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Genome-Wide Gene Expression Profiling of Randall’s Plaques in Calcium Oxalate Stone Formers

† † Kazumi Taguchi,* Shuzo Hamamoto,* Atsushi Okada,* Rei Unno,* Hideyuki Kamisawa,* Taku Naiki,* Ryosuke Ando,* Kentaro Mizuno,* Noriyasu Kawai,* Keiichi Tozawa,* Kenjiro Kohri,* and Takahiro Yasui*

*Department of Nephro-urology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; and †Department of Urology, Social Medical Corporation Kojunkai Daido Hospital, Daido Clinic, Nagoya, Japan

ABSTRACT Randall plaques (RPs) can contribute to the formation of idiopathic calcium oxalate (CaOx) kidney stones; however, genes related to RP formation have not been identified. We previously reported the potential therapeutic role of osteopontin (OPN) and macrophages in CaOx kidney stone formation, discovered using genome-recombined mice and genome-wide analyses. Here, to characterize the genetic patho- genesis of RPs, we used microarrays and immunohistology to compare gene expression among renal papillary RP and non-RP tissues of 23 CaOx stone formers (SFs) (age- and sex-matched) and normal papillary tissue of seven controls. Transmission electron microscopy showed OPN and collagen expression inside and around RPs, respectively. Cluster analysis revealed that the papillary gene expression of CaOx SFs differed significantly from that of controls. Disease and function analysis of gene expression revealed activation of cellular hyperpolarization, reproductive development, and molecular transport in papillary tissue from RPs and non-RP regions of CaOx SFs. Compared with non-RP tissue, RP tissue showed upregulation (˃2-fold) of LCN2, IL11, PTGS1, GPX3,andMMD and downregulation (0.5-fold) of SLC12A1 and NALCN (P,0.01). In network and toxicity analyses, these genes associated with activated mitogen- activated protein kinase, the Akt/phosphatidylinositol 3-kinase pathway, and proinflammatory cytokines that cause renal injury and oxidative stress. Additionally, expression of proinflammatory cytokines, num- bers of immune cells, and cellular apoptosis increased in RP tissue. This study establishes an association between genes related to renal dysfunction, proinflammation, oxidative stress, and ion transport and RP development in CaOx SFs.

J Am Soc Nephrol 28: 333–347, 2017. doi: 10.1681/ASN.2015111271

The prevalence of kidney stone disease is nearly 9% in The other pathway involves overgrowth on interstitial the adult population and continues to increase world- apatite plaques, the so-called Randall plaques (RPs),9 wide.1,2 This condition has a medical and economic as observed in some idiopathic CaOx SFs. impact3 andisreportedtobeassociatedwithcompli- cations such as metabolic syndrome (MetS)4,5 and CLINICAL RESEARCH 6 ESRD. The pathogenesis of kidney stone formation Received November 27, 2015. Accepted May 4, 2016. has been investigated, and there are two major theories K. Taguchi and S.H. contributed equally to this work. for predicting lithogenesis.7 One pathway involves for- mation of intra-tubular crystals in the duct of Bellini, Published online ahead of print. Publication date available at www.jasn.org. the so-called Randall plugs, as observed with both ex- 8 Correspondence: Dr. Atsushi Okada, Department of Nephro-urology, perimental hyperoxaluria-induced animal models Nagoya City University Graduate School of Medical Sciences, 1 and human primary hyperoxaluria and with calcium Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. phosphate (CaP), struvite, in addition to some idio- Email address: [email protected] pathic calcium oxalate (CaOx) stone formers (SFs). Copyright © 2016 by the American Society of Nephrology

