respectively). The second objective of this study was to compare the renal function and metabolic status in adult lean versus obese hybrids. Our intention was to determine whether hypertension and metabolic abnormalities are present in lean animals, and how the presence of those risk factors affects renal function.

METHODS

Animals

A total of thirteen 45- week old obese, male ZDFxSHHF (hybrid) rats as well as six obese male and three obese female SHHF/Mcc-fa^cp rats were obtained from Genetic Model Inc. (Indianapolis, IN). Animals were housed in the University of Pittsburgh Medical Center animal care facility (temperature, 22 ⁰C; light cycle, 12 hours; relative humidity 55%) for at least one week before being used in acute experiments. Another group of 28 weeks old male, lean (n = 7) and obese (n = 6) ZDFxSHHF rats were also obtained from Genetic Models Inc. Eight 20 weeks old male Wistar Kyoto (WKY) and six 20 weeks old males Spontaneously Hypertensive rats (SHR) were obtained from Taconic Farms (Germantown, NY) and were kept in the animal care facility for 26 weeks before being used in experiments. Rats were fed Pro Lab RMH 3000 rodent diet (PMI Nutrition Inc., St Louis, MO) containing 0.26% sodium and 0.82% potassium and were given water ad libitum. Institutional guidelines for animal welfare were followed.

Protocol 1 (11 months old ZDFxSHHF)

Metabolic cages and renal function measurements

At the age of 45 weeks, animals (obese ZDFxSHHF, obese SHHF, SHR and WKY rats) were placed in metabolic cages for a one day acclimation period before 24-hour food and water intake and urine volume were measured. Urine samples were analyzed for creatinine, protein, sodium and potassium concentrations. A volume of blood (0.75 mL) was drawn from the tail vein, and obtained plasma was used for determination of creatinine, sodium and potassium levels. Creatinine and electrolytes were measured with a creatinine analyzer (Creatinine Analyzer 2, Beckman Instruments, Inc, Fullerton, CA, USA) and a flame photometer (model IL943 flame photometer, Instrumentation Laboratory, Lexington, MA, USA), respectively. Total protein concentration was measured in the urine samples by a spectrophotometric assay using bicinchoninic acid reagent (Pierce; Rockford, IL) and a modification of the method described by Lowry [[15]]. Twenty-four- hour urinary sodium and potassium excretion, creatinine clearance and 24hr-urinary protein excretion (normalized by body weight, kidney weight and creatinine clearance) were calculated. During metabolic cage studies, two ZDFxSHHF animals became anuric and died. No signs of congestive heart failure (i.e., dilated cardiomyopathy as seen in SHHF) were observed in those two animals that most likely died from renal failure.

Blood pressure measurements and in situ heart performance analysis

A subset of 46 weeks old ZDFxSHHF rats (n = 6) was used to assess heart performance in situ. Animals were anesthetized with pentobarbital (45 mg/kg i.p.) and placed on a Deltaphase Isothermal Pad (Braintree Scientific, Braintree, MA). Body temperature was monitored with a rectal temperature-probe thermometer (Physiotemp Instruments, Clifton, NJ) and maintained at 37 ± 0.5 ⁰C by adjusting a heat lamp positioned above the animal. The trachea was cannulated with PE-240 to facilitate respiration, and the right jugular vein and right carotid artery were exposed and separated from the surrounding tissue. A PE-50 catheter was placed into the right jugular for supplemental pentobarbital. Another PE-50 catheter, filled with 10 % heparin solution, was inserted into the right carotid artery and was connected to a heart-performance analyzer (HPA-210, Micro-Med, Inc, Louisville, KY). Thirty minutes later systolic (SBP) and diastolic (DBP) blood pressures were measured (by recording maximal and minimal pressure on the HPA) and mean arterial blood pressure (MABP) was calculated (MABP = (SBP + 2 DBP)/3). Next, in order to monitor heart performance in situ, the right carotid artery catheter was advanced into the left ventricle. Assessment of left ventricular function in situ included measurement of seven time-pressure variables: heart rate (HR), ventricular peak systolic pressure (VPSP), rate of maximal change in pressure during ventricular contraction (+dP/dt_max), rate of maximal change in pressure during ventricular relaxation (−dP/dt_max), ventricular end diastolic pressure (VEDP), ventricular minimal diastolic pressure (VMDP) and the calculated parameter of ventricular contraction +dP/dt normalized by pressure (+dP/dt/P). The measured variables were recorded at 2-minute intervals and 30-minute average values were calculated for the measured parameters. Next, a midline abdominal incision was made, the right kidney was removed and placed in 10% formalin buffer for histological analysis. In addition, the ZDFxSHHF rats that underwent the surgical procedure were instrumented for measurement of glomerular filtration rate (inulin clearance) and renal hemodynamics as described previously [[15]]. However, during recovery the period an insignificant drop in MABP (∼5-10 mm Hg) was followed by a marked reduction in urine output (i.e., animals became severely oliguric or anuric during clearance measurement period) preventing accurate measurements of renal excretory function.

