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Autosomal Dominant Polycystic Kidney Disease (ADPKD) is characterized by the development of numerous fluid-filled cysts in the kidneys of patients. We recently published our description of the proteome of renal cyst fluid in ADPKD. As a follow-up experiment, we hypothesized that the protein-bound subfraction consists of molecules of mechanistic or diagnostic interest in ADPKD. Using a manual biomarker enrichment kit, we have identified 44 distinct proteins in human cyst fluid.
Mutations in the gene that codes for the protein polycystin result in the multi-organ disease Autosomal Dominant Polycystic Kidney Disease (ADPKD). This genetic disorder causes renal failure in adults secondary to polycystic kidneys and associated renal fibrosis . We are characterizing the proteome of cyst fluid in polycystic kidney disease (PKD) to define proteins of mechanistic or diagnostic importance in this disease.
Our previously published experiments  with human renal cyst fluid identified over 390 proteins. A higher yield was obtained using the ProteoPrep® 20 (Sigma-Aldrich) immunodepletion kit to remove albumin (ALB) and 19 additional high abundance proteins present in the cyst fluid, thereby enhancing the ability to detect lower abundance proteins. Since ALB and other carrier molecules bind proteins and peptides in the serum  we hypothesized that the protein-bound subfraction of renal cyst fluid would contain proteins of pathophysiologic interest in PKD. A recent paper by Gundry  highlights the plethora of proteins that bind to carrier molecules in human sera. Using the ProXPRESSION™ Biomarker Manual Enrichment kit (PerkinElmer), we sought to define the protein-bound fraction of cyst fluid. The kit contains Cibacron Blue, a nonselective ALB-binding dye. Although the enrichment kit is designed to bind ALB, we understand from the manufacturer (Vivascience) that the column may bind other proteins to a lesser degree, therefore we describe our identified proteome as “biomarker enriched” rather than “ALB-bound” .
Our cyst samples were identical to the four samples (CF02–CF05) used in the previously published experiment . These de-identified human samples are cyst fluid collections that were managed according to the bioethical recommendations of the Institutional Review Board. The cyst fluid was aspirated postoperatively from excised kidneys in patients with end-stage renal disease.
Since the binding capacity of the Vivapure™ Blue column in the ProXPRESSION™ kit is 4 mg, we collected a calculated volume of cyst fluid that would result in approximately 3 mg of protein entering the column. Cyst fluid was centrifuged and the calculated volume of super-natant was diluted 1:10 with binding buffer. After loading the diluted supernatant onto a Vivapure™ Blue spin column, the column was washed three times with binding buffer to remove unbound proteins. The column contains a patented chromatography membrane. This membrane combined with the proprietary buffer and elution chemistries reproducibly elutes peptides and proteins bound to carrier proteins. To elute the peptides, 200 μL of elution buffer was introduced onto the Vivapure™ columns. The column was centrifuged at 250 × g for 2 min and the elution step was repeated per the ProXPRESSION™ kit instruction manual.
To prepare samples for analysis by tandem MS, we performed a Bradford assay to determine the concentration of each eluent. Using 6 M urea we diluted the sample to reach 200 μL of total volume, resulting in an approximate protein concentration of 500 μg/mL per sample. Next 200 μL of a reduction/alkylation cocktail was introduced into each sample; this cocktail contains ACN, iodoethanol, and triethylphosphine. After 90 min incubation the samples were vacuum dried overnight. Each sample was resuspended in NH4HCO3 and 150 μL of a 20 μg/mL sequence grade trypsin (Princeton Separations) solution was added prior to incubation at 37°C. After 3 h another 150 μL of trypsin was added and the sample incubated overnight.
The samples were analyzed in replicate using a Thermo-Finnigan linear ion-trap mass spectrometer with a Surveyor autosampler and MS HPLC system (Thermo-Finnigan). The scan was completed in “Triple Play” (MS scan, Zoom scan, and MS/MS scan) mode. The acquired data was searched against the international protein index (IPI) human database (ipi.HUMAN.v3.34) using SEQUEST (v.28 rev.12) algorithms in Bioworks (v.3.3). Parameters for the search were set to: fragment ion tolerance 1.0 amu, peptide tolerance 2.0 amu, enzyme limits set as “fully enzymatic – cleaves at both ends”, and cleavage sites set at 2. These search results were validated using PeptideProphet  and ProteinProphet  in the trans-proteomic pipeline (TPP, v. 3.3.0) (http://tools.proteomecenter.org/software.php). The identified proteins were entered into the Pathway Studio© 5.0 software for analysis (Fig. 1).