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In a recent study using genome-wide analysis and genome- the pathologic united theory of RP formation,16 molecular- recombined mice, we found OPN expression in renal tubular level analysis of cellular function is necessary for better cells and macrophage (Mw) migration in the interstitial space understanding of the role of RPs. The recently developed around crystals to be essential for stone formation.10–12 We found nephroureteroscopic technique permits more detailed analy- that the anti-inflammatory phenotype Mw played a suppressive sis of RPs involving both microscopic and genomic analyses.17 role in kidney stone formation via renal crystal phagocytosis.13 Therefore, to establish molecular-targeted therapies for Differentiation and induction of anti-inflammatory Mw are kidney stones, we investigated the gene expression profiles of considered a potential therapeutic approach for kidney stone RP sections from human papillary tissues and studied the disease; however, this evidence is only applicable to Randall plugs, factors controlling the development of RPs using microarray which have similarities with the hyperoxaluric mouse model. and immunohistochemical analyses. Understanding of RPs is also essential to clarify the potential of molecular therapies, such as OPN and Mw-related genes. Regarding the origin of some idiopathic CaOx kidney RESULTS stones, Evan et al. made major contributions to the study of the microscopic structure of RPs, which begin in the basement Patient Background membranes of thin loops of Henle with calcium deposits.14 Patients who underwent percutaneous nephrolithotomy or Despite numerous studies involving animal hyperoxaluric retrograde intrarenal surgery for calcium-based stones were stone models and human samples,15 the exact role of RP in the formation of CaOx crystals remains unknown. Since mor- phologic, mineral, and matrix-based investigations provided

Table 1. Patient backgrounds Control CaOx SFs Characteristics P Value (n=7) (n=23) General Age, y 56618 59613 NSa Gender, M/F 4:3 14, 9 NSc Side, Rt/Lt 3:4 13, 10 NSc BMI, kg/m2 22.162.9 23.364.7 NSa Stone Stone composition CaOx, % — 91.469.1 — CaP, % — 5.567.5 — Stone volume, mm3 — 76161528 — Stone density, HU — 8206375 — Hydronephrosis, grade 0,I,II 5,1,1 12, 6, 5 NSd Serum BUN, mg/dl 12.863.7 14.563.8 NSb Cre, mg/dl 0.960.4 0.960.2 NSb Ca, mg/dl 9.460.7 9.460.6 NSb P, mg/dl 3.261.0 3.160.3 NSb UA, mg/dl 4.961.2 5.861.6 NSb WBC, cells/ml 651461748 678361853 NSb CRP, mg/dl 1.961.7 0.360.1 NSb Urine NSb pH 6.860.6 6.660.7 NSb WBC, cells/HPF 9610 19628 NSb Figure 1. Endoscopic and microscopic distribution of RPs. Rep- RBC, cells/HPF 16637 19629 NSb resentative photographs show renal papillary tissues from both 6 Data are presented as the mean SD. Grade of hydronephrosis was cate- normal and RP mucosa. The endoscopic image shows renal papilla gorized by Society for Fetal Urology. NS, not significant; M, male; F, female; Rt, right; Lt, left; BMI, body mass index; —, inapplicable data; HU, Hounsfield mucosa in the upper calyx during retrograde intrarenal surgery. unit; BUN, blood urea nitrogen; Cre, creatinine; Ca, calcium; P, phosphorus; The normal papilla shows fleshy smooth mucosa without bleeding UA, uric acid; WBC, white blood cell; CRP, c-reactive protein; HPF, high or calcification. Some RPs are showing as a white patchy lesion fi power eld; RBC, red blood cell. (arrow heads) as well as a ductal plug (arrow) within the same aStatistical analyses performed by t test. b papilla. Micro tissues were stained with hematoxylin-eosin, von Statistical analyses performed by Mann–Whitney U test. cStatistical analyses performed by Fisher exact test. Kossa (for detection of CaP crystals), and Pizzolato (for detection dStatistical analyses were performed by Kruskal–Wallis test. of CaOx crystals) staining. *Location of RP. Magnification, 3400.

334 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 333–347, 2017 www.jasn.org CLINICAL RESEARCH enrolled in this study. There were no statistical differences in the indicating that RPs contained CaP but did not have a CaOx general background among the seven controls and 23 CaOx SFs component (Figure 1). suchasage,gender,sideoftreatment,andbodymassindex.Based Energy dispersive x-ray (EDX) microanalysis revealed that the on the composition of the stone fragments obtained during spectra of both calcium and phosphorus matched those for the lithotripsy, CaOx SF was defined as a patient with kidney and/or RP region; other regions did not show spectra for both (Figure ureter stones that had .80% content of CaOx crystals. There 2A). Transmission electron microscopy (TEM) showed that were no significant differences in serum and urine parameters there were numerous collagen fibers in both interstitial cellular among the three groups (Table 1). spaces around RPs and outside interstitial spaces around the basement membranes of renal tubular cells (Figure 2B). Immu- Observation of RPs and the Surrounding Tissue nohistochemical TEM showed much more diffuse and higher During the endoscopic intrarenal operation, RPs were ob- expression of OPN, considered to represent the matrix of CaOx served as plain white calcification regions that were covered and CaP stones, in RPs compared with both renal tubular cells with the papillary epithelium when viewed through a neph- and interstitial cells without RPs (Figure 2C). roureteroscope. Some RPs coexisted with ductal plugs in the same renal calyx papilla. Hematoxylin-eosin staining showed Gene Profiling of Papillary Tissue of CaOx SFs and destruction of the papillary epithelium layer and interstitial Controls cellular disorder surrounding RPs. The RPs were positive for Microarray analysis was performed to compare the gene ex- von Kossa staining but negative for Pizzolato staining, pression profiles of papillary tissue from nonstone patients