In another subset of ZDFxSHHF rats an attempt was made to more accurately determine blood pressure. Five obese ZDFxSHHF underwent a surgical procedure to implant transducers for continuous monitoring of blood pressure and heart rate in the conscious unrestrained animals by radiotelemetry as described previously [[16]]. However, during or immediately after recovering from anesthesia three out of five animals became hemodynamically unstable and died within 24 hours. Therefore, telemetry measurements of blood pressure were conducted only in two animals.

Protocol 2 (Adult lean and obese ZDFxSHHF rats)

At the age of 29 weeks, seven lean and six obese male ZDFxSHHF rats were placed in metabolic cages and the metabolic and renal functional studies were conducted as described previously (supra vide). In addition, tail vein blood samples were taken for measurement of lipids, glucose and insulin. Serum cholesterol and triglyceride levels were measured by enzymatic colorimetric methods (Cholesterol and Triglyceride kits, Sigma, ST Louis, MO) whereas glucose and insulin levels were measured by glucose colorimetric kit (Sigma, St Louis, MO) and radioimmunoassay kit (Insulin rat RIA kit, Linco Research, Inc St Louis, MO), respectively. On the day following the metabolic cage study, the animals were anesthetized and instrumented for measurements of renal hemodynamics and excretory function. Briefly, animals were tracheotomized by placing a polyethylene (PE)-240 catheter into the trachea to facilitate breathing. The left jugular vein was cannulated with two PE-50 catheters (Clay Adams, Becton Dickison, MD) for supplemental anesthetic. An additional cannula (PE-50) was inserted into the left carotid artery and connected to a Micro-Med digital blood pressure analyzer (Micro-Med. Inc., Louisville, KY) for continuous monitoring of mean arterial blood pressure (MABP) and heart rate (HR). The blood pressure analyzer was set to time-average MABP and HR at 5-min intervals. Through a midline incision, the abdominal cavity was exposed and the right kidney was removed. The excised kidney was stored in 10% formalin buffer for histopathological analysis. Next, the left renal artery was exposed and a transit-time blood flow probe (Transonic System Inc., Ithaca, NY) was placed on the artery to continuously monitor renal blood flow with a model T206 flow meter. The left ureter was cannulated with a PE-10 catheter for collection of urine. Next, an intravenous infusion of (carboxyl- ¹⁴C) inulin (0.5 μCi bolus + 0.035 μCi/min) was initiated, and the animal was allowed to stabilize for 45 minutes.

Urine was collected during a 30-minute clearance period, and a 20 μL sample of urine was placed in a scintillation vial to measure ¹⁴C radioactivity. The remaining urine was used to measure the urinary concentration of sodium and potassium by flame photometry. At the midpoint of the clearance period, a 0.5 mL arterial blood sample was collected for plasma ¹⁴C radioactivity, hematocrit, sodium and potassium measurements. Saline volume equivalent to the withdrawn blood volume was infused intravenously after the blood samples had been removed from the rat. Urine and plasma samples were analyzed, and glomerular filtration rate (GFR), urine flow (UV), sodium excretion, potassium excretion, creatinine excretion, filtration fraction, urinary protein excretion, renal blood flow and renal vascular resistance were calculated. The left kidney was removed and renal hemodynamic parameters were expressed per gram of kidney weight.

