Urine Proteomics Revealed a Significant Correlation Between Urine-Fibronectin Abundance and Estimated-GFR Decline in Patients with Bardet-Biedl Syndrome Open Access

Kidney dysfunction is a well-known clinical feature of BBS patients; however, its pathogenesis is still largely unknown. The syndrome has a genetic origin, and to date 21 genes have been associated with human BBS but it is presumable that other unknown genes are yet to be discovered [1]. Even though many efforts have been devoted to address genotype-phenotype correlation, there is still little information on this issue [2-4].

Renal abnormalities in BBS patients are both structural and functional. The majority of BBS patients show structural defects of the kidney and the urinary tract [5]; kidney dysfunction ranges from renal hyposthenuria to the end stage renal disease (ESRD) [6-9]. Whether the defective ability to concentrate the urine is an early marker of kidney dysfunction is currently unknown [10-13]. The molecular basis of kidney dysfunction is poorly understood. The BBS genes encode proteins that are mainly localized to the centrosome and regulate the function of the primary cilium (PC) [14]. Several studies have linked the disruption of cilia homeostasis with defects in renal development and cysts formation, thus BBS is considered the result of ciliary dysfunction and is included in the group of diseases named ciliopathies. Mouse models of BBS have shown that BBS2, BBS4 and BBS10 depletion lead to systemic-related renal disease [15, 16]. In addition, in vitro studies have shown that BBS10 is implicated in the mechanisms mediating water re-absorption along the collecting duct (CD) [17, 18]. The role of BBS genes in human kidney disease is largely unknown. In the recent years, urinary proteomic investigation has been applied to clinical medicine, allowing identification of up- or down-regulated proteins, that may help to clarify the pathophysiology of kidney dysfunction and to discover disease biomarkers [19]. The present study is the first investigation on urine proteome in BBS patients. Our results demonstrate that these patients have a unique urine protein profile. In addition, the correlation between altered proteins and kidney dysfunction may shed light in understanding the patho-physiology of renal disease and may help to predict renal outcome.

This study was performed in adherence to the Declaration of Helsinki and was approved by Institution Review Board. All patients or their legal representatives gave their written informed consent. A group of 14 adult individuals with a clinical diagnosis of BBS according to Beales criteria [5] were enrolled in the study. Retinal degeneration was assessed by ophthalmological testings. Obesity was defined as body mass index over 30 kg/m². Learning disabilities included defects in writing, reading, speaking, besides deficits of memory and coordination. The patients underwent biochemical analyses to address renal function; in addition, kidney ultrasound was performed. The GFR was estimated with CKD-EPI formula using standardized serum creatinine measurement, a procedure of creatinine testing calibrated to a traceable reference method (Isotope Dilution Mass Spectrometry or IDMS). Mean annual decline in GFR was estimated by the ratio between retrospective data indicating changes in GFR and follow-up duration (recent eGFR–eGFR at initial visit)/follow-up duration (years). Mean follow-up period was 4 years (3-7 years).

All patients underwent a screening for diabetes mellitus by measuring fasting serum glucose (FPG) and glycated hemoglobin (HbA1c). A repeated FPG ≥126 mg/dl (7.0 mmol/l) and/or HbA1c levels of 6.5% or greater were considered diagnostic of diabetes. Hypertension was defined as a systolic blood pressure of 140 mm Hg or more, or a diastolic blood pressure of 90 mm Hg or more.

Patients with an eGFR lower than 55mL/min/1.73m², with chronic infection and/or malignancy were excluded. Twenty healthy volunteers, age- and gender-matched (ten females and ten males) were used as controls.

Urine samples were collected as described [20]. After overnight fasting, the second void urine sample was collected from each patient and control and immediately centrifuged at 2000 rpm for 10 min. Urine creatinine concentration was measured by the analytical BM/Hitachi 904 as described. Urine samples from healthy volunteers were pooled in order to obtain two gender-specific control groups, composed of equal urine volumes from each subject. Urinary creatinine concentration was measured in samples from each single control-subject and in pooled controls. Small molecules and salts were removed by ultra-filtration and subsequent precipitation steps.