Figure 2. Ultrastructural observations obtained using EDX microanalysis and TEM. (A) EDX microanalysis of RPs. Upper images are microphotographs of non-, calcium (Ca)-, and phosphorus (P)-staining tissues. Lower images show spectra of carbon (C), oxygen (O), sodium (Na), Osmium (Os), Ca (arrow head), and P (arrow) for each tissue. L1, lesion 1 (the nonplaque area); L2, lesion 2 (another nonplaque area); RP, RP area. (B) Ultrastructural details of collagen fibers surrounding RP and normal renal tubular cells from non-RP lesion detected in papillary tissue by TEM. N, nucleus; arrow, basement membrane. Scale bar, 2 mm. (C) Immunoelectron microscopy analysis of OPN by TEM. OPN-positive area is indicated by black dots (arrow) on RPs and a renal tubular cell. Arrow, basement membrane. Scale bar, 1 mm.

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(C group) and non-RP (N group) and RP (P group) tissue from groups had 6019 and 8274 genes with increased expression, CaOx SFs. Cluster analysis demonstrated that gene expression respectively, and 70%–97% (5860 genes) of them were mutu- profiles of CaOx SFs (including both N and P groups) markedly ally shared with both P and N groups. In contrast, compared differed from those of nonstone patients (C group) (Figure with the C groups, P and N groups had 451 and 577 genes with 3A). The scatter diagram showed significantly different gene decreased expression, respectively, and 71%–91% (412 genes) expression between the C and N/P groups and smaller differ- of them were common with P and N groups. Additionally, 21 ences in gene expression between the N and P groups (Figure and ten genes in the P group showed increased and decreased 3B). expression compared with the N group, respectively (Figure Figure 4A shows the microarray comparison results for 4). genes that showed a twofold increase or decrease in gene ex- Ingenuity canonical pathway analysis indicated significant pression among P and N groups in CaOx SFs, with the C group activation of the following pathways in the N and P groups as control patients. Compared with the C groups, the P and N compared with the C group: cAMP-mediated signaling,

Figure 3. Comparison of gene expression in renal papillary tissues among RP and normal tissue from CaOx SFs and normal tissue from control patients by using microarray analysis. (A) Cluster analysis of the expression of all 50,599 genes on a human array chip. C group, normal papillary tissues from control patients; N group, normal papillary tissues from CaOx SFs; P group, RP papillary tissues from CaOx SFs. (B) Scatter plots of gene expression difference between C and N (left), C and P (center), and N and P groups (right).

336 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 333–347, 2017 www.jasn.org CLINICAL RESEARCH

