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Current Proteomics, 2020, 17, 88-94

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ISSN: 1570-1646
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The journal for current and in-depth reviews on proteomics

Proteomics Study in Urolithiasis Impact

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BENTHAM
SCIENCE

Manavi Jain1, Paramveer Yadav1 and Priyadarshini1,*

1
Department of Biotechnology, Jaypee Institute of Information Technology, Noida, India

ARTICLE HISTORY
Received: April 20, 2019
Revised: May 23, 2019
Accepted: July 09, 2019
DOI:
10.2174/1570164616666190722161823
Abstract: Urolithiasis, which is the presence of stones in the urinary tract, has long been linked with a
higher risk of causing chronic kidney diseases and associated illnesses, such as diabetes-affecting 12%
of the world population. This clinical condition arises due to the supersaturation of urine and altera-
tions in the expression of cellular and urinary proteins. The renal stone mineral composition has been
well understood and incorporated as a routine part of stone removal, however, the protein composition,
an essential fraction of the stone matrix has been inadequately understood and not adeptly established.
Stone proteomics consists of a number of techniques including crystal analysis using X-ray diffrac-
tometry and IR spectroscopy, sample purification, identification and characterization of proteins using
high throughput mass spectrometric methods. However, not many studies have utilized the data ob-
tained from these experiments to assign functional significance to associated identified proteins. Pro-
tein network analysis using bioinformatic tools such as STRING to study protein-protein interactions
will enable researchers to get better insight into stone formation mechanics. Hence, a comprehensive
proteomic study of kidney stone matrix will help in deciphering protein-crystal pathways generating
novel information useful for clinical application.

Keywords: Urolithiasis, X-ray diffractometry, IR spectroscopy, protein-protein interaction, string, protein network analysis.

1. INTRODUCTION affected by the anomalies in the urinary chemical constitu-

tion [6]. Based on various compositional experiments kidney
Urolithiasis is the term used to describe a condition in
stones are known to be a complex mixture of inorganic and
which the stones are developed within the urinary tract lead-
organic substances resulting in the formation of organic ma-
ing to health threats throughout the globe because of its fre- trix due to the trapping of urinary biomolecules within the
quent occurrence rates and also due to the absence of ade-
inorganic mineral layers at some stage during stone for-
quate cure. The stone formation may be a systemic disorder
mation [7]. About 75% of stones are principally made of
associated with metabolic syndrome [1] or it may also be
calcium oxalate crystals, but up to 50% of these comprises
closely associated with diabetes, hypertension and obesity
calcium hydroxyl phosphate (brushite or calcium hydroxyap-
with lower dependence on the gender gap and higher impli-
Current Proteomics

atite) in trace or greater amounts; 10-20% constitutes magne-

cation towards diet and environmental factors [2]. Urolithia-
sis affects 12% of the world population-all ages, sexes and
races-at some period during their lifetime by various stone
types [3, 4].
Urine, in physiological state, is supersaturated with salts
which in a normal individual does not lead to stone formation.
However, in a pathological condition, these supersaturated
salts cause ectopic biomineral crystallization eventually lead-
ing to stone formation. Different phases of stone formation are
nucleation, growth, and aggregation [5]. Large aggregates
remain in the kidney and interfere with the urinary system
process. The type of renal calculi, which may also vary in
size and shape, depends on the mineral composition which is
*Address correspondence to this author at the Department of Biotechnology,
Jaypee Institute of Information Technology, Noida, India;
Tel: 01202594211(0); E-mail: priya.juit@gmail.com
sium ammonium phosphate (struvite or triple phosphate); 5%
of urate; and 1-2% are composed of cystine [8, 9].
The organic matrix constitutes 2-5% of the total stone
weight and is significantly composed of proteins, lipids, gly-
cosaminoglycans and carbohydrates [7]. Several studies had
illustrated the crucial role played by kidney stone matrix
proteins in the process of crystallization. Proteins are the
large and complex biological molecules, which have an im-
portant role in the form of catalysts, cellular messengers or
structural elements and are needed for the proper functioning
of our body [10]. However, under certain biological envi-
ronments, these proteins influence the development of kid-
ney stones. They serve as stimulating or inhibiting agents in
the calculi formation [5, 9]. Various in-vitro studies have
revealed the presence of many proteins with such stones like
albumin, osteopontin, Tamm-horsfall protein, prothrombin
related proteins and uromodulin [5, 7, 11, 12]. Mostly, the

