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. 2018 Jul 15;10(7):2115-2125.
eCollection 2018.

Urinary proteomics analysis based on mass spectrometry and identification of therapeutic targets of Shenkangling interventions in rats with adriamycin nephropathy using iTRAQ

Si Ai  1   2 , Jian Zheng  1   3   2 , Cai-Xia Qiu  1 , Xiao-Lu Lu  1 , Xu-Wei Li  1
Affiliations

Urinary proteomics analysis based on mass spectrometry and identification of therapeutic targets of Shenkangling interventions in rats with adriamycin nephropathy using iTRAQ

Si Ai et al. Am J Transl Res. .

Abstract

Objective: The isobaric tags for relative and absolute quantification (iTRAQ) technique for proteomic analysis was employed to identify diagnostic markers and therapeutic targets of Shenkangling intervention or prednisone tablets in rats with adriamycin nephropathy (AN).

Methods: Fifty healthy, clean-grade Sprague-Dawley rats were selected, with 10 rats in the normal group and the remaining 40 rats receiving a tail vein injection of 5.5 mg/kg of adriamycin (ADR) to induce AN. Treatment began 1 week later. The normal group received gastric administration of normal saline. Forty rats with induced AN were further randomly divided into the AN modeling group (n = 10), AN modeling + prednisone treatment group (n = 10), AN modeling + Shenkangling intervention group (n = 10), and AN modeling + prednisone + Shenkangling intervention group (n = 10). iTRAQ was employed in combination with mass spectrometry to analyze the differentially expressed proteins in the urine after 3 weeks of treatment (in the fourth week of the experiment).

Results: Compared with normal rats, AN rats had 6 down-regulated proteins and 1 upregulated protein. Compared with AN rats, prednisone rats had 2 down-regulated and 6 upregulated proteins. Compared with AN rats, combined treatment rats had 2 down-regulated and 8 upregulated proteins. Compared with the AN model group, the Shenkangling treatment group had 3 down-regulated and 9 upregulated proteins. Gro, Afamin, Cystatin-related protein 2, Afamin, and isoform CRA_a were considered diagnostic markers of primary nephrotic syndrome (PNS). Telomerase was considered the therapeutic target of prednisone. Urinary protein 2, Apolipoprotein A-II, 45 kDa calcium-binding protein, Vitronectin, and Osteopontin were the therapeutic targets of the Shenkangling intervention. Afamin, isoform CRA_a, Apolipoprotein A-IV, Coagulation factor XII, Prolactin-induced protein, and Coagulation factor XII were the therapeutic targets of the Shenkangling intervention combined with prednisone.

Conclusion: The feasibility of urinary proteomics analysis in rats using a large number of proteins with finite molecular weights is controversial. The markers screened in this study may be of clinical value for the diagnosis and treatment of nephropathy. However, these findings should be confirmed in future cohort studies.

Keywords: Shenkangling intervention; adriamycin nephropathy; iTRAQ; prednisone; proteomics.

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Conflict of interest statement

None.

Figures

Figure 1
Figure 1
Urine protein concentrations (mg/day, mean ± SD) in the normal group, AN model group, Shenkangling intervention group, prednisone treatment group, and combined treatment group at weeks 1, 2 and 3 after modeling.
Figure 2
Figure 2
ALB and CHOL levels at the fourth week of modeling. Blue indicates the normal group, red the AN model group, green the Shenkangling intervention group, purple the prednisone treatment group, and light blue indicates the combined treatment group.
Figure 3
Figure 3
A: PI distribution. B: MW distribution. C: Residue number distribution. D: GRAVY value distribution.
Figure 4
Figure 4
GO term distribution; from left to right, the X axis reflects biological process, cellular component, and molecular function. The Y axis represents the number of functional proteins. Note: (1) GO: Gene Ontology; BP: Biological Process; MF: Molecular Function; CC: Cellular Component; (2) Go slim was used by UniProtKB-GOA goslim (N. Mulder, M. Pruess PMID:12230037 Nov 2002).
Figure 5
Figure 5
Significant enrichment of identified proteins to the corresponding pathways; curves were plotted using the negative logarithm with 10 as the base for the pathways and higher-level pathways.
See this image and copyright information in PMC

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