Figure 4. Scheme of the microarray analysis demonstrated diversity of gene expression difference among each group. (A) Comparison of each group and gene expression changes. Each extracted value represents the number of statistically different genes with .2.0- or ,0.5-fold difference in expression between groups. P, RP papillary tissue group from CaOx SFs; N, normal papillary tissue group from CaOx SFs; C, normal papillary tissue group from control patients. (B) Venn diagram of a select number of genes that showed .2.0-fold difference in expression in both normal and RP papillary tissue of CaOx SFs compared with normal papillary tissue of control patients (left red figure), and the number of genes that showed ,0.5-fold difference in expression in both normal and RP papillary tissue of CaOx SFs compared with normal papillary tissue of control patients (right blue figure). coagulation system, ga signaling, extrinsic prothrombin activa- PG-endoperoxide synthase (PTGS) 1, monocyte to macro- tion pathway, and calcium signaling (Table 2). phage differentiation (MMD), and chromosome 8 open Per disease and function analyses, upregulated genes reading frame 4, and downregulation of potassium channel common to both P and N groups were categorized based on inwardly rectifying subfamily J member 1, solute carrier cell/neuron hyperpolarization, fertilization, ion/carbohydrate/ family 12 member 1 (SLC12A1), and sodium leak channel monosaccharide transport, duct cell differentiation, andros- nonselective (NALCN) were related to extracellular proin- tenedione modification, and endocrine cell depolarization. flammatory cytokine and intracellular signal pathways (Fig- Downregulated genes common between P and N groups were ure 5). categorized to obesity, cell attachment, tensile strength of the IL1b and TNF were determined to be upstream cytokines skin, leukemia, endocrine gland hypoplasia, anion homeosta- upregulated among genes in the P group compared with the N sis, phosphatidylserine distribution, glycemic control, and group (Supplemental Table 5). metabolic bone disease (Table 3). Toxicity analysis showed that LCN2, IL11, glutathione The top 100 up- or downregulated genes and top networks peroxidase (GPX) 3, and aquaporin (AQP) 1 were responsible in the N and P groups compared with those in the C group are for ARF, renal ischemia-reperfusion injury, cardiac hypertro- shown in Supplemental Tables 1–3. phy, and oxidative stress (Table 5).

Gene Profiling of RP Tissue in CaOx SFs Validation of Up/Downregulated Molecules in RP The top eight genes that were upregulated by .2-fold or Papillary Tissues of CaOx SFs downregulated by ,0.5-fold in the P group compared with To validate the mRNA and protein expression results, we used the N group of CaOx SFs are listed in Table 4. quantitative RT-PCR (qPCR) and immunohistochemical Network analyses of genes whose expression differed by staining. The mRNA expression of LCN2, IL11, SLPI, .2-fold or ,0.5-fold in the P group compared with the N PTGS1, GPX3, and MMD in the P group was significantly group are shown in Supplemental Table 4. The top-scored higher and that of secretoglobin family 1D member 2 network demonstrated upregulation of lipocalin (LCN) 2, IL (SCGB1D2), SLC12A1, and NALCN was significantly lower 11, secretory leukocyte peptidase inhibitor (SLPI), mucin 4, than that in the N group (Figure 6, A and B).