Renal histopathology

The right kidney tissue sample stored in 10% formalin buffer was sectioned (sections included the full thickness of the kidney from capsule through pelvis) and was processed into paraffin blocks for light microscopy. Three histologic sections (3 microns) were cut and stained with hematoxylin-eosin and methenamine silver-trichrome(MST). Kidney slices were examined by light microscopy and scored in a blinded fashion by one of the investigators (S.B.). A total of at least 150 glomeruli from each kidney were studied and the percentage of glomeruli showing segmental (FSGS) and global (FGGS) glomerulosclerosis was counted. Other histopathologic features assessed semiquantitatively included tubular atrophy (0-3⁺), tubular dilation (0-4⁺), presence of proteinaceous casts (0-3⁺), interstitial inflammation (0-3⁺), interstitial fibrosis (0-3⁺), arterial medial hypertrophy (0-3⁺), and arteriolar sclerosis (0-3⁺).

All results are presented as mean ± SEM. Statistical analyses were performed using the Number Cruncher Statistical System (Kaysville, UT), and significance was defined as p; 0.05. Group comparisons of data from experiments in aged animals was performed by one- (1F) or two- (2F) nested (hierarchical) analysis of variance (ANOVA) as appropriate. If this analysis indicated a significant difference among the means, specific comparisons were made with a Fisher's LSD test. Unpaired, 2-tailed Student's t-tests and non-parametric Mann Whitney tests were employed for group comparisons of data as appropriate.

RESULTS

Protocol 1

As presented in Figure 1, 11-month old obese ZDFxSHHF rats have marked hypertension, similar to that in obese SHHF, whereas the age-matched SHR are even more hypertensive. The blood pressure values in the anesthetized ZDFxSHHF rats are similar to blood pressure values that were obtained in the two conscious animals by radiotelemetry (MABP = 171 ± 2 mmHg, average over 7 days).

Figure 1. Blood pressure in 46 weeks old ZDFxSHHF, SHHF, SHR and WKY rats (1-F Anova, p < 0.05; a - vs SHR and WKY).

Data regarding the in situ heart performance are presented in Table 1. With the exception of −dP/dt, the values for the other time-pressure parameters of left ventricular function are intermediate between those for SHHF and SHR, suggesting relatively preserved left ventricular function in obese ZDFxSHHF hybrids compared with obese SHHF controls. Reduction in left ventricular relaxation (i.e., −dP/dt) is most likely due to the marked cardiac hypertrophy seen in ZDFxSHHF, (heart/brain ratio 1.21 ± 0.06, Table 1).

Table 1. Heart weights and heart performance in vivo in 11 months old WKY, SHR, SHHF/Mcc-fa^cp and ZDFxSHHF-hybrid rats

Parameters	WKY	SHR	SHHF	ZDFxSHHF
(M ± SE)	(n = 8)	(n = 6)	(n = 6)	(n = 6)
Heart weight (g)	1.66 ± 0.06	1.75 ± 0.06	2.01 ± 0.15^d	2.19 ± 0.11^a
Heart/B.W. (mg/g)	2.57 ± 0.09	4.31 ± 0.20^d	3.03 ± 0.25^e	3.41 ± 0.39^a
Heart/brain ratio	0.83 ± 0.03	1.06 ± 0.06	1.17 ± 0.07^d	1.21 ± 0.06^a
Heart rate (b/min)	345 ± 14.6	379 ± 10.3	358 ± 15.8	349 ± 9.3
VPSP (mmHg)	125.5 ± 4.4	252 ± 15.8^d	195 ± 15.3^a	169.8 ± 17.7^a
+dP/dtmax (mmHg sec^−1)	7651 ± 1319	13551 ± 1505^d	11219 ± 1068	10153 ± 2074
−dP/dtmax (mmHg sec^−1)	4642 ± 756	10103 ± 599	7690 ± 1217	5200 ± 1258^e
+dP/dtmax/P (sec^−1)	355.3 ± 87.6	352.7 ± 62.5	223.5 ± 38.3	221.5 ± 28.8
VEDP (mmHg)	6.90 ± 1.87	2.98 ± 2.42	10.85 ± 3.77^e	7.12 ± 2.30
VMDP (mmHg)	−1.90 ± 1.79	−9.23 ± 4.04	−4.55 ± 3.33	−2.06 ± 0.94

VPSP = ventricular peak systolic pressure

+dP/dt_max = maximum dP/dt during ventricular contraction

−dP/dt_max = maximum dP/dt during the ventricular contraction

VEDP = ventricular end diastolic pressure

VMDP = ventricular minimum diastolic pressure

1-F ANOVA; p < 0.05

a = vs. WKY and SHR

b = vs. SHR and SHHF/Mcc-fa^cp

c = vs. WKY and SHHF/Mcc-fa^cp

d = vs. WKY

e = vs. SHR.

