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Cystinuric Renal Stones Linked to Fibrosis: Proteomics Based Evidence
Larisa Kovacevic, MD1, Hong Lu, PhD1, Joseph A. Caruso, PhD2, David S. Goldfarb, MD3, Yegappan Lakshmanan, MD1.
1Children's Hospital of Michigan, Detroit, MI, USA, 2Proteomics Facility Core, Detroit, MI, USA, 3NYU Langone Medical Center, New York, NY, USA.

Background: Cystinuria has significant morbidity due to large and recurrent kidney stones, and need for multiple procedures. Renal function in patients with cystinuric kidney stones is affected compared to those with non-cystinuric stones, but the cause of this decline in not entirely clear. Biopsies of the papillae performed in patients with cystine kidney stones show interstitial fibrosis. Therefore, preventive strategies and early diagnosis and treatment are crucial in this condition.
Objectives: We assessed (1) the differences in the urinary proteins between children with cystinuria and kidney stones (CYS), and healthy controls (HC), with particular attention to the fibrosis-related proteins, and (2) the presence of diagnostic biomarkers for CYS.
Methods: We compared urinary proteomes of 2 newly diagnosed children with CYS and 2 age- and gender-matched HC, using liquid chromatography-mass spectrometry (LC-MS/MS). Both CYS children had normal kidney function, and were on no CYS treatment. Relative protein abundance was estimated using spectral counting. Proteins of interest were selected using the following criteria: 1) ≥5 spectral counts; 2) ≥2-fold difference in spectral counts; and 3) ≤0.05 p-value for the Fisher’s Exact Test. Protein function was analyzed using the DAVID online bioinformatics resource, and Cytoscape was used to investigate the key nodes of unique proteins.
Results: Of the 623 proteins identified by proteomic analysis, 180 exhibited at least 2-fold difference between CYS and HC. Of those, 94 proteins were up-regulated in CYS, 26 of which were involved in response to wounding, 21 in inflammatory response, 18 in immune response, and 4 in cellular response to oxidative stress. 86 proteins were down-regulated in CYS, 26 of which were involved in cell adhesion. 140 proteins were found only in children with CYS, 33 of which met the selection criteria. Protein-protein interaction modeling of CYS unique proteins identified actin, vimentin, heat shock 70 kDa protein, inter-alpha-trypsin-inhibitor heavy chain, matrix metalloproteinase-9, and serum paraoxonase as the key nodes, proteins that are associated with fibrosis pathways (Table).
Accession Number*ProteinMW (kDa)No. of assigned peptides**No. of unique peptides**Sequence coverage
HSP70Heat shock 70 kDa protein7024/187/514/15
ITIH2Inter-alpha-trypsin inhibitor10633/8112/1127/16
MMP9Matrix metalloproteinase 97826/159/718/12
PON1Serum paraoxonase4023/749/1448/55

*Human; **Values presented in each patient
*** % of sequence that has been identified for each protein
Conclusion: We provide proteomic evidence of oxidative injury, inflammation, wound healing and fibrosis in children with cystinuria and kidney stones. We speculate that oxidative stress and inflammation may cause remodeling via actin and vimentin pathways, leading to fibrosis. Additionally, we identified ITIH2 and MMP-9 as potential diagnostic biomarkers and novel therapeutic targets in CYS. These unique proteins merit further investigation.

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