Samples were 50-fold-concentrated using a 3kDa cut-off ultrafiltration unit (3000 NMWL, Amicon® Ultra-15 Centrifugal Filters, Merck Millipore, Tulla green Carrigtwohill, Co. Cork, Ireland) at 3000 rpm for 60 min. The recovered urine was precipitated adding cold acetone and stored at -20°C overnight. Pellets were then separated from supernatant, air-dried and re-suspended in a buffer solution consisting of 7M Urea, 2M Thiourea, 4% Chaps, 30mM Tris-HCl pH=7, 8. Two independent 10% SDS-preparative gel (16 x 20 cm) were used for female and male groups, respectively. Protein extracts from patients and control pools were resolved on a 10% 1D-SDS-polyacrylamide gel and stained as previously described [21, 22]. Each lane was cut, digested with trypsin and analyzed by LC–MS/MS [23, 24].

The peptide mixtures were analyzed by LC–MS/MS using a Q-ExactiveTM (ThermoScientific, Bremen, Germany) coupled to an UltiMate 3000 RSLCnano LC system (ThermoScientific), including a nano-pumps binary system and a third low-pressure micro-pump. The peptide mixtures were concentrated on a trapping pre-column (Acclaim PepMap C18, 300µm × 5 mm nanoViper, 5 µm, 100 Å, Thermo Scientific), using 0.05% formic acid and 2% acetonitrile. The peptide separation was performed using a C18 column (Acclaim Easy Spray PepMap RSLC C18, 75 µm × 15cm nanoViper, 3µm, 100 Å, Thermo Scientific). Peptides were eluted with a nonlinear gradient set as follows: 4% B for 5 minutes, from 4 to 40% B in 45 minutes and from 40 to 90 % B in 1 minute at flow rate of 300nL/min. A and B mobile phases were 0.1% formic acid and 80% acetonitrile, 0, 08% formic acid respectively. The mass spectrometer Q-ExactiveTM was setup in a data dependent Full MS/ddMS2, enabling the acquisition of MS/MS spectra for the ten most intense precursor ions (top ten) and dynamic exclusion of 30 sec. Resolution was set to 70000 for MS spectra acquisition and 17500 for MS/MS spectra acquisition.

Proteomic analysis was performed using ThermoProteomeDiscoverer^TM platform (version 2.1.0.81; ThermoScientific), interfaced to the Sequest HT Search Engine server (University of Washington, United States) for protein identification [25]. Proteins identified by a minimum of two peptides were accepted. Only proteins identified in at least 70% of patients (5/7) underwent further analyses. The spectral counting approach was used to compare protein expression profile in patients versus control groups [26, 27]. The relative protein abundance was evaluated by the SpC log ratio (R_SC) [28]. To ensure the quantitative accuracy, two methods were used. To calculate each protein Normalized Spectral Abundance Factor (NSAF), the number of spectral counts (SpCs) was divided by protein length (SAF) and normalized to the sum of SAFs in a given lane. The NSAF of each ‘i’ protein was then normalized against urine creatinine concentration (cNSAFi). The NSAF of each protein from the control pools was normalized by urine creatinine concentration (cNSAF_poolfemale and cNSAF_poolmale). Finally, to each ‘i’ protein the R_NSAF was calculated as the binary logarithm of a ratio of cNSAFi versus cNSAF_pool.

The dataset of differentially represented proteins was analyzed using ‘STRING: functional protein association networks’ software 7.0 (http://string-db.org/) [29, 30].

The statistical analysis were carried out by means of GrapfPadPrism software. The protein fold changes, expressed as R_NSAF, were correlated with eGFR and annual eGFR (∆eGFR) using Pearson Correlation test. Differences were considered statistically significant for p-values  < 0.05.

Fourteen adult BBS patients with clinical diagnosis of BBS have been selected for the study. The main features are listed in Tables 1 and 2. Patients were aged 19-43 years old; all patients with the exception of one had retinal dystrophy; BMI ranged between 23 and 43.7 kg/m². Polydactyly and learning disabilities were present in10/14 and 9/14 patients respectively; diabetes was present in only 1 male and 1 female patient. 2 male patients (and no any female patient) were hypertensive. Renal structural abnormalities on ultrasound were present in 10/14 patients, with calyceal and parenchymal cysts, unilateral renal hypoplasia, ureteric tract malformation being the most common defects. Protein to creatinine ratio (PCR) was over 150 mg/g in one male patient. The estimated GFR ranged from 55 to 130 mL/min/1.73m². Annual GFR decline varied among subjects; interestingly, the majority of patients showed a progression of kidney disease.