Obese, 11-month old hybrids are hyperglycemic (Figure 2, upper panel) and, in contrast to age matched obese SHHF controls that are hyperinsulinemic, have insulin levels in the range seen in SHR and WKY. Obese hybrids have hypercholesterolemia similar to obese SHHF and moderate hypertriglyceridemia as compared to obese SHHF rats (Figure 2, lower panel).

Figure 2. Metabolic status in 46 weeks old ZDFxSHHF, SHHF, SHR and WKY rats. (1-F Anova, p < 0.05; a - vs SHR and WKY; b - vs all other groups).

Metabolic cage studies (Table 2) revealed severely reduced renal function in obese hybrids (i.e., 3- and 5-fold increase in plasma creatinine and 3- and 7-fold decrease in creatinine clearance compared with SHHF and WKY, respectively). ZDFxSHHF rats excrete significant amounts of protein though proteinuria is less pronounced than in obese SHHF. However, it should be expected that severe reduction in GFR would result in a reduced driving force for protein filtering. Therefore, when normalized by creatinine clearance, proteinuria in ZDFxSHHF animals is even more pronounced compared to obese SHHF controls.

Table 2. Metabolic and renal function in 11 months old WKY, SHR, SHHF/Mcc-fa^cp and ZDFxSHHF-hybrid rats

Parameters	WKY	SHR	SHHF	ZDFxSHHF
(M ± SE)	n = 8	n = 6	(n = 6)	(n = 11)
Body weight (g)	645 ± 14.4	407 ± 9.5^c	672 ± 39.8	653.6 ± 45.8
Food intake (g/kg/day)	41.7 ± 1.3	51.4 ± 2.1	40.6 ± 3.9	35.1 ± 3.6^b
Fluid intake (ml/kg/day)	73.0 ± 5.9	109.8 ± 7.7	108.2 ± 14.6^a	138.2 ± 13.5^a
Urine volume (ml/kg/day)	46.0 ± 7.6	47.3 ± 6.4	81.1 ± 16.2	125.0 ± 12.7^a
Na + excretion (mEq/kg/day)	3.51 ± 0.25	3.33 ± 0.26	3.18 ± 0.36	3.37 ± 0.37
K + excretion (mEq/kg/day)	10.5 ± 0.56	10.5 ± 0.88	9.6 ± 0.87	8.1 ± 0.83
Plasma Creatinine (mg/dl)	0.66 ± 0.03	0.97 ± 0.07^*	1.03 ± 0.3	3.62 ± 0.59^a7∮
Creatinine Clearance (L/kg/day)	5.73 ± 0.34	3.89 ± 0.39^*	2.41 ± 0.78^a	0.67 ± 0.12^a7∮
Urinary protein excretion UPE (mg/kg/day)	44.5 ± 7.6	109.0 ± 21.0^*	2335 ± 427^a	1668 ± 208^a7∮
UPE/Kidney (mg/g kidney/day)	23.4 ± 4.0	75.2 ± 14.5^*	1179 ± 215^a	536.3 ± 66.9^a7∮
UPE/Creatin Clearance (mg/L)	7.8 ± 1.32	29.8 ± 5.4^*	1297 ± 171^a	1939 ± 310^a7∮

1F-ANOVA (p < 0.05)

a = vs. WKY and SHR

b = vs. SHR

c = vs. all other groups student “t” (p < 0.05)

∮ = vs. SHHF/Mcc-fa^cp

^* = vs. WKY.

Renal morphologic changes at 46 weeks of age are summarized in Table 3. Obese ZDFxSHHF exhibited prominent renal hypertrophy both in absolute terms and when expressed as kidney weight normalized by body weight or brain weight. More importantly, renal hypertrophy is significantly greater in obese hybrids as compared with obese SHHF rats that have similar blood pressure and metabolic and lipid status. It should be emphasized that no signs of hydronephrosis were observed in old obese hybrids. Light microscopy analysis of at least 150 glomeruli per animal revealed a high incidence of segmental and occasional global focal glomerulosclerosis, with ∼60% of examined glomeruli being affected in obese ZDXxSHHF rats. The incidence of glomerulosclerosis in obese hybrids was significantly greater than in obese SHHF controls (59.2 ± 6.9 vs 33.2 ± 3.0%, ZDFxSHHF vs SHHF, p < 0.05). Obese, 46 weeks old hybrids also exhibited severe tubulointerstitial (TI) injuries (tubular atrophy and marked tubular dilatation with proteinaceous fluid, interstitial inflammation and fibrosis) and marked chronic vascular changes (arterial medial hypertrophy and arteriolar sclerosis; Figure 3). More importantly, those injuries were more pronounced in ZDFxSHHF rats as compared to obese SHHF controls (Table 3). The increased incidence of segmental glomerulosclerosis and severity of TI and vascular changes in obese hybrids correlated with the reduction in renal function as measured in conscious animals (Table 2).