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Proteomics Study in Urolithiasis
studies are associated with the isolation, identification and
characterization of stone associated proteins but the func-
tional significance of these proteins remained unexplained
[13]. This review discusses the methods by which kidney
stones are analyzed, the matrix proteins are extracted and
identified, utilizing the obtained information to gain protein-
crystal interactions and aid in disease management as well as
preventing the recurrence. However, the basic mechanism
behind the adverse yet extraordinary effects of these matrix
proteins is not well recognized, which needs to be explored.
2. EPIDEMIOLOGY AND ETIOPATHOGENESIS OF
UROLITHIASIS
Prevalence of urolithiasis has changed in the last several
decades. Factors which commonly affect urolithiasis are age,
eating habits, geographic distribution, climate, lifestyle, drugs,
genetic and metabolism [14].
Urine is supersaturated in nature and this condition leads
to crystallization. The process of kidney stone formation
starts from crystal nucleation of supersaturated urine. These
crystal nuclei serve as nidus for the stone formation, which
further grows in size. These grown crystals aggregate to-
gether to form the kidney stone. We all have supersaturated
urine but only a few develop kidney stones. In healthy indi-
viduals, due to the presence of inhibitor molecules, these
crystals are flushed off from the urine. Kidney stone patients
lack these inhibitors and have a large number of promoters.
Biomolecules serve as inhibitor or promoter are proteins,
lipids and glycosaminoglycans [7, 14, 15].
3. CHEMICAL COMPOSITION OF RENAL CALCULI
Most abundant kidney stone is calcium oxalate followed
by calcium phosphate, uric acid and cysteine [14, 16]. Urine
is supersaturated with calcium, oxalate, phosphate, uric acid
or cysteine. Inorganic and organic composition of kidney
stone depends on the type of crystals and modulators present
in the urine.
Composition of drug-induced renal calculi is of two types.
First one is formed by drug or its metabolites, while the se-
cond one is formed by common composition. The cause of
later one is the metabolic effect of the drug on urinary pH and
high excretion of calcium, phosphate and citrate [17].
4. STONE COMPOSITIONAL ANAYSIS
Kidney stones comprise crystalline or inorganic phase and
the non-crystalline or organic phase. The inorganic compo-
nents comprise 90% of the stone and form the crystal struc-
ture. These include calcium oxalate, calcium phosphate, uric
acid, struvite and cystine [18]. Commonly, a compound mix-
ture of these crystalline constituents forms a stone. On the
other hand, progress in determining the matrix composition
has been slow partly due to its insolubility and partly be-
cause of its variable nature within and between two stones.
The organic phase of the stone matrix comprises the macro-
molecules, which are primarily found in the urine [19, 20].
Boyce characterized the organic matrix as 64% protein, 9.6%
non-amino sugars, 5% hexosamine, 10% bound water, and
the remaining as inorganic ash [21]. In addition to these,
Current Proteomics, 2020, Vol. 17, No. 2 89
lipids such as cholesterol, triglycerides have also been shown
to be a notable fraction of the matrix [22].
Stone mineral composition analysis is a necessary meas-
ure for determining the aetiology and the desired medical
intervention for the medication of the patient’s disease.
However, it is also essential for selecting an appropriate pro-
tein extraction method. Several biophysical techniques are
used for accurate renal stone analysis, such as infrared spec-
troscopy, X-ray crystallography, micro Computed Tomogra-
phy (micro-CT) and electron microscopy. These techniques
along with the well-constructed reference libraries, form the
basis for complete stone analysis [23]. Mineral constituents
can be analyzed by Infrared spectrometry (IR spectrometry),
but the crystal structure cannot be established. X-ray diffrac-
tometry can determine the mineral composition based on
crystal structure without damaging the stone sample. High-
resolution X-ray powder diffraction crystallography is con-
sidered a gold standard in identifying the crystal but has cer-
tain shortcomings including the amount of sample require-
ment, and time taken to acquire diffraction data [24]. Fourier
Transform spectroscopy is a well-recognized method of
choice that allows speculation, which alone will certainly not
unravel the mystery of why proteins reside in the stone ma-
trix, however a comprehensive proteomic analysis may help
arrive at some answers. The first step in the proteomic analy-
sis involves stone demineralization in which using a glass
mortar and pestle, stones are pulverized into fine powder. To
enhance the extraction of proteins various extraction buffers
can be used utilizing: Ethylene-Diamine-Tetra-Acetic Acid
(EDTA), Sodium Dodecyl Sulphate (SDS), urea, Dithio-
threitol (DTT), formic acid or a combination of these in ad-
dition to Tris HCl, glycerol and β -mercaptoethanol and in-
termittent sonication steps [25-27]. Then removal of extrac-
