Urinary Proteomics Yield Pathological Insights for Ureteropelvic Junction Obstruction

Abstract

Prenatal hydronephrosis is a common condition that may spontaneously resolve after birth. However, this condition can result in renal damage and requires surgical correction in a number of cases. Preventing renal damage is paramount, but existing diagnostic technology is invasive, exposes infants to radiation, is costly, and is often indeterminate. A better understanding of the pathophysiology of renal obstruction as reflected in the urinary proteome may provide new insights into the disease that could potentially alter the clinical management of hydronephrosis. We performed a quantitative proteomics study of urine that was surgically obtained from eight clinically significant, unilaterally obstructed infants versus eight healthy controls, with the goal of identifying quantitatively varying proteins and the biological networks associated with them. Notably, urine was obtained from both the obstructed kidney and the bladder. Over 1100 proteins were identified, and a total of 76 quantitatively varying proteins were identified. Proteins involved in oxidative stress, inflammation, and renal disease pathways showed the most significant abundance differences. This study gives a deeper understanding of the critical proteomic changes associated with renal obstruction and represents the deepest proteomic profile of renal obstruction to date.

Fig. 1.

An overview of the sample preparation in which samples from BU, KU, or CU were prepared according to the spin-filter-based one-step protocol (10). Albumin was depleted, protein amounts were determined, and proteins were digested with trypsin. After TMT labeling, two groups of three samples each were mixed and analyzed by LC-MS/MS.

Fig. 2.

Volcano plots of the KC (A) and BC (B) ratios determined for all proteins quantified. The KC ratios were shifted toward smaller Q-values and larger fold changes overall, suggesting that KU samples were more altered than BU samples, as expected. This highlights the value of analyzing urine directly from the obstructed kidney.

Fig. 3.

Fold-changes of statistically significant proteins in the KU and BU samples are plotted. Although many proteins have similar fold-changes in both samples, there are several entries that are more enriched in one sample. In addition, off-diagonal proteins are differentially expressed in each sample.

Fig. 4.

The putative biomarker panel shows many highly networked proteins associated with oxidative stress. Entries shown in blue were down-regulated, while entries in green were up-regulated. The orange entries were differentially regulated in BU and KU samples. Highly networked small molecules and a regulatory protein, NFE2L2 are also shown to highlight underlying relationships.

Fig. 5.

Western blotting validation of MS data. A representative blot is shown for (A) GSTM1, validating the increase in KU relative to CU observed by MS. In (B), validation of the observed increase of HSP70 in KU is shown. In (C), a bar graph showing the averaged changes of these two proteins in three samples by WB is shown.