Figure 3. Obese 46 weeks old male ZDFxSHHF rat - Low power magnification of superficial through deep renal cortex showing prominent dilated tubules containing proteinaceous fluid, and many abnormal glomeruli (methenamine silver-trichrome (A)(MST), 26x). Abnormal glomeruli at higher magnification (B) showing segmental collapse/sclerosis (MST, 257x), and global collapse/mesangial expansion with hypertrophied podocytes containing protein resorption droplets (arrow) (c) (MST, 257x).

Table 3. Kidney weight and renal hystology in 46 weeks old male, WKY, SHR, SHHF and ZDFxSHHF-hybrid rats

	WKY	SHR	SHHF	ZDFxSHHF
	n = 6	n = 6	n = 9	n = 11
Kidney (gr)	1.90 ± 0.05	1.45 ± 0.09^¶	1.98 ± 0.14	3.11 ± 0.14^¶
Kidney/body weight (mg/g)	2.95 ± 0.08	3.67 ± 0.16^†	2.94 ± 0.11	4.75 ± 0.45^x, ‡
Kidney/Brain weight ratio	0.96 ± 0.02	0.98 ± 0.09	1.16 ± 0.07	1.71 ± 0.09^x, ‡
FSGS + FGGS (%)	0	1.1 ± 0.5	33.2 ± 3.0^x	59.2 ± 6.9^x, ‡
Tubular Atrophy (0-3^+)	0	0	1.45 ± 0.17^a	2.3 ± 0.2^a,**
Tubular Dilatation (0-4^+)	0.08 ± 0.07	1.0 ± 0^*	3.4 ± 0.12^a	3.3 ± 0.15^a
Cast (0-3^+)	0.08 ± 0.07	1.0 ± 0^*	3.4 ± 0.1^a	3.2 ± 0.2^a
Interst. Inflammation (0-3^+)	0	0	1.85 ± 0.28^a	2.3 ± 0.2^a
Interstitial Fibrosis (0-3^+)	0	0	1.25 ± 0.1^a	2.7 ± 0.2^a, ^**
Medial Hytpertrophy (0-3^+)	0.25 ± 0.16	1.6 ± 0.19^*	2.35 ± 0.27^a	2.4 ± 0.2^a
Arteriolar Sclerosis (0-3^+)	0	1.5 ± 0.16^*	2.1 ± 0.28^a	2.6 ± 0.2^a

1F-ANOVA (p < 0.05)

x = vs. WKY and SHR

¶ = vs. all other groups; student “t” test (p < 0.05)

† = vs. WKY

‡ = vs. SHHF; Mann-Whitney test (p < 0.05)

a = vs. WKY and SHR

^* = vs. WKY

^** = vs. SHHF.

Protocol 2

Metabolic status of both lean (+/fa or +/+) and obese (fa/fa^cp) 29 weeks old ZDFxSHHF rats are presented in Table 4. Body weight, as well as the mean daily food intake and fluid intake, were significantly higher in obese animals. Obese animals had hyperglycemia, hyper-insulinemia, hypercholesterolemia and extremely elevated plasma triglycerides levels.

Table 4. Metabolic profile of 29 weeks old obese (fa/fa^cp) and lean (+/fa, +/+) ZDF-SHHF-hybrid rats

Parameters	Lean (n = 6)	Obese (n = 7)
Body Weight (g)	503.3 ± 12.0	614 ± 17.9^*
Food Intake (g/kg/day)	48.0 ± 2.1	63.0 ± 3.3^*
Fluid Intake (mL/kg/day)	69.4 ± 2.9	153.9 ± 19.0^*
Glucose (mg/dL)	115.0 ± 10.9	424.0 ± 37.1^*
Insulin (μU/mL)	42.3 ± 3.5	117.2 ± 8.8^*
Triglycerides (mg/dL)	194.3 ± 23.4	5200 ± 702^*
Total Cholesterol (mg/dL)	109 ± 4.1	632.1 ± 38.9^*
HDL-Chol (mg/dL)	48.2 ± 2.3	68.1 ± 12.2^*
VLDL + LDL-Chol	61.4 ± 3.0	464.1 ± 41.8^*

^* p < 0.05 two groups “t” test.