Two independent gender comparative analyses were performed. Urine proteomes of 7 female and 7 male BBS subjects were compared with the proteome of the gender-matched control pool. Urinary proteins were resolved on a 10% 1D-SDS-polyacrylamide gel as shown in Fig. 1. Gel bands were cut and proteins identified by LC-MS/MS. The analysis revealed a dataset constituted by 73 and 66 proteins in female and male patients, respectively. The details of protein identification is reported in supplementary Tables 1S and 2S. For all supplemental material see www.karger.com/doi/10.1159/000488096.

Label-free proteomic analysis was performed to compare proteome composition between patients and controls. In order to estimate the differences in urine proteome, the relative abundance of each protein was calculated by two spectral counting parameters, R_SC and R_NSAF. Female and male datasets are reported in Table 3 and Table 4, respectively. Fig. 2 shows the urine protein abundance (fold changes) based on Rsc and R_NSAF in female (Fig. 2A) and male (Fig. 2B) BBS individuals. The results were similar in both conditions, confirming the reliability of the quantitative analysis of this study. Female dataset showed 23 over-represented proteins and 42 under-represented proteins compared with the control pool. Male dataset showed 27 over-represented proteins and 36 under-represented proteins compared with the control. 42 proteins were de-regulated in both male and female BBS individuals compared with controls (Table 5). Fig. 3 shows that the majority of these proteins had the same behavior in both female and male BBS; however, some of them showed an opposite abundance in males and females, thus suggesting an inter-gender variability.

Urinary extracellular matrix organization (ECM) proteins and cell adhesion molecules (CAM) were highly deregulated in BBS patients

Protein association networks were analyzed using STRING database (http://string-db.org/). Proteins were grouped according to the biological and functional connections found by Gene Ontology (GO) Database (Fig. 4). The most representative biological processes were cell adhesion (13 nodes, 0.00513 FDR) and extracellular matrix organization (8 nodes, 0.00776 FDR). The network distributions of the differentially represented BBS urinary proteins revealed the central role of Fibronectin.

We verified whether urine proteins that were over or under-represented in BBS patients showed any correlation with the eGFR. Statistical analysis revealed that urine FN, CD44 antigen and lysosomal alpha-glucosidase were significantly correlated with the eGFR, as shown in Fig. 5. Interestingly, also vitronectin, IgK chain V, Ig lambda chain V and Isoform 2 of Tyrosine-protein phosphatase non-receptor type substrate 1 (SIRP-α) were significantly correlated with the eGFR in each group of female and male patients, but not when we considered the cohort in its totality (supplementary Table 3S).

Urine proteome of BBS patients was correlated with retrospective data indicating the rate of annual change in the eGFR (ΔeGFR). The rationale of the study was to address whether patients undergoing a faster progression of renal dysfunction may show a specific set of urine proteins. This analysis revealed that urine FN was significantly correlated with the ΔeGFR. This finding is consistent with the correlation of FN with the eGFR, further supporting the hypothesis that it may be considered as a marker of kidney disease (Fig. 6).

This is the first study to our knowledge that analyzes urine proteome in a population of adult BBS patients and correlates the spectrum of urinary proteins with renal function.

There is no doubt that urine proteomics is an emerging field that has provided new insights in the understanding of the patho-physiological mechanisms of renal and extra-renal diseases [31]. Urine is a biological fluid that can be easily collected in a non-invasive manner, and it is an important source of renal and extra-renal proteins and peptides [19]; this has raised much attention on the potentiality of this tool in the discovery of disease biomarkers [32].

Given the large genetic heterogeneity of BBS, laborious genetic tests are required for a molecular screening of patients. However, to date the diagnosis is mainly based on clinical findings [5]. Proteomics screening may be potentially of help in (I) identifying disease-specific biomarkers, useful for a more accurate diagnosis, and (II) in identifying the subset of patients at high risk of developing kidney disease, with a great impact on patients care.

The present study has investigated urine proteomic profile in adult BBS patients in comparison with age and sex-matched healthy volunteers. The results have been then stratified based on the eGFR, to address whether some classes of proteins correlated with kidney dysfunction. Finally, urine proteome of BBS patients has been correlated with retrospective data indicating the annual rate of eGFR decline.

The study shows that BBS patients have a particular urine proteome, differing from that of normal subjects. Fourtytwo proteins were deregulated in both male and female patients compared with control; the analysis of protein association networks revealed two networks consisting of 8 and 13 nodes respectively, extracellular matrix organization (ECM) and cell adhesion.