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Table 2. High/Lowest activation z-score canonical pathways in both RP and normal papillary tissue, where gene expression differs from that in the control mucosa by at least twofold Ingenuity Canonical P Value Ratio z-Score Molecules in Pathway Pathways acAMP-mediated signaling 0.001 0.17 3.307 AKAP12,HTR5A,PTGDR,MC3R,PTGER3,PDE3A,PDE4A,TAAR1,CHRM3, HRH3,OPRL1,PPP3R2,MC5R,AKAP14,PPP3R1,CNR2,PDE4D,PDE11A,DRD3, MC2R,HTR6,RGS2,CALML5,GRM8,OPRM1,SSTR3,GNAI1,DRD5,RAPGEF3, GRM4,MC4R,XCR1,P2RY14,AKAP4,CNGB1,LPAR1,PDE5A,ADCY10 aCoagulation system 0.002 0.29 2.530 F11,BDKRB2,PLG,F9,PROS1,PROC,VWF,F7,TFPI,FGG aGas signaling 0.01 0.18 2.500 HTR6,RGS2,ADD2,HTR5A,MC3R,PTGDR,TAAR1,GNG13,DRD5,RAPGEF3,MC4R, CHRM3,GNB1,MC5R,GNB4,CNGB1,MRAS,ADCY10,GNG12,MC2R aExtrinsic prothrombin 0.01 0.31 2.236 PROS1,PROC,F7,TFPI,FGG activation pathway CREB signaling in neurons 0.40 0.11 2.138 POLR2F,CALML5,GRM8,GRID1,GRIA1,GRIK3,GNAI1,GNG13,GRM4,PRKCG, GNB1,GRM5,GNB4,PLCG2,MRAS,PLCB1,ADCY10,PLCL1,GNG12 aGai signaling 0.02 0.17 1.807 OPRM1,PTGER3,GRM8,SSTR3,GNAI1,GNG13,GRM4,HRH3,OPRL1,XCR1,GNB1, GNB4,P2RY14,LPAR1,CNR2,CAV1,MRAS,ADCY10,DRD3,GNG12 Dopamine receptor signaling 0.28 0.13 1.633 MAOB,PPP1R14D,PPP2R5B,NCS1,DRD5,PPP1CB,SLC18A1,DRD3,ADCY10,CALY Glutamate receptor signaling 0.06 0.18 1.342 GRM5,GNB1,CALML5,SLC17A7,GRM8,GRIA1,GRID1,GRIK3,DLG4,GRM4 Phospholipase C signaling 0.27 0.12 1.279 MYL10,PPP1CB,GNG13,PPP3R2,GNB1,GNB4,PPP1R12B,PPP3R1,MRAS,PLCB1, ARHGEF2,GNG12,CALML5,ARHGEF4,MEF2A,PLA2G1B,RAPGEF3,ARHGEF17, CD79A,MYL1,PRKCG,MYL9,RHOQ,PLCG2,MEF2D,ADCY10,ARHGEF10,FNBP1 Chemokine signaling 0.19 0.14 1.265 ROCK2,CCR3,CALML5,PPP1R12B,PLCG2,MRAS,GNAI1,PLCB1,PPP1CB,NOX1 Synaptic long term 0.29 0.12 1.213 GRM8,GRID1,GRIA1,PPP2R5B,PLA2G1B,GNAI1,PPP1R17,GRM4,PRKCG,GRM5, depression PRKG1,PLCG2,PLB1,GUCY1A2,MRAS,PLCB1,PLCL1 aCalcium signaling 0.02 0.15 1.155 CHRNA1,CALML5,TRPC1,GRIA1,MYH14,TRDN,MEF2A,TNNI3,CHRNB4,SLC8A2, TPM1,TPM2,ATP2A2,MYL1,TRPC7,ATP2B2,ATP2A1,MYL9,PPP3R2,CHRNG, ACTA2,ATP2B3,MEF2D,PPP3R1,TNNI1,SLC8A1,ATP2B4 PPARa/RXRa activation 0.48 0.11 1.147 CYP2C18,ACVR1,ADIPOQ,CYP2C9,BMPR2,ABCA1,CYP2C19,GK,PLCG2,INS, MRAS,TGFB2,TGFB3,SMAD4,PLCB1,PLCL1,ADCY10,ACVR1C,MAP4K4 Ga12/13 signaling 0.05 0.15 21.414 VAV2,F2RL2,MYL10,CDH4,MEF2A,MYL1,CDH11,ROCK2,MYL9,CDH9,CDH12, LPAR1,VAV3,MEF2D,MRAS,CDH17,CDH8,CDH13 CREB, cAMP responsive element binding protein; PPARa/RXRa, peroxisome proliferator activated receptor alpha/retinoid X receptor alpha. aP,0.05 indicates significant difference.

Based on the qPCR results, we examined the protein plasma cells, and neutrophils, respectively, increased in the P expression of differentially regulated genes by immunohisto- group compared with the other groups (Figure 8). chemistry. Widespread LCN2, IL11, GPX3, and SCGB1D2 protein expression was noted in urothelial cells, tubular epithelial cells, and the interstitial spaces of renal papillae. DISCUSSION PTGS1, MMD, SLC12A1, and NALCN proteins were mainly expressed in the cells of the epithelium, tubules, and interstitial Several studies have reported both basic and clinical significance spaces. LCN2, IL11, GPX3, and MMD expressionwas relatively ofRPs for idiopathic CaOxSFs.18–21 Recent reportsregarding RPs strong, whereas SCB1D2, SLC12A1, and NALCN expression have tended to use either computed- and microscopic-technical was weaker in the P group than in the other groups (Figure 7, A or clinical characterization with 24-hour urine samples. Using and B). microcomputed tomography (micro-CT), Williams et al. re- viewed the concept of stone growth in RPs, in which CaOx Validation of Proinflammation and Apoptosis-Related crystals start to adhere to RPs that originated from papillary Molecules Between RPs and Normal Papillary Tissues interstitium apatite through the loss of the papillary epithelium According to network, upstream regulator, and toxicity and were then excreted in the renal collecting system.18 Nuclear analyses, we further compared proinflammatory gene expres- magnetic resonance spectroscopy revealed that the apatite in sion and apoptosis among C, N, and P groups. Expression of RPs was composed of CaP and that RPs included variable pro- IL1B in the P group and that of nitric oxide synthase 2 and TNF portions of protein, glycosaminoglycan, lipid, and carbonate.19 in the N and P groups was markedly higher than those in the C Another study using x-ray microanalysis and electron group. The number of cells stained positively for CD68, CD138, microscopy indicated that RPs had high zinc levels and were neutrophil elastase, and terminal deoxynucleotidyl transferase associated with calcifying collagen fibers as well as crystals pre- dUTP nick end labeling (TUNEL), which represented Mws, sent in membrane-bound vesicles.20,21