Table 5 summarizes hemodynamic and renal functional parameters measured in anesthetized lean and obese 29 weeks old ZDFxSHHF rats. Both lean and obese animals were hypertensive and had mean blood pressure similar to old (46 wks of age) obese ZDFxSHHF rats. Obese, adult hybrids had increased RVR, decreased inulin clearance (GFR), and more pronounced proteinuria compared with lean littermates. Table 6 and Figures 4 and 5 show data regarding renal pathology. Obese, 29 weeks old hybrids exhibited significant renal hypertrophy (Table 6) that was associated with the significant presence of segmental and occasional global glomerulosclerosis (∼20% of the examined glomeruli) and significant TI damage and vascular changes (Figure 4). Lean animals with the exception of mild vascular changes, have normal renal histology (Figure 5, Table 5). No hydronephrosis was noted in either lean or obese animals.

Figure 4. Obese 29 weeks old male ZDFxSHHF rat - Low power magnification of superficial through mid renal cortex showing focally dilated tubules containing proteinaceous material and scattered abnormal glomeruli (A) (MST, 26x). At higher magnification, there is a spectrum of glomerular pathology ranging from mild mesangial matrix expansion (arrow) to marked mesangial and segmental sclerosis (arrowhead) (B) (MST, 129x).

Figure 5. Lean 29 weeks old male ZDFxSHHF rat - Low power magnification of entire renal cortex through superficial medulla showing no discernible abnormality (A) (MST, 26x). At higher magnification, glomeruli show mild diffuse mesangial matrix expansion (B) (MST, 129x).

Table 5. Hemodynamic and renal function parameters in anesthetized (A) and conscious (B), 29 weeks old obese (fa/f^cp) and lean (+/fa, +/+) ZDF-SHHF-hybrid rats; ^* p < 0.05

Parameters	Lean (n = 6)	Obese (n = 7)
A
Mean Arterial Blood Pressure (mmHg)	164.7 ± 2.0	165.3 ± 5.2
Heart Rate (b/min)	367 ± 11.9	348 ± 11.9
Renal Blood Flow (mL/g kid/min)	8.68 ± 0.63	3.44 ± 0.35^*
Renal Plasma Flow (mL/g kid/min)	4.79 ± 0.33	2.13 ± 0.20^*
Renal Vascular Resistance (mmHg/mL/min/g kid)	1.93 ± 0.14	2.97 ± 0.32^*
Glomerular Filtration Rate (mL/g kid/min)	1.59 ± 0.18	0.61 ± 0.08^*
Filtration Fraction	0.356 ± 0.08	0.286 ± 0.04
B
Urine Volume (mL/kg/day)	31.8 ± 2.7	127.9 ± 16.9^*
Urine Na+ (mEq/kg/day)	4.00 ± 0.22	5.37 ± 0.26^*
Urine K+ (mEq/kg/day)	12.5 ± 0.52	16.3 ± 0.7^*
Urine Creatinine (mg/kg/day)	45.7 ± 1.6	32.0 ± 2.5^*
Creatinine Clearance (L/kg/day)	4.71 ± 0.75	2.63 ± 0.6^*
Urine Glucose (g/kg/day)	0.01 ± 0	6.08 ± 2.40^*
Urine Protein (mg/kg/day)	500 ± 20	2320 ± 90^*
U-Protein/Cr Clearance (mg/L)	119.8 ± 16.9	1182 ± 222^*
U-Protein/U-Creatinine Ratio	11.0 ± 0.01	74.0 ± 5.0^*

^* p < 0.05 two groups “t” test.