The association between ECM proteins and CKD has been already described in literature [33]. The putative underlying mechanism is the imbalance between ECM synthesis and degradation with subsequent fibrosis, a general pathophysiological mechanism in all types of kidney disease, independently of the initial injury [34]. Recently, Hijmans et al. have demonstrated that components of ECM proteins are detectable in the urine before the histological evidence of renal fibrosis, and have indicated urinary collagen degradation markers as potential diagnostic and predictive biomarkers of renal fibrosis [35]. In our study, ECM proteins showed different urinary abundance in BBS patients compared with controls, even in the absence of GFR decline. As all BBS patients had renal abnormalities on ultrasound, it is possible that this finding may indicate these molecules as early markers of kidney defects, that proceed GFR decline. Longitudinal studies, as well as metabolomic analysis [34], will enable us to address whether this data may predict the progression of renal failure.

CAMs are critical for either cell-cell and cell-extracellular matrix interactions [36]. We found significant differences in urinary abundance of cadherins and integrins in BBS patients. The meaning of this association remains to be elucidated. In the best studied experimental model of ciliopathy, the autosomal dominant polycystic kidney disease (ADPKD), it has been largely demonstrated the role of polycistin-1 (PC-1) in cell-matrix and cell-cell interactions [37]. Recently, Castelli et al. have shown that PC-1 overexpression in cells leads to enhanced adhesion to the substrate, while PC-1 downregulation reduced this ability via microtubule cytoskeleton regulation [38]. It is now clear that PC-1 has also extra-cilia localization, raising the hypothesis that some features of ADPKD are based on cilia-independent cellular defects. The role of BBS genes in biological functions other than PC homeostasis has been suggested by some studies. Tobin et al. demonstrated that BBS8 knocking down in zebrafish caused defective neural crest migration [39]. Accordingly, Hernandez et al. have demonstrated that BBS4 and BBS6 null cells showed deficient migration and adhesion abilities, disruption of actin stress fiber formation and focal adhesions [40], indicating a possible role of BBS genes in cytoskeleton organization and related biological processes. In a recent study we showed that BBS10 silencing in a mouse cortical collecting duct-derived cell line led to a dramatic reduction of tubulin acetylation, associated with an impaired AQP2 trafficking to the apical membrane [9]. Whether other apical channels and/or transporters are affected remains to be elucidated [41, 42]. The molecular mechanisms linking BBS genes to cytoskeleton organization and its role in the pathophysiology of BBS defects requires additional studies. We may speculate that cytoskeleton defects can cause aberration in ciliogenesis and signaling pathways, that eventually lead to kidney and other organs defects.

The analysis of urine proteome profile with respect to the eGFR demonstrated a significant increased abundance of fibronectin, CD44 antigen and lysosomal alfa glucosidase when the GFR declines. Fibronectin is a component of the extracellular matrix, whose deposition is believed to lead to glomerular and tubular basement membrane thickening and ultimately to glomerulosclerosis and interstitial fibrosis in almost all kidney diseases [43]. The correlation between its abundance and the eGFR strongly supports the hypothesis that it may reflect renal pathology.

CD44 antigen is a transmembrane protein undetectable or barely detectable in the normal kidney, that is expressed in the proximal tubule upon ischemic injury and in renal cell tumors [44]. In mice, it is expressed during T-cell mediated tubule-interstitial nephritis, and it is believed to play a role in cell repair through cell binding to the ECM [45]. Previous proteomics studies have identified the association between CD44 antigen and renal disease in several clinical conditions, including renal transplant rejection, IgA nephropathy and focal segmental glomeruloscherosis (FSGS) [46, 47].Our findings confirm that this protein correlates with renal dysfunction also in BBS. The increased urinary abundance of the lysosomal alfa glucosidase is more difficult to interpret. The protein has a crucial role in glycogen degradation to glucose, and its dysfunction due to mutations in the GAA gene leads to the glycogen storage disease type II (Pompe disease).The protein is largely expressed in the proximal tubule (PT). Whether the increased urinary abundance in patients with lower GFR is a generic marker of nephron disruption remains to be elucidated. The current data do not support a possible role of general or specific PT dysfunction in BBS patients, but further studies are needed to answer this questions [48].

