PreClinOmics - ZDSD Diabetic Nephropathy

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Presentation of PreClinOmics ZDSD rat for diabetic nephropathy .

Text of PreClinOmics - ZDSD Diabetic Nephropathy

  • 1.Diabetic Nephropathy in the ZDSD Rat PreClinOmics, Inc. Contact Richard G. Peterson for more information. rpeterson@preclinomics.com 317-872-6001 x 13

2. Obesity and metabolic syndrome are clear predictors of chronic kidney disease largely due to the potentiation of chronic inflammation by insulin resistance. In addition, the lipoprotein abnormalities, increased hemodynamics, hypercoagulability and vascular dysfunction associated with metabolic syndrome have all been implicated as causative for renal disease. Biomarkers for renal dysfunction (i.e., IL6, TNF- ,NGAL,KIM-1, VEGF etc.) as well as significant albuminuria , elevated free fatty acids with oxidative stress, and histological analysis have shown the ZDSD rat to exhibit nephropathy that closely mimics that observed in obese insulin resistant patients. Renal Injury 2 3. Diabetic Nephropathy in the ZDSD Rat Increased kidney weight Increased urinary markers for kidney disease Increased serum markers for kidney disease Glomerular sclerosis Nodular sclerosis, KW nodules Thickening basement membrane of glomerular capillaries Podocyte effacement on capillaries 3 4. Terminal kidney weights are highest in the ZDSD rat groups. These increased kidney weights and high urinary volume along with increased micro-albumin concentration and the total amount of micro-albumin indicate that there may be significant diabetic nephropathy in the ZDSD rat model. Terminal Comparison Kidney Weight Urine Analysis 0 1 2 3 4 5 6 7 CRL-SD, CD +/fa ZDF ZDSD, Diabetic 12-21 weeks ZDSD, Diabetic 7-11 weeks Weight(g) 4 0 50 100 150 200 250 300 CRL-SD, CD +/fa ZDF ZDSD, Diabetic 12-21 weeks ZDSD, Diabetic 7-11 weeks 5. Experiment 1 ZDSD Diabetic Nephropathy Spontaneous Diabetes ELISA Analysis of Markers 5 6. 6 Weight 10 12 14 16 18 20 22 24 26 28 30 300 400 500 600 SD ZDSD Age (weeks) Weight(g) Glucose 10 12 14 16 18 20 22 24 26 28 30 0 200 400 600 SD ZDSD Age (weeks) Glucose(mg/dL) Urine Volume 10 20 22 24 26 30 0 50 100 150 200 SD ZDSD Age (weeks) UrinaryVolume(ml/24hr) 7. 7 Urinary albumin 10 20 22 24 26 30 0 25 50 75 100 125 150 SD ZDSD Age (weeks) Urinaryalbumin(mg/day) beta-2 microglobulin 10 20 22 24 26 30 0 500 1000 1500 2000 SD ZDSD Age (weeks) Urinary b -2microglobulin( m g/day) Cystatin C 10 20 22 24 26 30 0 10 20 30 SD ZDSD Age (weeks) UrinarycystatinC( m g/day) KIM-1 10 20 22 24 26 30 0.0 2.5 5.0 7.5 10.0 12.5 15.0 SD ZDSD Age (weeks) UrinaryKIM-1(ng/day) 8. Experiment 2 Urine BioMarkers of Renal Disease Study Details Male ZDSD rats were allowed to become diabetic spontaineously on Purina 5008 and aged to 33 weeks. Two groups of animals were selected for further study: animals that were diabetic for longer than 16 weeks and animals that were diabetic for less than 8 weeks. Mesoscale (MSD) urine panels were run on urine (Argutus AKI test, Kidney Injury Panel 1 and Rat Clusterin) Pathological evaluation of the kidneys was done. 8 9. Data From Urinary Excretion Study 9 10. Urinary Excretion of Kidney Markers 10 11. Urinary Excretion of Kidney Markers 11 12. 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 Bowmans 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. 12 13. 13 14. Kidney Histopathology of the ZDSD Rat A Novel Animal Model of Diabetes 14 Glomerulopathy Tubular dilation Protein casts Inflammation HistopathologyScore(0-5) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Non-diabetic Diabetic * * * * *compared to Non-diabetic animals (t-test) /degeneration 15. Experiment 3 Diabetic Nephropathy, EM of Glomerular Pathology 15 16. Experimental details Male ZDSD rats allowed to become spontaneously diabetic. Animals were terminated and perfused fixed at about 35 weeks of age. Control CD rats ZDSD rats that had been diabetic from 12-13 weeks ZDSD rats that had been diabetic from 16-17 weeks Took pictures of glomerular capillaries and BM Measured GBM thickness Evaluated podocyte morphology 16 17. Glomerular Capillary, Basement Membrane Control, Age Matched Diabetic, 12 Weeks 17 18. Glomerular Capillary, Basement Membrane Control, Age Matched Diabetic, 16.5 Weeks 18 19. 