Table 6. Renal hystology in male, 29 weeks old, lean (+/fa) and obese (fa/fa^cp) ZDFxSHHF-hybrid rats ^* p < 0.05, Mann-Whitney test

	Lean 29 wks	Obese 29 wks
Kidney Weight (g)	1.61 ± 0.06	2.56 ± 0.06^*
Kidney/Body Weight (mg/g)	3.18 ± 0.07	4.18 ± 0.13^*
FSGS + FGGS (%)	0.1 ± 0.1	20.1 ± 3.2^*
Tubular Atrophy (0-3^+)	0	1.1 ± 0.1^*
Tubular Dilatation (0-4^+)	0.1 ± 0.1	2.7 ± 0.2^*
Cast (0-3^+)	0.1 ± 0.1	2.7 ± 0.2^*
Interst. Inflammation (0-3^+)	0	1.42 ± 0.2^*
Interstitial Fibrosis (0-3^+)	0	0.25 ± 0.2^*
Medial Hytpertrophy (0-3^+)	1.2 ± 0.2	1.75 ± 0.1^*
Arteriolar Sclerosis (0-3^+)	0.9 ± 0.2	1.3 ± 0.1

^* p < 0.05, Mann-Whitney test.

DISCUSSION

Elucidation of the pathophysiology of diabetic nephropathy in non-insulin-dependent diabetes mellitus (NIDDM) requires appropriate animal models. Over the past three decades efforts have been made to develop and/or identify appropriate models of NIDDM nephropathy [18-19]. However, in the most of these models, marked hyperglycemia and renal damage were not present simultaneously. In addition, a very recent evaluation of renal changes in the ZDF rats (a model that appears to mimic the metabolic status of human NIDDM) failed to identify this strain as a model of diabetic nephropathy due to the presence of significant hydronephrosis [[11]]. The present study is the first to characterize the hybrid offspring of ZDF mated with SHHF as a animal model which expresses multiple risk factors of renal disease (i.e., hypertension, hyperglycemia, hyperinsulinemia, hyperlipidemia,) resulting in severe renal damage.

Obese ZDFxSHHF rats are hypertensive and have blood pressure in the range similar to that of lean and obese hypertensive controls (SHR and SHHF, respectively). Lean ZDFxSHHF rats are also hypertensive and have blood pressure similar to their obese littermates. This is in contrast to Zucker strains, of which obese animals have been shown to be normotensive or mildly hypertensive and lean littermates to be normotensive [[11]], [20-22]. However, elevated blood pressure in both obese and lean ZDFxSHHF rats is in concert with elevated blood pressure seen in strains of obesity and genetic hypertension, i.e., obese SHR, SHR/N cp and obese and lean SHHF rats [8-9], [[14]], [[16]], [23-24]. Blood pressure levels are much higher in hybrids than those reported for Zucker strains, suggesting that ZDFxSHHF hybrids inherited elevated blood pressure from the SHHF parent. The fact that, in ZDFxSHHF hybrids, hypertension is present in both the lean and obese phenotype suggests that increased blood pressure is not specifically associated with the obese genotype. Although the latter suggests that this model may be unsuitable for studying the pathogenic relationship between obesity and hypertension, the model may be useful for studying the effects of elevated blood pressure on renal function in the presence of other risk factors for renal disease (i.e., hyperglycemia, hyperinsulinemia, hyperlipidemia and obesity). In obese ZDFxSHHF rats, hypertension is accompanied by marked cardiac hypertrophy. However, assessment of heart performance in situ suggests that obese ZDFxSHHF at the age of 11 months have relatively preserved cardiac function. In the present study, since death was not the end-point, we were not able to more precisely determine the cause of death in aged animals. During the study-period two animals became anuria and died without signs of heart failure, suggesting renal failure as the most probable cause of death. Although concurrent heart failure in some of the animals can not be ruled out, it seems that toward the end of life in obese ZDFxSHHF, the animals' renal pathology predominates. The severity of glomerular (∼60% glomerulosclerosis) and TI damages, and the marked reduction of renal function in old ZDFxSHHF strongly suggest that obese ZDFxSHHF rats develop and ultimately die from end-stage renal failure.

