Proteomic analysis of inhibitory protein profiles in the urine of children with urolithiasis: Implication for disease prevention
Larisa Kovacevic, MD1, Natalija Kovacevic, MD2, Yegappan Lakshmanan, MD1.
1Children's Hospital of Michigan, Detroit, MI, USA, 2Henry Ford Hospital, Detroit, MI, USA.
Background: It is increasingly recognized that urine contains proteins that act in the urinary tract’s defense against crystallization of calcium salts and formation of stone. Therefore, the identification of these urinary proteins represents an important step for renal stone prevention. Using a proteomic approach, we aimed to screen for the presence of biomarkers that are down regulated in children with urolithiasis (RS) compared to healthy controls (HC). We hypothesized that RS and HC would display unique inhibitory protein profiles that could be used for comparative pathway analysis.
Methods: Prospective, controlled, pilot study of pooled urine from RS (N=30, 24 females, mean age 12.95±4.03 years) versus age- and gender-matched healthy controls (HC), using liquid chromatography-mass spectrometry (LC-MS/MS). Patient inclusion criteria consisted of age 5-18 years, history of renal stone, at least one 24-hour satisfactory urine collection, absence of hematuria or pyuria, and normal renal function. Children who were not toilet trained, or those with bladder stones, nephrocalcinosis, neuropathic bladder, major congenital bladder abnormality, active urinary tract infection, chronic kidney disease, previous major reconstructive bladder surgery, and significant cardiac, pulmonary, gastro-intestinal, and neurological problems were excluded. Relative protein abundance was estimated using spectral counting. The criteria for protein selection were: 1) patient/control abundance ratio of <0.5 as a threshold to be well above observed technical variations in MS experiments; and 2) ≤0.05 p-value for the Fisher’s Exact Test. Results were confirmed by ELISA testing. Statistical analyses were conducted with IBM SPSS® version 20.
Results: Overall, we identified 1813 proteins, with 1639 proteins found in children with urolithiasis, and 1396 proteins found in controls. Of those, 417 were found only in patients and 174 only in controls. Using the above mentioned criteria, 67 proteins were down-regulated in RS group, and 17 of those were significantly different (Table). Protein-protein interaction modeling of significant proteins identified syndecan-1 as the key node, a protein associated with adhesion pathways. ELISA analysis by subgroups showed statistically significant difference in the urinary excretion of osteopontin (5.1± 3.22 vs 14.1 ±9.5 ng/mg creatinine, p=0.046), and nearly significant difference in the urinary excretion of keratin (0.42± 0.54 vs 2.02 ± 2.8 ng/mg creatinine, p=0.090) between stone patients with hypocitraturia and controls.
Conclusions: Children with RS have a different urinary inhibitory polypeptide profile compared to HC. Decreased urinary excretion of these proteins indicates their inhibitory role in renal stone formation, especially of the adhesion phase. Lower concentrations of urinary osteopontin and keratin in patients with urolithiasis and hypocitraturia suggest their involvement in the pathogenesis of this disease. Further characterization of these proteins in a larger sample is imperative.
| Name | Accession Number | Assigned peptides [Patient - Control] | Ratio (Patient/ Control) | Fisher''s Exact Test (p-Value) |
| Actin, alpha cardiac muscle 1 | ACTC | 0/43 | 0 | < 0.00010 |
| Actin, cytoplasmic 1 | ACTB | 0/66 | 0 | < 0.00010 |
| Annexin A5 | ANXA5 | 0/5 | 0 | 0.033 |
| Keratin, type II cytoskeletal 6B | K2C6B | 0/21 | 0 | < 0.00010 |
| Serpin B4 | SPB4 | 0/25 | 0 | < 0.00010 |
| Syndecan-1 | SDC1 | 1/21 | 0.05 | < 0.00010 |
| Annexin A2 | ANXA2 | 5/28 | 0.18 | < 0.00010 |
| Annexin A1 | ANXA1 | 8/37 | 0.22 | < 0.00010 |
| Serpin B3 | SPB3 | 8/33 | 0.24 | < 0.00010 |
| Osteopontin | OSTP | 46/151 | 0.30 | < 0.00010 |
| Trefoil factor 2 | TFF2 | 12/35 | 0.34 | 0.00070 |
| Vasorin | VASN | 23/64 | 0.36 | < 0.00010 |
| Collagen alpha-1(III) chain | CO3A1 | 17/46 | 0.37 | 0.00023 |
| Cadherin-1 | CADH1 | 10/27 | 0.37 | 0.0046 |
| Granulins | GRN | 81/204 | 0.40 | < 0.00010 |
| Actin, cytoplasmic 2 | ACTG | 23/56 | 0.41 | 0.00019 |
| Cubilin | CUBN | 83/182 | 0.46 | < 0.00010 |
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