Abstract: PO0419
The Kidney Produces a Non-Polymerizing Form of Uromodulin That Associates with Reduced Risk of AKI
Session Information
- AKI: Repair and Progression
November 04, 2021 | Location: On-Demand, Virtual Only
Abstract Time: 10:00 AM - 12:00 PM
Category: Acute Kidney Injury
- 103 AKI: Mechanisms
Authors
- Micanovic, Radmila, Indiana University School of Medicine, Indianapolis, Indiana, United States
- LaFavers, Kaice A., Indiana University School of Medicine, Indianapolis, Indiana, United States
- Patidar, Kavish Rohit, Indiana University School of Medicine, Indianapolis, Indiana, United States
- Khan, Shehnaz, Indiana University School of Medicine, Indianapolis, Indiana, United States
- El-Achkar, Tarek M., Indiana University School of Medicine, Indianapolis, Indiana, United States
Background
Uromodulin (Tamm-Horsfall protein, THP) is a glycoprotein uniquely produced in the kidney. It is released by cells of the thick ascending limbs (TAL) apically in the urine, and basolaterally in the renal interstitium and systemic circulation. Processing of mature urinary THP, which polymerizes into supra-molecular filaments, requires cleavage of an external hydrophobic patch (EHP) at the C terminus. However, THP in the circulation is not polymerized, and it remains unclear if non-aggregated forms of THP exist natively in the urine. We propose that an alternative processing path, which retains the EHP domain, can lead to a non-polymerizing form of THP.
Methods
We have generated an antibody that specifically recognizes THP with retained EHP (THP+EHP). Liquid chromatography, mass spectrometry and C terminal sequencing were performed to delineate potential sites of enzymatic cleavage of immunoprecipitated THP+EHP. Immunofluorescence confocal microscopy was used to localize this isopform in the human kidney. Finally, we finally developed a customized ELISA to measure THP+EHP and used it on urine and plasma samples from a small cohort of patients hospitalized with liver cirrhosis.
Results
Using native and denaturing Western blots, we established the presence of THP+EHP in the urine in a non-polymerized native state. Proteomic studies confirmed the identity of THP+EHP and suggested that enzymatic cleavage occurred at Arg615, which is one amino acid beyond the GPI anchoring site. In the human kidney, THP+EHP predominantly co-localized with urinary THP in TAL cells, but it was also detected, at lower levels, in other tubular segments. In a cohort of patients with liver disease, admission urinary THP+EHP, but not total THP, was significantly lower in patients who subsequently developed acute kidney injury during hospitalization. THP+EHP was also detected in the plasma, albeit at very low concentrations.
Conclusion
Our findings uncover novel insights into uromodulin biology by establishing the presence of an alternative path for cellular processing. Our proof-of-principle findings in patients warrant further investigations to establish the utility of THP+EHP as a sensitive biomarker of kidney health and susceptibility to injury.
Funding
- NIDDK Support