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Table 3. High/Lowest activation z-score categories of disease and functions in both RP and normal papillary tissue gene expressions differed from control mucosa by twofold Diseases or Functions Activation Predicted Numbers of Categories P Value Annotation z-Score Activation State Molecules Most increased Cellular function and maintenance Hyperpolarization 0.001 3.307 Increased 14 Cellular function and maintenance Hyperpolarization of cells 0.001 2.752 Increased 8 Cellular function and maintenance Hyperpolarization of neurons 0.002 2.591 Increased 7 Embryonic development, organismal development, Fertilization 0.01 2.425 Increased 28 reproductive system development and function Molecular transport Transport of ion 0.002 2.343 Increased 86 Carbohydrate metabolism, molecular transport Transport of carbohydrate 0.01 2.319 Increased 33 Carbohydrate metabolism, molecular transport Transport of monosaccharide 0.01 2.243 Increased 29 Cellular development, tissue development Differentiation of duct cells 0.001 2.236 Increased 5 Endocrine system development and function, lipid Modification of 0.01 2.169 Increased 5 metabolism, small molecule biochemistry androstenedione Cell morphology, cellular function and maintenance, Depolarization of endocrine 0.003 2.000 Increased 4 endocrine system development and function cells most decreased Nutritional disease Obesity 0.004 22.730 Decreased 80 Cell-to-cell signaling and interaction Attachment of cells 0.003 22.566 Decreased 26 Hair and skin development and function Tensile strength of skin 0.002 22.195 Decreased 7 Cancer, hematologic disease, immunologic disease, Leukemia 0.001 22.183 Decreased 233 organismal injury and abnormalities Developmental disorder, endocrine system Hypoplasia of endocrine 0.006 22.157 Decreased 8 disorders gland Cellular function and maintenance, small molecule Homeostasis of anion 0.001 22.078 Decreased 15 biochemistry Carbohydrate metabolism, lipid metabolism, Distribution of 0.01 22.000 Decreased 4 molecular transport, small molecule biochemistry phosphatidylserine Organismal functions Glycemic control 0.001 22.000 Decreased 6 Connective tissue disorders, metabolic disease, Metabolic bone disease 0.001 22.000 Decreased 18 skeletal and muscular disorders

The clinical association between RPs and risk of CaOx amounts of collagen fibers were found in the interstitial space stone formation is yet controversial. The urinary volume, surrounding the basement membrane, both with and with- urinary calcium and citrate excretion, and serum osteocal- outRPs.TheRPsshoweddiffuseOPNexpression.Our results cin levels seem to be related to RPs22–24; however, reports are consistent with previous reports suggesting that RPs are indicate that ductal tubular plugging, and not RPs, is more composed of CaP and that their origin is correlated with relevant for kidney stone risk as predicted by 24-hour urine collagen fibers and OPN expression.21,28 Increased collagen and CT imaging.25,26 Although the rate of urinary meta- fiber and OPN expression plays a crucial role in RP growth; bolicabnormalityismuchlowerinJapanthaninthe they also contribute to dramatic changes in the expression of United States, the urolithiasis prevalence in both countries other molecules, such as those involved in inflammation and is approximately 10%.27 This finding shows that not only immunity, oxidative stress, and sodium/potassium trans- metabolic but also molecular-based investigations are es- porter and channels. sential to characterize kidney stone pathogenesis. ComparisonwiththeCgroupofnon-SFsshowedthatthe Since both morphologic and molecular analyses are re- N and P groups of CaOx SFs had many common up- or quired to analyze the relevance of RPs for CaOx SFs in order downregulated genes. Canonical pathway analysis showed to determine the pathogenesis of RPs, we performed the activation of signaling pathways including cAMP, co- genomic and immunohistochemical analyses using papil- agulation, Ga, and calcium in both RPs and normal papil- lary tip samples from patients with kidney stones in this lary tissues of CaOx SFs compared with normal papillary study. tissues of control patients. According to disease and func- Here, we examined RPs that were of sufficient size to be tion analyses, cellular hyperpolarization, reproductive de- visualized by endoscopy. Positive results were obtained for velopment, differentiation, and molecular transport also the large RPs in von Kossa but not Pizzolato staining, increased, but nutrition levels, cell-to-cell attachment, indicating that they contained CaP and not CaOx. Large and organismal development decreased in the papillae of