A series of proteins, including vitronectin, Ig kappa chain V-III Isoform 2 and Tyrosine-protein phosphatase non-receptor type substrate 1 (SIRP-α) were significantly correlated with the eGFR separately in woman and men but not when we considered the whole cohort, maybe for the inter-gender variability [49, 50]; it is not excluded that they may be part of a panel of proteins that reflect kidney dysfunction.

Finally, urine proteomes were analyzed based on mean annual eGFR decline. Interestingly, the majority of BBS patients showed a progressive decline of the eGFR, suggesting that those patients are at high risk to develop kidney failure. Whether intrinsic factors related to the genetic disease or common risk factors, as hypertension, diabetes and obesity may play a role in GFR decline is currently unknown and is object of intensive investigations. In our cohort there is a little presence of diabetic patients, and only 2 of 14 patients were hypertensive; surely these conditions may play a role in kidney disease progression, as in general population, but the evidence of GFR decline in non hypertensive and non diabetic patients suggests that other factors may participate in the pathogenesis of GFR decline. Larger studies are needed to address whether obesity may play a more significant role.

Interestingly, our data demonstrate a significant correlation between fibronectin and ΔeGFR. This finding further confirms the importance of this protein as a marker of kidney disease.

Besides the proteins previously described, a series of other proteins have been found over or under- represented in the urine of BBS patients compared with controls, that may be of potential interest in the understanding the pathogenesis of renal and extra renal dysfunction. Some of those proteins are surely of renal origin. It is the case of Uromodulin, whose abundance is reduced in female BBS patients, confirming our previous data [6]. The protein is kidney-specific, and is the most abundant tubular protein in normal urine [51, 52]. Recently, much attention has been paid on its urinary levels given the correlation with incident CKD and kidney disease progression [53]. Other proteins may derive from the blood, considering that whole urine samples have been used in our study. Even though we focused on kidney disease, some of our findings may shed light on extra-renal dysfunctions of BBS patients. Male BBS individuals showed a reduced urine excretion of the lysosomal enzyme N-acetylglucosamine-6-sulfatase. Protein deficiency results in the accumulation of under-degraded substrate, in mucopolysaccharidosis type IIID (Sanfilippo D syndrome), a clinical condition characterized by intellectual disability and behavioral disturbances [54]. Whether this finding may have a correlation with learning disabilities, a common feature of BBS patients, is currently unknown. In addition, both male and female BBS patients showed a reduced urine excretion of the Zinc-alpha-2-glycoprotein (ZAG). This protein is implicated in multiple biological processes, including lipid mobilization, cell cycling, tumor proliferation, and transport of nephritic by-products [55]. It has been recently recognized as a negative regulator of fibrosis progression [56], and our finding may indicate a role in renal and/or extra-renal disease in BBS.

We are aware that the current study has a number of limitations. First, pooled urine was used for proteomics analysis of the controls, and this clearly limited the interpretation of data. Second, whole urine samples have been used as source of proteins in both patients and controls, as stated above, while the recent literature has shown the advantages of using exosomes in studying renal diseases, as exosomes extraction reduces the complexity of urine and ensures the renal and urinary tract origin of the found molecules. In addition, due to sample preparation, low molecular weight proteins could have been missed, limiting the information on the whole proteome profile. Moreover, even though the number of patients with low eGFR is limited, the absence of a control group of (early stages) CKD subjects does not allow a firm conclusion on the meaning of over and under- represented proteins, as this effect may be the result of impaired renal function in general. Finally, even if the disease is rare, the number of patients is quite limited, and a larger study is required to confirm the current findings and to address whether additional pathways may be identified in the subset of patients with kidney dysfunction. To this purpose, including BBS patients with more advanced renal disease will be of help in understanding whether derangement of metabolic pathways common to CKD from any cause (as oxidative stress and sulfur aminoacid metabolism [57]) or disease-specific biological processes underlie renal disease progression and its complications [58].

However, given the rarity of the disease, the correlations we found may serve as a pilot study in the discovery of urinary markers of kidney dysfunction.

Our data indicate the potentiality of urine proteomics in the discovery of early markers of eGFR decline in BBS. Larger prospective studies are required to confirm these findings. The possibility to assess the risk to develop chronic kidney disease will impact the management of BBS patients, enabling to start preventive strategies in time.

All authors declared no competing interests.

This work was supported in part by a grant from the Italian Association of Bardet-Biedl Patients (ASBBI).

M. Caterino and M. Zacchia contributed equally to this paper.