tion buffer components and desalination is done using cen-
trifugation and dialysis steps. Following which proteins are
separated using chromatographic techniques, eluted using a
desirable elution buffer, concentrated using lyophilization
and filtrates stored at -80ºC. Total protein content can be
determined using Bradford assay, Lowry assay, complete
acid hydrolysis as well as various other assays [25-28]. It is
possible that a sufficiently large fraction of stone matrix pro-
teins is resistant to extraction using the above methods [29].
This may be due to the low abundance of these proteins or
their tight interaction with the crystal surface.
5. PROTEIN IDENTIFICATION
Mass spectrometry technology has been widely used to
discern proteins by connecting the peptide tandem mass
spectra with the mass of the theoretical peptide fragments
[25, 26, 30, 31]. Proteins are digested to small peptides of a
predictable sequence using a specific proteolytic enzyme,
then are sequenced into fragment ion masses. These are
matched with the theoretical masses using a suitable data-
base with a sophisticated algorithm to communicate a certain
level of confidence to the matches allowing protein identifi-
cation. Several studies have used one and two dimensional
gel electrophoresis followed by in-gel digestion with trypsin
and subsequent Liquid Chromatography (LC)/Mass Spec-
troscopy (MS) or Matrix Assisted Laser Desorption Ioniza-
tion (MALDI)-MS/MS to identify proteins [32-35]. Some
90 Current Proteomics, 2020, Vol. 17, No. 2
other studies have used LC-MS/MS or MALDI-MS/MS of
the entire extracts [25, 36-38], using LC gradient profiles to
separate the concentrated, reduced and alkylated tryptic pep-
tides. The peptide elution pattern is assessed, peptide retention
time is determined, ion peak areas are calculated from extract-
ed peptide ion chromatogram generated from liquid chroma-
tography in combination with mass spectrometry [28].
The acquired spectrometric data from the experiment can
be searched against the protein sequence database of
HUMAN using X!Tandem algorithms as shown by Witz-
mann et al. [28]. General parameters, such as parent monoi-
sotopic mass error, static modification etc. were set and the
results were further validated using PeptideProhet [39] and
ProteinProphet [40]. MS spectra can also be analysed using
SEQUEST, MASCOT and studied through a database
search. In addition to this a software can be used with an
algorithm for matching the observed peptide spectra with the
theoretically derived one in the tandem mass spectrum data-
base and assigning scores [33]. These scores in combination
with other predictors, such as the calculated Grand Average
of Hydropathicity (GRAVY) score, aliphatic index, number
of negatively and positively charged residues for the identi-
fied proteins are used by the algorithm to assign each peptide
with an overall score [41]. Only validated proteins with
probability of greater than or equal to 90% and peptides with
probability of greater than or equal to 80% significance are
reported. Identity can be further validated by using immuno-
logical assays specific for the identified proteins [28].
Fig. (1). Schematic representation of proteomic analysis of sample.
For urolithiasis study, sample can be renal cells, calculi or urine.
Suitable method can be adopted for extraction and estimation of
protein followed by purification characterization and PAGE analy-
sis. Purified proteins can be identified and characterized by Mass
Spectrometric analysis and database search from search engines.
Interaction between identified proteins can be studied through pro-
tein network analysis.
Jain et al.
Corresponding author (Priyadarshini et al. 2009) had
done the proteomic analysis of renal stone where the protein
was extracted through EGTA method. Extracted proteins
were purified from anion exchange chromatography. This
protein was found to be the inhibitor of calcium oxalate
growth process. Homogeneity of purified protein was studied
by RP-HPLC. Peptide mass fingerprinting of purified protein
was done by MALDI-TOF analysis and mass spectrometric
data obtained were searched on Mascot search engine. A
novel calcium oxalate inhibitory protein, phosphate cytidyl-
yltransferase 1 from the organic matrix of human calcium
oxalate stone was purified [26].
In another study, corresponding author along with co
workers (Singh SK et al. 2012) reported cationic proteins
Histone lysine N-methyl transferase, Inwardly rectifying K
channel and Wnt2 protein as inhibitor of calcium oxalate
crystal nucleation and growth [42].
6. PROTEINS IN UROLITHIASIS
Source of the matrix proteins is a complicated issue gov-
erned by several separate and overlapping events. First be-
ing, the surrounding milieu in the early stages of crystal
growth in the renal tubule leading to the inclusion of macro-
molecules present in the urine based on their crystal binding
capacity. Second, proteins present due to the abrasion of the
urothelial lining on crystal obtaining a microscopic size dur-
ing further growth, aggregation and getting lodged in the
kidney tubule. Due to simple perfunctory trauma and the
