Abstract: SA-PO759
Carbamylation of Albumin Results in Structural Changes That Minimize Binding to Cubilin and FcRn Resulting in More Rapid Serum Clearance
Session Information
- CKD: Mechanisms - III
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: CKD (Non-Dialysis)
- 2103 CKD (Non-Dialysis): Mechanisms
Authors
- Yadav, Shiv Pratap Singh, Indiana University School of Medicine Division of Nephrology, Indianapolis, Indiana, United States
- Kumar, Sudhanshu, Indiana University School of Medicine Division of Nephrology, Indianapolis, Indiana, United States
- Sandoval, Ruben M., Indiana University School of Medicine Division of Nephrology, Indianapolis, Indiana, United States
- Campos-bilderback, Silvia B., Indiana University School of Medicine Division of Nephrology, Indianapolis, Indiana, United States
- Rhodes, George, Indiana University School of Medicine Division of Nephrology, Indianapolis, Indiana, United States
- Wagner, Mark C., Indiana University School of Medicine Division of Nephrology, Indianapolis, Indiana, United States
- Molitoris, Bruce A., Indiana University School of Medicine Division of Nephrology, Indianapolis, Indiana, United States
Background
Chronic kidney disease results in high serum urea concentrations and leads to increased cardiovascular disease. An increase in urea results in excessive protein carbamylation which has been associated with increased mortality. We hypothesized carbamylation would alter the structure of albumin leading to reduced binding to cubilin and or FcRn resulting in increased serum clearance.
Methods
To test this hypothesis rats were injected at time 0 with RSA or RSA modified with KCN 30min, 2hr or 4hr to induce increasing amounts of carbamylation. Increased serum clearance was observed in a dose dependent fashion. To determine the mechanism we quantified the number of lysine residues modified on RSA using MALDI TOF and showed increasing Lys residues modified with longer KCN incubation times. We then asked whether increased clearance was the result of decreased binding to cubilin, which would result in reduced PTC uptake, and or FcRn which would reduce transcytosis of albumin. Using Microscale Thermophoresis to quantify changes in binding affinity values, Kd, we observed a reduction in both cubilin and FcRn binding with carbamylated RSA, compared to RSA.
Results
Specifically, the Kd of cubilin 7,8 increased from 0.01 mM for unmodified RSA to 1.2 mM for 30min, 1.7 mM for 2hr and 10 mM for 4hr carbamylated RSAs, respectively. For FcRn the Kd for albumin was10 mM for unmodified albumin, while 2hr carbamylated RSA was >100 mM and 4hr had no binding. The decreased binding to both cubilin 7,8 and FcRn can explain the increased serum clearance and is also consistent with our observed increase in carbamylated albumin in the urine. To better understand the changes to albumin mediated by carbamylation we performed a structural characterization analysis of modified albumin using small-angle X-ray scattering (SAXS) to evaluate shape changes with carbamylation. Guinier and P(R) analyses yielded the radius of gyration (Rg) and Dmax with longer carbamylation time.
Conclusion
These data indicate carbamylation of albumin results in an altered structure that mediates reduced binding to cubilin and FcRn. This results in reduced proximal tubule uptake and transcytosis of albumin resulting in more rapid serum clearance and lysosomal accumulation of carbamylated albumin.
Funding
- NIDDK Support