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Table 4. Top upregulated and downregulated genes comparing RP with normal papillary tissue Gene Fold Agilent ID Entrez Gene Name P Value Location Type Symbol Change Genes with increased expression in plaque mucosa A_23_P169437 LCN2 Lipocalin 2 6.167 0.02 Extracellular space Transporter A_33_P3243887 IL11 IL 11 3.949 0.01 Extracellular space Cytokine A_24_P190472 SLPI Secretory leukocyte peptidase inhibitor 3.407 0.03 Cytoplasm Other A_24_P208825 MUC4 Mucin 4, cell surface associated 2.826 0.04 Plasma membrane Other A_24_P64167 PTGS1 PG-endoperoxide synthase 1 2.824 0.02 Cytoplasm Enzyme A_33_P3369371 GPX3 Glutathione peroxidase 3 2.717 0.03 Extracellular space Enzyme A_23_P164047 MMD Monocyte to macrophage differentiation- 2.390 0.01 Plasma membrane Kinase associated A_23_P253350 C8orf4 Chromosome 8 open reading frame 4 2.022 0.04 Other Other Genes with decreased expression in plaque mucosa A_23_P150555 SCGB1D2 Secretoglobin, family 1D, member 2 22.322 0.001 Extracellular space Other A_33_P3252003 KCNJ1 Potassium channel, inwardly rectifying 22.317 0.01 Plasma membrane Ion channel subfamily J, member 1 A_21_P0007591 NAV2 neuron navigator 2 22.255 0.02 Nucleus Other A_23_P84666 GDPD1 Glycerophosphodiester phosph 22.213 0.02 Other Enzyme odiesterase domain containing 1 A_24_P136029 SLC12A1 Solute carrier family 12 (sodium/ 22.112 0.03 Plasma membrane Transporter potassium/chloride transporter), member 1 A_23_P29057 KCNJ6 Potassium channel, inwardly rectifying 22.110 0.001 Plasma membrane ion channel subfamily J, member 6 A_33_P3230166 NALCN Sodium leak channel, nonselective 22.068 0.001 Plasma membrane ion channel A_33_P3253723 AQP1 Aquaporin 1 (Colton blood group) 22.005 0.001 Plasma membrane transporter

CaOx SFs compared with those of controls. Additionally, IL11, a member of the IL6 family, is associated with oxidative other networks related to the cardiovascular system, im- stress and compensatory proliferation.36 Some studies report- mune response, and inflammatorydiseasewereraisedas ed that both neutrophil gelatinase-associated LCN and IL11 the top associated networks in differences between papillae are important biomarkers for AKI.37–39 of CaOx SFs and controls. The diversity of the results was PTGS1, also known as cyclooxygenase-1, acts as a vaso- derived from the heterogeneity of sample tissues, which constrictor in the kidney and contributes to development of consisted of a large amount of renal tubular and interstitial arterial hypertension.40 Stoller et al. have supportively hy- cells and a small amount of urothelial and immune cells as pothesized the theory that kidney stones and RPs could be shown in Figure 8B and Supplemental Figure 1. However, caused by renal vascular injury.29,30 GPX3 is found in the the predicted association between CaOx SFs and non-SFs as renal tubular cells in basement membranes, which indicates described above supports the united RP formation the- the existence of oxidative stress.41 Toxicity analysis showed ory16,29–31 involving disorders of cellular structure, signal- that ARF, ischemia-reperfusion injury, and oxidative stress ing, differentiation, mineral density, tissue inflammation, were associated with gene expression of LCN2, GPX3, IL11, and vascular formation. and AQP1, such that not only renal tubular and urothelial Microarray, network, and validation analyses showed that cell damage but also some kinds of vascular injury resulted the RP papillary tissue of CaOx SFs had higher LCN2, IL11, in RP formation. Although the Akt/PI3K pathway is related PTGS1, GPX3, and MMD expression and lower SLC12A1 and to suppression of renal cell apoptosis and facilitation of NALCN expression. Network analysis demonstrated that these vascularization, MMD positively regulates Akt/PI3K activa- genes were directly related to each other via extracellular tion in Mw.42,43 Since MMD expression occurs via stimu- signal-regulated kinase 1/2,32,33 Akt/phosphatidylinositol lation of proinflammatory Mw,43 MMD upregulation 3-kinase, protein kinase a (Pka), and proinflammatory cyto- resulted in activation of proinflammatory cytokines and kines. LCN2, which is also known as neutrophil gelatinase- oxidative stress. associated LCN, is expressed in tubular cells, Mw, and neutrophils The downregulated genes SLC12A1 and NALCN encode a and is related to cellular apoptosis and inflammation.34,35 membrane transporter and channel, respectively. SLC12A1

