Differential levels of CHMP2B, LLPH, and SLC25A51 proteins in secondary renal amyloidosis

ABSTRACT

Objectives

Renal amyloidosis (RA) is a rare disease, typically manifested with proteinuria, nephrotic syndrome, and ultimately leads to renal failure. The present study aims to profile the proteomes of renal amyloidosis patient’s serum and healthy controls, along with relative quantification to find out robust markers for RA.

Methods

In this study, 12 RA patients and their corresponding age and gender-matched healthy controls were recruited from the Nephrology department of Max Super Specialty Hospital, New Delhi. We employed gel-based proteomic approach coupled with MALDI-TOF MS to compare protein expression patterns in RA patients and controls. Furthermore, validation of differential proteins (selected) was done using bio-layer interferometry.

Results

Eleven proteins showed remarkably altered expression levels. Moreover, expression modulation of three proteins (LLPH, SLC25A51, and CHMP2B) was validated which corroborated with two-dimensional gel electrophoresis (2-DE) results showing significant upregulation (p < 0.05) in RA patients followed by ROC analysis which demonstrated the diagnostic potential of these proteins. A protein-protein master network was generated implicating the above identified proteins along with their interactors, fishing out the routes leading to amyloidosis.

Conclusion

This study indicates that the identified serum proteomic signatures could improve early diagnosis and lead to possible therapeutic targets in RA.

KEYWORDS:

• 2D gel electrophoresis
• bio-layer interferometry
• serum biomarkers
• early diagnosis
• pathogenesis
• MALDI-TOF MS

Authors contribution

N. Gupta collected the samples and related clinical information; N. Gupta, T. Sahar, S. Khowal, I.A. Ganaie, M. Mughees, and S. Wajid conducted the experiments; N. Gupta, T. Sahar, S. Khowal and S. Wajid tabulated and analyzed the data; N. Gupta and T. Sahar did statistical and bioinformatics analysis; N. Gupta and T. Sahar wrote the manuscript; D. Khullar was the co-investigator and clinical collaborator of the study; S.K Jain was the co-investigator of the study and reviewed the manuscript; S. Wajid designed and conceptualized the study, provided research setup/instrumentation facilities, interpreted the data, was the principal investigator of the study, reviewed and finalized the manuscript.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Abbreviations

AA: Amyloid A; Aβ: Amyloid β; ATTR: Amyloidogenic Transthyretin; APP: Amyloid precursor protein; AL: Light chain amyloidosis; ALS: amyotrophic lateral sclerosis; °C: degree Celsius; DTT: Dithiothreitol; 1DE: One-dimensional electrophoresis; 2DE: Two-dimensional electrophoresis; EDC: 1-ethyl-3-dimethylaminopropyl carbodiimide; ESCRT: Endosomal sorting complex required for transport pathway; ESRD: End-stage renal disease; IPG: Immobilized pH gradient; KD: Kilo Dalton; kg: Kilogram; MALDI-TOF MS: Matrix-assisted laser desorption ionization time of flight mass spectrometry; mg: Milligram; mins: minutes; mL: milliliter; mM: milli molar; µL: microliter; µM: micro molar; NCBInr: National Centre of Biotechnology nonredundant; NHS: N-Hydroxysuccinamide; PBST: Phosphate-buffered saline with tween; p.m.t: Peptide mass tolerance; PMF: Peptide mass fingerprinting; RA: Renal Amyloidosis; RT: Room temperature; SD: Standard deviation; SSP: Standard Spot Parameter; SDS: Sodium dodecyl sulfate; TTR: Transthyretin

Supplementary material

Supplemental data for this article can be accessed here

Additional information

Funding

This was supported by grants from the Indian Council of Medical Research (ICMR), Government of India, Major Research Project grant numbers: 5/4/7-8/2012/NCD-II (to S. Wajid), 3/1/2(4)/Nephrology/2019-NCD-II (to N. Gupta), and 45/25/2018-NAN/BMS (Research Associate Fellowship, to M. Mughees). This paper was also supported by fellowships from the Government of India, University Grants Commission: 22/12/2013(ii)EU-V (to T. Sahar), F.4-1/2006(BSR) 7-357/2011(BSR); 22 October 2013 to (S. Khowal), and F. No. 7-357/2011(BSR) (to I.A. Ganaie).