Proteomic analyses of urinary exosomes identify novel potential biomarkers for early diagnosis of sickle cell nephropathy, a sex-based study

Balamurugan Packialakshmi; Emily Limerick; Hans C Ackerman; Xionghao Lin; Sergei Nekhai; James D Oliver 3rd; Ian J Stewart; Mark A Knepper; Courtney Fitzhugh; Xiaoming Zhou

doi:10.3389/fphys.2024.1300667

Proteomic analyses of urinary exosomes identify novel potential biomarkers for early diagnosis of sickle cell nephropathy, a sex-based study

Front Physiol. 2024 Feb 15:15:1300667. doi: 10.3389/fphys.2024.1300667. eCollection 2024.

Authors

Affiliations

¹ Department of Medicine, Uniformed Services University of Health Sciences, Bethesda, MD, United States.
² Cellular and Molecular Therapeutic Branch, National Heart Lung and Blood Institute, Bethesda, MD, United States.
³ Physiology Unit, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, Rockville, MD, United States.
⁴ Department of Medicine, Howard University, Washington, DC, United States.
⁵ Nephrology Service, Walter Reed National Military Medical Center, Bethesda, MD, United States.
⁶ System Biology Center, National Heart Lung and Blood Institute, Bethesda, MD, United States.

Abstract

Sickle cell nephropathy (SCN) is a leading cause of morbidity and mortality in sickle cell disease (SCD). Early intervention is crucial for mitigating its effects. However, current diagnostic methods rely on generic tests and may not detect SCN until irreversible renal damage occurs. Therefore, specific biomarkers for early diagnosis of SCN are needed. Urinary exosomes, membrane-bound vesicles secreted by renal podocytes and epithelial cells, contain both common and cell type-specific membrane and cytosolic proteins, reflecting the physiologic and pathophysiologic states of the kidney. Using proteomics, we analyzed the proteomes of urinary exosomes from humanized SCD mice at 2 months (without albuminuria) and 4 months (with albuminuria) of age. Excretion of 164 proteins were significantly increased and 176 proteins was significantly decreased in the exosomes when mice developed albuminuria. Based on the relevance to SCD, chronic kidney disease and Western blot confirmation in mice, we analyzed protein abundance of heparanase, cathepsin C, α2-macroglobulin and sarcoplasmic endoplasmic Ca²⁺ ATPase-3 (SERCA3) in the urinary exosomes and urine of 18 SCD subjects without albuminuria and 12 subjects with albuminuria using Western blot analyses. Both male and female subjects increased or tended to increase the excretion of these proteins in their urinary exosomes upon developing albuminuria, but female subjects demonstrated stronger correlations between the excretion of these proteins and urine albumin creatinine ratio (UACR) compared to male subjects. In contrast, exosomal excretion of Tamm-Horsfall protein, β-actin and SHP-1 was independent of albuminuria. These findings provide a foundation for a time-course study to determine whether increases in the levels of these proteins precede the onset of albuminuria in patients, which will help determine the potential of these proteins as biomarkers for early detection of SCN.

Keywords: albuminuria; cathepsin C; chronic kidney disease; gender difference; heparanase; sarcoplasmic endoplasmic Ca2+ ATPase-3; sex difference; α2-macroglobulin.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was funded by the grant (MED-83-3923 to XZ) from the Collaborative Health Initiative Research Program between the National Heart Lung and Blood Institute and the Henry M. Jackson Foundation for the Advancement of Military Medicine. HA was funded by the Intramural Research Programs of National Heart Lung and Blood Institute and National Institute of Allergy and Infectious Diseases (HL006196, AI001150).