The ZDSD/Pco Rat, a Type II Diabetic Model for Diabetic Nephropathy without Leptin or Leptin Receptor Mutations

Abstract – Most rodent models for obesity and diabetes have monogenetic mutations that are responsible for the obesity. The ZDSD rat was developed by crossing a diet induced obese (DIO) rat that was derived from the Crl:CD(SD) strain with a homozygous lean ZDF/Crl rat and selective inbreeding for obesity and diabetes.

Male ZDSD rats (n=16), approximately 33 weeks of age, were evaluated for spontaneous development of diabetes and accompanying renal dysfunction. Ten of the 16 rats were designated diabetic as evidenced by significantly higher fed glucose (> 500 mg/dL), HbA1c values and glucose AUC calculated from an oral glucose tolerance test (OGTT) when compared to their normoglycemic littermates. Urine volume was approximately 10 fold compared to non-diabetic animals. The diabetic animals lost body weight. Grossly, kidneys were 26% larger in diabetic animals and analysis of 24 hour urine revealed significant increases in several biomarkers routinely used to assess renal injury (NGAL, osteopontin, Tim-1, GSTYb1, clusterin and renal papillary antigen-1). A functional assessment of kidneys from diabetic ZDSD rats revealed a significant 7 fold increase in albumin excretion (albumin/creatinine ratio) with a corresponding decrease in circulating albumin.

Kidneys were fixed in buffered formalin, embedded and stained with H&E, trichrome and PAS. The nephropathy was characterized primarily by glomerular changes. The animals which were diabetic had significant mesangial expansion. More advanced glomerular pathology was characterized by nodular, focal and global sclerosis.

In summary, the ZDSD/Pco rat is an important new model that expresses obesity and diabetes without apparent alterations in the leptin pathway. This model demonstrates changes which are consistent with diabetic kidney disease. The nature of this model is more likely to make it a more useful than other rat models in testing compounds that are effective in treating diabetes, obesity and the complications of diabetes such as nephropathy. Partially supported with SBIR grant from NIH (1 R43 DK074242)

• Most rodent models of type 2 diabetes have a monogenic mutation that is responsible for the initiation of obesity and its subsequent insulin resistance.
• The most common mutations are in the leptin receptor itself (Zucker Fatty rat, Zucker Diabetic rat and db/db mouse) or in the leptin molecule as in the ob/ob mouse.
• Human mutations in the leptin pathway are very rare and thus are not thought to contribute to obesity and its related sequelae.

• Nephropathy is a serious complication of impaired glucose metabolism. Current rodent models are reported to exhibit varying degrees of renal dysfunction, however the absence of a functional leptin pathway may cause divergence from characteristic human nephropathy.
• In an effort to mimic the renal effects of impaired glucose metabolism, the ZDSD rat was developed with the following phenotype:
  • Functional leptin pathway with polygenic obesity.
  • Slow development of insulin resistance that culminates in beta cell failure.
  • Demonstration of progressive renal dysfunction which is characteristically similar to human diabetic nephropathy.

• This strain was produced by crossing diet induced obese (DIO) rat derived from the CRL:CD(SD) strain with homozygous lean ZDF/Crl.
• The strain is selectively bred for obesity and diabetes.
• Further selection of genetically matched breeders provides phenotypic homogeneity.

• Characterize the hyperglycemia-induced renal dysfunction in ZDSD rats
  • Compare ZDSD to other relevant strains at 30-33 weeks of age: includes obese ZDF, lean ZDF, CD controls
  • Evaluate effect of short term feeding of diabetogenic diet on serum biomarkers
  • Evaluate age-related changes in renal biomarkers and histology compared to CD controls

Pathophysiology

• Long term evaluation of male ZDSD rats
• Terminal comparison, males, @ 30-33 wks of age
  • Obese ZDF
  • Lean ZDF +/fa
  • CRL:CD
  • ZDSD, diabetic 7-11 wks
  • ZDSD, diabetic 12-21 wks

 

Serum BioMarkers of Renal Disease

     Study Details

• Male ZDSD Rats were placed on a diabetogenic diet (RD12468) between 17 and 19 wks of age.
  15 out of 21 animals in this experiment developed diabetes during this period; the animals that responded, remained diabetic when they were put back on chow diet (LabDiet 5008)
• RulesBased Medicine panels (Rat Metabolic MAP, Rat Kidney MAP, RodenMAP) were run on serum samples that were collected
  • before diabetes developed (14 wks)
  • while diabetic on the diabetogenic diet (18 wks) and
  • one week after they were taken off the diabetogenic diet (20 wks)

 

Urine BioMarkers of Renal Disease

     Study Details

• Male ZDSD Rats were allowed to become diabetic spontaineously on LabDiet 5008 and aged to 33 wks.
  Two groups of animals were selected for further study:  animals that were diabetic for longer than 16 wks and animals that were diabetic for less than 8 wks.
• MesoScale (MSD) urine panels were run on urine samples collected (Argutus AKI test, Kidney Injury Panel-1 and Rat Clusterin)
• Pathological evaluation of the kidneys was done

 

Pathological Evaluation of Kidney

• Glomerulopathy:  Changes in the renal glomeruli consisted of one or more of the following: increased cellularity in the mesangium; increased in mesangial connective tissue; thickening of Bowman’s capsule; hypertrophy of capsular epithelium; dilation of the capsular space. Individual glomeruli appeared moderately enlarged.  The lesions were highly variable within individual glomeruli and between glomeruli within a kidney.  The changes were most usually segmental, although a rare glomeruli was fibrotic (condensed).  Expanded mesangial material stained positively with the PAS stain and to a lesser extent with the Trichrome stain.
• Tubular dilation/degeneration:   This change was mainly in the cortex and consisted of irregularly dilated, empty tubules, that sometimes were lined by cuboidal epithelium that stained basophilic compared to the expected normal eosinophilic tubular epithelium.  In some individual tubules the epithelium were flattened.  These dilated/degenerate tubules were randomly scattered throughout the cortex, and sometimes were associated with protein casts and/or non-suppurative inflammation (see below).  Focal mild increases in fibrous connective tissue within the interstitial space was present, frequently in association with the interstitial inflammatory response, but not restrictively so.  
• Protein casts:  Individual tubules contained acellular, uniformly staining eosinophilic material consistent with protein.  These protein casts were present in the cortex and in the medulla, as well as at the cortico-medullary junction in various sections.  Often, several such dilated tubules containing protein casts were clustered together, usually in the cortex.
• Inflammation:  The inflammatory process consisted of focal collections of lymphocytes and macrophages, which were seen in the cortical interstitial space, adjacent to individual glomeruli and individual blood vessels, and in association with the renal pelvic epithelium. 

 

• Polygenic obesity that can be modulated by diet
• No leptin or leptin receptor defects
• Diabetes develops spontaneously on Purina 5008  and synchronously with a diabetogenic diet (Purina 5SCA or Research Diets D12468) when the animals are 16-17 weeks old
• Slower onset of diabetes than in the ZDF rat
• Insulin resistance develops as animals age and gain weight
• Pancreatic failure may go more slowly
• Reference compounds are effective in treating the diabetes (Rosiglitazone and Metformin) and obesity (Rimonabant).
• Demonstrates diabetic complications such are diabetic nephropathy

• The ZDSD Rat model of type 2 diabetes/obesity more closely resembles the human condition than other rodent models with no apparent alterations in the leptin pathway.
• Diabetic nephropathy is evidenced by tissue and BioMarker changes.  In addition, the severity of renal injury correlates with the duration of hyperglycemia.