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Identification of Potential Megalin/Cubilin Substrates Using Extensive Proteomics Quantification from Kidney Megalin-Knockdown Mice

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Abstract

Megalin and cubilin, endocytic proteins present in the proximal tubule of the kidney, are responsible for reabsorbing filtered proteins from urine. Our hypothesis was that potential substrates of megalin/cubilin could be identified by examining urinary protein differences between control (WT) mice and kidney-specific megalin knockdown (KD) mice. Using the IonStar proteomics approach, 877 potential megalin/cubilin substrates were discovered, with 23 of these compounds representing known megalin/cubilin substrates. Some of the proteins with the largest fold changes in the urine between KD and WT included the known megalin substrates retinol-binding protein and vitamin D-binding protein. Of the total proteins identified as novel substrates, about three-quarters of compounds had molecular weights (MWs) below 69 kDa, the MW of albumin, and the remaining had higher MWs, with about 5% of the proteins having MWs greater than 150 kDa. Sex differences in the number of identified substrates occurred, but this may be due to differences in kidney megalin expression between both male and female megalin KD and WT animals, with the ratio of megalin between WT and KD being 2.76 and 2.14 for female and male mice, respectively. The top three ingenuity canonical pathways based on the urinary proteins in both female and male KD mice were acute phase response signaling, liver X receptor/retinoid X receptor activation, and intrinsic prothrombin activation pathways. In conclusion, analysis of urine samples from kidney-specific megalin KD and WT mice was found to be useful for the identification of potential endogenous substrates for megalin and cubilin.

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Abbreviations

DBP:

Vitamin D binding protein

FDR:

False discovery rate

FXR:

Farnesoid X receptor

IPA:

Ingenuity pathway analysis

KD:

Knockdown

LC–MS/MS:

Liquid chromatography mass spectrometry

LMW:

Low molecular weight

LXR:

Liver X receptor

MUPs:

Major mouse urinary proteins

MW:

Molecular weight

NPXY:

Asn-Pro-any amino acid-tyrosine motifs

PSM:

Peptide-spectrum match

PT:

Proximal tubule

RBP4:

Retinal binding protein

RXR:

Retinoid X receptor

SPR:

Surface plasmon resonance

WT:

Wild type

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Funding

Financial support by the Center of Protein Therapeutics, University at Buffalo (MEM).

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, NY, 14214-8033, USA

    Bei Zhao, Chengjian Tu, Shichen Shen, Jun Qu & Marilyn E. Morris

  2. New York State Center of Excellence in Bioinformatics and Life Sciences, 701 Ellicott Street, Buffalo, New York, 14203, USA

    Chengjian Tu, Shichen Shen & Jun Qu

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  1. Bei Zhao
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Contributions

Participated in research design: Morris, Qu, and Zhao.

Conducted experiments: Morris, Zhao, Tu, and Shen.

Contributed new reagents or analytic tools: Morris and Qu.

Performed data analysis: Morris, Tu, Shen, and Zhao.

Wrote or contributed to the writing of the manuscript: Morris, Qu, Tu, Shen, and Zhao.

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Correspondence to Marilyn E. Morris.

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Zhao, B., Tu, C., Shen, S. et al. Identification of Potential Megalin/Cubilin Substrates Using Extensive Proteomics Quantification from Kidney Megalin-Knockdown Mice. AAPS J 24, 109 (2022). https://doi.org/10.1208/s12248-022-00758-2

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