We identified 142 proteins from the biomarker-enriched subfraction that had at least 90% confidence via TPP. Of these, 44 proteins were found to be (i) common to at least two cysts and (ii) distinct, meaning that each identified protein had at least one unique peptide (Table 1). The table outlines how many cysts each protein was identified in, ranging from 2/4–4/4 cysts. Ten of these proteins (bold font) were identified only in the biomarker-enriched sample, suggesting a highly protein-bound state in cyst fluid. We hypothesize that many of the proteins identified in both experiments are fluctuating between a protein-bound and unbound state. A Supporting Information Table lists these 44 proteins along with the peptide sequences used for identification and their probability. Proteins of interest include apolipoprotein A-1 (APOA-1), complement C3, pigment epithelium-derived factor (PEDF), along with many others. APOA-1 has been found to be overly expressed in previous studies of PKD in rodent models . Complement C3 and other innate immunity components have recently been implicated in the pathogenesis of cysts in ARPKD, and the authors believe there is a similar pathogenetic mechanism in ADPKD . PEDF appears to be highly protein-bound and its presence in all sample injections in this study has prompted us to propose experiments studying this protein's effect on cyst growth in vitro. The Supporting Information Table can be found on our website (http://anatomy.iupui.edu/mason/pkd-proteomics.htm) or at the online repository PRIDE (www.ebi.ac.uk/pride).
Figure 1 displays the results of a Pathway Studio® analysis (www.ariadnegenomics.com) conducted to assess potential functional significance of the detected proteins. Pathway Studio® finds common regulators and associates pathway components with like-behaving biological entities and processes. Protein identification numbers (Swiss-Prot) are submitted and a flow diagram is produced demonstrating protein interactions and pathways of interaction. It is noted that APOA-1 and antithrombin III (SERPINC1) are known to complex with ALB . The most notable interactions are between plasminogen (PLG) and several other identified proteins. These interactions do not appear to be specific to the pathology of PKD.
We compared our renal cyst subfraction to the ALB-enriched serum sample as defined by Gundry et al. . Our study identified 44 whereas Gundry identified 120 distinct proteins. Interestingly, our sample contains eight unique complement components (redundant isoforms and precursors excluded) as opposed to seven in the Gundry serum sample (clusterin is included as a complement component). An enrichment of these innate immunity proteins in cyst fluid relative to the Gundry serum sample is interesting. We hypothesize that this enrichment of complement components in our subfraction is characteristic of PKD, and corroborates the recent finding of Mrug et al. in the cpk mouse model of recessive polycystic disease. In that study, complement C3, adipsin (factor D), clusterin, and ten other complement component genes are overexpressed in cystic renal tissue . We have confirmed the presence of complement C3 and factor D in this subfraction of ADPKD cyst fluid.
In our previous paper , we had hypothesized that epidermal growth factor (EGF) would be identified in the biomarker enriched subfraction of cyst fluid. However, EGF was not identified in this experiment. A likely explanation lies in the severity of the renal disease in our patient samples. Since these cyst fluid samples were obtained at the time of dialysis initiation, all surgically excised kidneys were necessarily from end-stage disease patients. Weinstein et al. found low urinary excretion of EGF in ADPKD patients at an early stage in the disease . It has been shown that urine EGF virtually disappears in chronic renal failure patients .
This study follows our characterization of the renal cyst fluid proteome . We have identified the biomarker-enriched proteome of ADPKD fluid. We identified 44 distinct proteins; including molecules of mechanistic interest, such as APOA-1, PEDF, and complement C3. The relative enrichment of protein-bound complement components highlights a difference between the serum and cyst fluid proteomes. Future studies defining the role of innate immunity components in cystogenesis and cyst growth could lead to therapeutic intervention in PKD.
We thank David Hong for assisting with the operation of the mass spectrometer.
The authors have declared no conflict of interest.