subjection of crystal to negatively charged side chains of
proteins, certain typical organizational proteins such as col-
lagens, and fibrinogen, are likely to get entrapped between
the developing stone surface and epithelium layer [25]. Mac-
romolecular modulators of crystallization are highly promi-
nent in the local environment at the time of cell injury due to
crystal adhesion, inflammation and cell recovery. For in-
stance, Thongboonkerd et al. (2008) revealed that the pres-
ence of COM crystals altered the levels of 53 proteins, out of
which levels of 25 proteins were increased and levels of re-
maining 28 protein were reduced in renal epithelial cells
[43].
Uromodulin, albumin, calgranulin and hemoglobin are
commonly detected proteins irrespective of the crystal con-
stituents [33]. Albumin, uromodulin and hemoglobin are
abundantly found in blood and urine, as a result also found in
the stones [34]. Albumin and uromodulin have been reported
to have both stimulatory and inhibitory activity in crystal
aggregation of calcium stones [44, 45]. Albumin serves as
Inhibitor of aggregation of calcium oxalate crystals and pro-
moter of nucleation of calcium oxalate crystals [12, 46, 47].
Osteopontin which is present in the organic matrix of kidney
stone is basically found in kidney, bone and breast milk. This
protein is reported to be the inhibitor of crystallization of
calcium phosphate and calcium oxalate [48].
Osteopontin (OPN) which is known to partake in the reg-
ulation of mineralization, both physiological and pathologi-
cal, is produced by the kidney and is commonly found in
urine. Several studies indicate that osteopontin plays an in-
hibitory role throughout the crystal formation process of cal-
Proteomics Study in Urolithiasis
cium oxalate in vitro; also hindering the adhesion of crystals
to cultured epithelial cells [49].
Calcium binding proteins, calgranulin A and B produced
mainly by leukocytes and monocytes which are gathered to
suppress inflammation, play an inhibitory role in the crystal-
lization of calcium oxalate monohydrate [33]. Cathepsin G
protein belonging to peptidase S1 family participates in the
engulfment, eradicating and digestion of pathogens and helps
in recasting the connective tissue at the inflammatory site
[50]. Another inflammatory protein found to be involved in
the process was Azurocidin secreted from granulocyte [51].
However, it remains uncertain as to what roles these proteins
play in the process of kidney stone formation-as an inducer
or simply getting engulfed into the process by means of in-
flammation.
The peptide chain of inter-α inhibitor and acidic protein
nephrocalcin are reported to inhibit the calcium oxalate crys-
tallization [52, 53]. The role of proteins in different phases of
calcium crystallization is presented in Table 1.
Tamm-Horsfall protein is a glycoprotein, which has its
origin from an ascendant limb of loop of Henle. THP is an
inhibitor of calcium oxalate crystal aggregation. Effect of THP
is dependent on its own concentration, urinary pH and ionic
strength. It plays dual role in calcium oxalate crystallization
depending on the renal environmental conditions [54].
Some of the other abundantly found inhibitors of crystal-
lization include Chondroitin Sulphate and Heparan Sulphate.
Some known promoters include CD44, Monocyte Chemoat-
tractant protein-1, and Annexin II [7, 52].
7. PROTEIN NETWORK ANALYSIS
Protein network refers to the network of interacting pro-
teins responsible for the occurrence of various biological
processes. Most of these processes are dependent on the ac-
tivity of more than one protein. Hence studying these Pro-
tein-Protein Interactions (PPIs) creates an understanding
about modification of kinetic properties of enzymes, any
Table 1. Role of proteins in involved in nucleation, growth and aggregation of kidney stone formation.
Sr. No. Name of Protein
Inhibitor of aggregation of calcium oxalate crystals, promoter of nucleation of calci-
1 Albumin
2 Osteopontin Inhibitor of crystallization of calcium phosphate and calcium oxalate,
3 Tamm-horsfall protein
4 Phosphate cytidylyltransferase 1
5 Histone lysine N-methyl transferase,
6 Inwardly rectifying K channel
7 Wnt2 protein
8 calgranulin A and B
9 Nephrocalcin
10 Inter-alpha-inhibitor
Current Proteomics, 2020, Vol. 17, No. 2 91
alteration (increase or decrease) in protein abundance levels
related to a process, and even about the changes that might
be occurring in the specific binding of a protein with its sub-
strate by interacting with different intermediate binding part-
ners [55].
Identification of a large number of proteins involved in
urolithiasis has inspired scientists to study their interaction.
Currently, reports on the role of individual protein on uro-
lithiasis are available. To determine and analyze the type of
functional association between the proteins identified and to
connect their role in crystal modulation and stone formation
various databases and tools have been used. Ingenuity Path-
way Analysis (IPA) software server can be used to order
stone-specific proteins into canonical pathways [28]. Inter-
ProSurf can be used for studying interacting amino acid resi-