The present study revealed that obese hybrids have more severe renal damage as compared with age matched obese animals with similar blood pressure and metabolic status (i.e. SHHF controls). It would appear that crossing of two strains of nephropathy (ZDF and SHHF rats), produces hybrids that carry additional genetic risk for renal dysfunction. In light of these findings, it should be mentioned that lean animals also excreted significant amount of proteins (Table 5). Urinary protein excretion (UPE) was five times greater in lean hybrids compared with UPE in 4 months older SHR (UPE = 500 ± 20 vs. 109 ± 21 mg/kg/day, 29 wks old lean ZDFxSHHF vs. 46 wks old SHR). Since lean hybrids have blood pressure and a metabolic profile similar to much older (lean) SHR, it seem that lean hybrids also carry greater risk for renal damage. However, in this study we were not able to separate lean homozygotes from heterozygotes, and it is possible that initial renal injury (proteinuria) seen in lean hybrids reflects the presence of the fa gene in heterozygote hybrids.

High glucose and insulin levels observed in fasted 29 weeks old obese ZDFxSHHF hybrids suggest that obese animals closely mimic the metabolic status of human NIDDM, a feature common to obese animals from both parental strains [[7]], [[10]]. In our study, we also observed that in 46-week old obese animals serum insulin levels decline to levels similar to age matched SHR and WKY. This probably reflects the failure of beta cells in obese animals to respond to glucose stimuli. In obese ZDF rats it has been shown that with aging, as beta cells become progressively more refractory to the glucose stimuli, initially elevated serum insulin levels decline. This is thought to be mainly due to the deficient expression of GLUT2 on beta cells [[25]]. Also, in old obese hybrids we detected elevated levels of triglycerides relative to age-matched SHR and WKY, though much lower that measured in the younger obese hybrids. At present, the cause of this difference between the 29-week-old and 46- week- old obese hybrids it is not clear. Interestingly, a similar decline in triglycerides levels was observed in obese Zucker rats, in which TG levels of > 5,000 mg/dL measured at 30 weeks of age, dropped to ∼1,000 mg/dL by the age of 45 week [[26]]. One possible cause for the decline in triglycerides levels is reduced food intake, since in the present study old obese animals, as they approached end-stage renal failure, tended to eat less.

At the kidney level elevated blood pressure in obese animals was accompanied by more prominent vascular changes (medial hypertrophy, and arteriolar sclerosis, Table 4), suggesting that severe glomerular and tubulointerstitial changes in old ZDFxSHHF may be hypertension-mediated. However, because no glomerular and TI changes were observed in age matched SHR (Table 4) as well as in lean hybrids (Table 5), it is unlikely that hypertension is the predominant factor for development of glomerular and TI injury in obese ZDFxSHHF rats. Severe (predominantly segmental) glomerulosclerosis and TI injury seen in obese ZDFxSHHF are in accordance with our current understanding of the role of proteinuria and hyperlipidemia in the development of TI and glomerular damages. Proteinuria is considered to be an important and independent risk factor for progressive renal failure that leads to interstitial inflammation and subsequent increases in extracellular matrix and scarring [27-28]. In young obese Zucker rats it has been shown that TI lesions occur before focal glomerulosclerosis and are closely related to the proteinuria [[29]]. In our study in adult obese ZDFxSHHF rats, significant predominantly segmental glomerulosclerosis (20 ± 3%) was associated with marked tubular dilatation and intratubular proteinaceous fluid. Hyperlipidemia has also been shown to accelerate the progression of human renal diseases [3-4] and the accumulated evidence suggests that hyperlipidemia is a major risk factor for development and progression of glomerulosclerosis in Zucker rat strains [[26]]. In obese Zucker rats, hyperlipidemia is believed to be associated with early influx of glomerular macrophages that precedes glomerulosclerosis [[30]] and the treatment of hyperlipidemia reduces glomerular injury [[20]]. In our study, obese adult animals had drastically elevated triglycerides levels (> 5,000 mg/dL) and it may be possible that hyperlipidemia significantly contributed to the progression of renal failure in these animals. However, longitudinal studies are required to more closely define the relationship between hyperlipidemia, proteinuria and progression of glomerular and TI injuries in this animal model. This study failed to demonstrate the development of hydronephrosis in any of the animals, though the cause of the renal tubular dilatation remains unestablished. This model is therefore suitable for studying the effects of different risk factors on renal structure.

In summary, the ZDFxSHHF(fa-fa^cp) rat model exhibits obesity, insulin resistance, hypertension and severe dislipidemia. Expression of these risk factors for kidney disease is associated with severe functional and structural changes and animals die at an early age (∼12 months) most likely from end-stage renal failure. These animals may be a useful model for studying the effects of expressed risk factors, as well as the effects of pharmacological interventions in chronic renal failure.