19 Glomerular Basement Membrane Thickness Time of Diabetes in the ZDSD Rat Thickness(mm) CD Control 12 Weeks 16.5 Weeks 0 100 200 300 400 500 20. 20 Scanning Microscopy Control Glomerular Capillary with Normal Podocyte Foot Processes 21. 21 Scanning Microscopy Diabetic Glomerular Capillaries Demonstrating Effacement 22. Experiment 4 Diabetic Nephropathy, Synchronized Diabetes: Clinical Data and LM of Glomerular Pathology 22 23. Experimental details Male ZDSD rats synchronized to become diabetic by feeding them Purina 5SCA. Animals were put on 5SCA at 19 weeks of age and were diabetic by 20 weeks of age. They were monitored until they were 47 weeks old. We evaluated the following groups: ZDSD rats that had been diabetic for 27 weeks (14) ZDSD rats that failed to become diabetic (4) Graphed terminal data and evaluated pictures of glomeruli and other kidney pathology 23 24. 24 Body weight 0 7 14 21 28 35 42 70 105 126 155 172 185 196 350 400 450 500 550 600 Diabetic Non-diabetic Day of study Bodyweight(g) Glucose 0 7 14 21 28 35 42 70 105 126 155 172 0 200 400 600 800 Diabetic Non-diabetic Day of study Glucose(mg/dL) Diabetic Non-diabetic 43 47 0 5 10 15 * t-test ** Age (weeks) HbA1c(%) HbA1c 43 0.0 0.2 0.4 0.6 0.8 1.0 Diabetic Non-diabetic Age (weeks) NEFA(mEq/L) NEFA 25. 25 Diabetic Non-diabetic 0 5 10 15 20 25 Diabetic Non-diabetic * * t-test Liverweight(g) 47weeksofage Liver Weight Diabetic Non-diabetic 0 2 4 6 Diabetic Non-diabetic Kidneyweight(g) 47weeksofage * t-test * Kidney Weight 43 47 0 100 200 300 Diabetic Non-diabetic Age (weeks) Urinevolume(mls/24hr) Urine Volume 43 0 20 40 60 80 Diabetic Non-diabetic * * t-test Age (weeks) Urinealbumin(mg/day) Urinary Albumin 26. Blood Chemistry 26 43 45 47 0 10 20 30 Diabetic Non-diabetic * t-test *** Age (weeks) SerumBUN(mg/dL) BUN 45 47 0.0 0.1 0.2 0.3 0.4 0.5 Diabetic Non-diabetic Age (weeks) Serumcreatinine(mg/dL) Creatinine 43 47 0 50 100 150 200 Diabetic Non-diabetic * t-test ** Age (weeks) Serumcholesterol(mg/dL) Cholesterol 43 47 0 500 1000 1500 Diabetic Non-diabetic * t-test ** Age (weeks) Serumtriglycerides(mg/dL) Triglyceride 27. 47 Week-old, 27 Weeks Diabetes Non-Diabetic Diabetic Diabetic Diabetic 27 28. 28 47 Week-old, 27 Weeks Diabetes 28 Non-Diabetic Diabetic 29. 47 Week-old, 27 Weeks Diabetes 29 Non-Diabetic Diabetic 30. 47 Week-old, 27 Weeks Diabetes 30 31. Follow-up study, Synchronized Diabetes was induced by putting ZDSD rats on 5SCA diet when they were 21 weeks of age. This can be done anytime after 16 weeks of age. Weight and glucose were followed periodically. 24 hour urine was collected and urinary albumin and creatinine were measured so that total 24 hour albumin and albumin/creatinine ratios could be determined. Terminal data were collected when the rats had been diabetic 13-14 weeks. 31 32. 32 Progression of Diabetes and Urinary Albumin in ZDSD Rats Diabetes was synchronized with 5SCA diet (3 weeks) when ZDSD rats were about 20 weeks old. Glucose rose rapidly while animals were on 5SCA diet and they remained hyperglycemic when they were taken off diet; overtly diabetic animals lost weigh. Urinary volume increased steadily while the urinary albumin levels had a rapid increase between 11 and 15 weeks after 5SCA was started. Glucose(mg/dL) 0 4 8 12 16 0 200 400 600 Weight(g) 0 4 8 12 16 0 200 400 600 Weeks After 5SCA UrinaryvolumemL 0 4 8 12 16 0 100 200 300 Weeks After 5SCA Urinaryalbumin(mg/day) 0 4 8 12 16 0 100 200 300 400 33. Glomerular Proteomics Nondiabetic, prediabetic and diabetic glomeruli were collected and analyzed for protein expression. Data were analyzed using volcano plotting to show the differences in expression of proteins in the three groups. The different groups were compared in the next three slides (33-35). Slide 36 demonstrates down-regulation of glyoxalase 1 with diabetes. 33 34. Protein Expression in the ZDSD Glomerulus 34 35. Protein Expression in the ZDSD Glomerulus 35 36. Protein Expression in the