dues in proteins [56]. In addition to these, Human Proteome
Map, Gene Ontology database and many more such tools can
also be used. Proteins can also be placed onto gene regula-
tion networks and multiple specific pathways using online
tools, one such being STRING.
Protein networks or protein-protein interaction networks
can be created by the help of STRING, which acts as a data-
base for predicting interactions between the proteins. In the
network, nodes represent different proteins of interest along
with their interaction which needs to be studied, and the edge
represents the interactions or associations between the nodes
or proteins. No edge means no interaction between the pro-
teins (Fig. 1). The nodes and edges are the basic components
of the network [56, 57].
In STRING, the user enters the proteins of interest either
by name or by sequence, for which the interactions are to be
predicted. STRING collects and combines all the publicly
available information related to the proteins including both
direct (physical) and indirect (functional) interactions [57,
58]. Depending upon various attributes selected by the user,
the interaction network is presented along with the predicted
association. This association can be used to infer functional
significance of the identified proteins in crystal modulation
Role in Kidney Stone Formation (Inhibitor or Promoter) Refs.
[12]
um oxalate crystals
[11, 48]
Inhibitor at high pH, promoter at low pH [11, 55]
Inhibitor of growth of calcium oxalate crystallization [26]
Inhibitor of growth of calcium oxalate crystallization [42]
Inhibitor of growth of calcium oxalate crystallization [42]
Inhibitor of growth of calcium oxalate crystallization [42]
Inhibitor of calcium oxalate crystallization [33]
Inhibitor of calcium oxalate aggregation [52, 53]
Inhibitor of calcium oxalate crystallization [52, 53]
92 Current Proteomics, 2020, Vol. 17, No. 2
and hence stone formation. Such an analysis was performed
by Peerapen et al. (2018) using the STRING tool which re-
vealed that the identified proteins were involved in important
functional networks relating to cell proliferation, wound
healing, oxidative stress and cellular junction integrity. As a
result, the study helped in determining the biological path-
ways through which the calcium oxalate monohydrate crys-
tals produced cytotoxicity in renal cells [13].
Several studies before this have also performed protein
network analysis as a part of their proteomic studies revealed
valuable information regarding effective modulators of kid-
ney stone disease. It was found that the hormone testosterone
altered the abundance of cellular proteins in treated MDCK
cells, promoting the development of crystals and increasing
the risk of disease. The protein-protein interaction generated
by STRING showed α-enolase as the central node, increased
levels of which were also observed in MDCK cells treated
with testosterone [59].
The findings in the study by Manissorn et al. (2016) indi-
cated that alpha-tubulin had preventive roles in urolithiasis,
i.e. it prevented the adhesion of cells and crystals resulting in
decreased cell death. In addition, it enhanced the tissue repair
function and cell proliferation [60]. Further, Pongsakul et al.
(2016) in their study demonstrated for the first time the in-
teraction and involvement of two potential targets for the
prevention of stone formation and its recurrence through
protein network analysis. It was reported that the strongest
interaction was found between two of the upregulated pro-
teins, lamin A/C (LMNA) and nesprin-1 (SYNE1) in COM
treated renal tubular cells. Si-LMNA leads to a decrease in the
number of calcium crystals adhered to the cell surface and
reduced the level of potential crystal receptors, thus indicating
its importance in tissue repair and cellular proliferation [61].
CONCLUSION
The role of proteomics in the study of urolithiasis is vast
and has helped to understand the pathophysiology of kidney
stone disease, biomarker discovery and identification of new
therapeutic targets. From determining the inorganic contents
of the calculi to the identification and characterization of
matrix proteins, the entire cascade of techniques mandates
the use of analytical methods with high reproducibility and
precision. The bioinformatic network analysis of associated
proteins enables risk assessment and helps in attaching func-
tional roles to proteins leading to disease. Thus, the prote-
omics analysis of renal stones presents a great potential for
dynamic monitoring of Kidney Stone Disease and in aiding
better individual response to therapy and prevention. How-
ever, there are still many challenges that must be addressed
before the possibility of effective and uniform clinical appli-
cation of stone proteome network analysis.
CONSENT FOR PUBLICATION
Not applicable.
FUNDING
None.
Jain et al.
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
otherwise.
ACKNOWLEDGEMENTS
Authors acknowledge Jaypee Institute of Information
Technology, Noida for providing infrastructure facility for
the study.
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