Abstract: PO1778
Experimental Membranous Nephropathy in a Novel Transgenic Rat Model of Decay-Accelerating Factor Deficiency Generated by CRISPR-Associated Protein 9 (Cas9) Genome Editing
Session Information
- Glomerular Diseases: Lupus and Membranous
October 22, 2020 | Location: On-Demand
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1202 Glomerular Diseases: Immunology and Inflammation
Authors
- Detsika, Maria, National and Kapodistrian Univeristy of Athens, School of Medicine, 1st Department of Critical Care Medicine and Pulmonary Services, GP Livanos and M Simou Laboratories, Evangelismos Hospital, Athens, Greece
- Gakiopoulou, Harikleia, National and Kapodistrian University of Athens, Pathology Department, Athens, Greece
- Grapsa, Eirini, National and Kapodistrian University of Athens - Nephrology Clinic, Aretaieio Hospital, Athens, Attica, Greece
- Lianos, Elias, Thorax Research Center of Intensive and Emergency Thoracic Medicine, Athens, Greece
Background
Decay accelerating factor (DAF), controls extent of formation of C3 and C5 convertases. Using clustered regularly-interspaced short palindromic repeats, CRISPR/ associated protein 9 (Cas9) genome editing a DAF deficient (daf-/-) rat model was generated. The present study describes the renal and extrarenal phenotype of this model and responses to podocyte injury in experimental membranous nephropathy (MN).
Methods
daf+/- rats were produced by injecting multiple CRISPRs targeting Cd55 exon 2 into Sprague-Dawley rat embryos. A founder harboring a net 4-bp deletion in exon 2 was backcrossed to the parental strain and litters were genotyped. 1 ml of anti-Fx1A serum was injected in daf+/+ and daf-/- rats to induce MN. Control rats received a single dose (1ml) of normal rabbit serum. DAF protein and mRNA levels were determined by western blotting and Real time PCR Renal function assessment involved measurement of serum urea and creatinine levels and urine albumin/creatinine ratio. C3 deposition was confirmed by immunofluorescence (IF) and western blot (WB) analysis.
Results
daf-/- rats were healthy, viable and able to reproduce normally. DAF was completely absent in renal and extrarenal tissues (lung, heart) at protein and mRNA level. There was no effect on glomerular Crry and CD59 protein expression. There were no glomerular or tubulointerstitial lesions in daf-/- rats compared to daf+/+ and no change in serum urea and creatinine or in urine protein excretion. There was a significant increase in proteinuria 14 days following anti-Fx1A injection in daf-/- rats accompanied by increased glomerular C3 deposition.
Conclusion
In experimental MN, DAF attenuates proteinuria. The daf-/- rat model provides a valuable tool to assess role of DAF in regulating complement activation in glomerular diseases, such as MN, which is best characterized in this species.