41-Week Study of Progressive Diabetic Nephropathy in the ZSF1 fa/fa CP Rat Model

Abstract

This symposium synopsis summarizes key results from a 41-week study of diabetic nephropathy (DN) in the type 2 diabetic ZSF1 fa/fa^CP rat model. During this study, we conducted longitudinal analysis of biomarkers, renal histopathology, ultrastructural assessment, renal quantitative image analysis, and transcriptome analysis of glomerular-enriched tissue. We concluded that there is translational value for using the ZSF1 rat model in mechanistic and therapeutic intervention efficacy studies for type 2 DN.

Animal models of diabetic nephropathy (DN) are utilized to assess mode of action and potential efficacy of a therapeutic molecule and to help identify any genetic, environmental, or other factors that lead to progression of DN. Criteria developed by the Animal Models of Diabetic Complications Consortium (AMDCC) for a translational animal model of DN include greater than a 10-fold increase in albuminuria, a greater than 50% reduction in glomerular filtration rate (GFR) over the course of DN, advanced mesangial matrix expansion, glomerular basement membrane thickening by greater than 50%, and tubulointerstitial fibrosis (Brosius et al. 2009).

ZSF1 rats exhibit spontaneous nephropathy that is considered secondary to obesity, hypertension, and diabetes. They are the F1 progeny of lean female Zucker diabetic fatty rats (+/fa) and male spontaneously hypertensive heart failure rats (+fa^CP; Tofovic et al. 2000). All offspring are hypertensive. Progeny that are homozygous for leptin receptor deficiency develop diabetes and DN. To help understand the value and utility of the ZSF1 rat model for future use in type 2 DN efficacy studies, this model was characterized by our group in a 41-week longitudinal study of renal disease progression (Dower et al. 2017). Primary end points included assessment of serum and urine biomarkers, microscopic assessment of kidney, and transcriptome analysis by next-generation RNA sequencing (RNA-Seq) of glomerular-enriched tissue. All procedures involving animals were reviewed and approved by the Pfizer Institutional Animal Care and Use Committee.

In obese rats, compared with lean rats, serum cholesterol, triglycerides, blood urea nitrogen, and urine neutrophil gelatinase–associated lipocalin normalized to urine creatinine were higher at 12 to 41 weeks. Urine microalbumin, N-acetyl-beta-D glucosaminidase and kidney injury molecule-1, all normalized to urine creatinine, were higher at 20 to 41 weeks. There were increased hyaline casts and proteinaceous material in renal tubules and increased infiltrates of lymphocytes and macrophages (mostly interstitial and occasionally glomerular) during the course of disease in obese rats. Using quantitative image analysis (QIA), mean glomerular tuft area and mean area of glomerular periodic acid Schiff-positive mesangial matrix were greater in obese rats at 12 to 29 weeks compared with lean rats. QIA of kidney sections stained for collagen IV or blinded semiquantitative scoring of trichrome-stained sections also revealed greater mean area of collagen IV per glomerulus and greater trichrome staining in glomerular basement membrane, Bowman’s capsule, mesangial space, and tubulointerstitium at 20 to 41 weeks in obese rats. Levels of urine collagen III breakdown product positively correlated with urine microalbumin, mean collagen IV staining area per glomerulus, and trichrome score. Ultrastructural changes, comprised of podocyte foot process effacement, increased mesangial matrix, and basement membrane thickening, generally increased in severity over time in obese rats but were not noted in lean rats. Levels of podocyte messenger RNAs (mRNAs) synaptopodin, glomerular epithelial protein 1, podocin, Wilms tumor 1 (WT-1), and nephrin, normalized to average expression in 12-week-old lean rats, declined in obese rats through week 41. However, this decline appeared to be due to a reduced abundance of podocyte mRNAs as opposed to a reduction in the number of WT-1 staining podocytes (Dower et al. 2017). At weeks 34 and 41, RNA-Seq and pathway analysis showed good concordance with a published data set of human DN glomeruli (Hodgin et al. 2013).

AMDCC-associated criteria observed in the ZSF1 diabetic rat included greater than 10-fold elevation of urine albumin, significant mesangial matrix expansion, glomerular basement membrane thickening, and tubulointerstitial fibrosis (GFR was not assessed in this study). These criteria along with additional changes in serum and urine biomarkers, renal histopathology, QIA, and glomerular transcriptome during the course of disease supported the translational value of the ZSF1 rat model of DN.