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Figure 5. Network function of inflammatory response, cellular movement, cell-to-cell signaling, and interaction between RP and normal papillary tissues in CaOx SFs (defined by either .2.0- or ,0.5-fold difference in expression; P,0.01). Red shapes indicate upregulated mRNAs, while gray shapes indicate downregulated mRNAs. Different shapes and prediction outlines are indicated in the legend box. is a sodium-potassium-chloride transporter found in the as- Finally, in addition to the detected inflammatory network cending limb of the loop of Henle and is responsible for Batter and validation of each candidate gene responsible using the RP syndrome type 1. SLC12A1 deficiency results in renal hypo- site, the upstream regulator analysis showed activation of IL1B kalemia, alkalosis, hypercalciuria, and nephrocalcinosis, and and TNF; therefore, we validated the major proinflammatory we previously found that CaOx SFs had single-nucleotide cascade and cell expression. As a recent study mentioned,47 polymorphisms of SLC12A1.44,45 NALCN is a nonselective these inflammation and cellular disorders might contribute to sodium leak channel that is related to osmoregulation mainly RP formation (Figure 9). in neuron cells.46 Although the contribution of NALCN to There are some limitations to this study. First, the lack of renal papillary tissue has not yet been identified, deficiency clinical data like those for 24-hour urine samples limits the of this gene might cause renal cell damage and alteration in clinical significance of the gene expression profiling of patients. intratubular mineral saturation with SLC12A1. Second, the heterogeneity and mechanical damage of biopsied papillary tissue may have resulted in inconsistency among analyses, with indication of an inflammatory response and Table 5. Toxicity analysis of genes which expressed over limited infiltration of immune cells as shown in immunohis- twofold differences between RP and normal papillary tissue tochemistry. Third, comparison of papillary tissues with or Ingenuity without RPs from the same kidney decreased the detection of P Value Ratio Genes in Lists Toxicity Lists other responsible candidate genes not statistically but clini- ARF panel (rat) 0.001 0.03 LCN2, AQP1 cally. Persistent renal ischemia- 0.02 0.03 LCN2 In conclusion, we found compelling evidence that genes reperfusion injury (mouse) related to renal injury, vasoconstriction, oxidative stress, Mw, Cardiac hypertrophy 0.04 0.01 GPX3, IL11 and sodium/potassium transporters and channels contribute Oxidative stress 0.04 0.01 GPX3 to RP development in CaOx SFs via proinflammatory

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Figure 6. mRNA validation by qPCR shows significant higher expression of LCN2, IL11, SLPI, PTGS1, GPX3, and MMD; whereas lower expression of SCGB1D2, SLC12A1, and NALCN is shown in P group compared with N group. mRNA validation of genes showing either (A) .2.0- or (B) ,0.5-fold difference in expression in RP papillary tissue compared with that in normal papillary tissue of CaOx SFs. The

342 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 333–347, 2017 www.jasn.org CLINICAL RESEARCH

Figure 7. Protein validation by immunohistochemistry shows strong expression of LCN2, IL11, PTGS1, GPX3, and MMD; whereas weak expression of SCGB1D2, SLC12A1, and NALCN is shown in P group compared with N group. Immunohistochemical distribution of genes showing either (A) .2.0- or (B) ,0.5-fold difference in expression in RP papillary tissue compared with that in normal papillary tissue of CaOx SFs. Genes were selected according to mRNA validation results obtained using qPCR. The locations of RPs are indicated as asterisks in each representative microphotograph. Magnification, 3400. activation through the mitogen-activated protein kinase and All the enrolled patients underwent physical examination, blood Akt/PI3K pathways. To our knowledge, this is the first study and urine tests, and ultrasonography and computed tomography reporting the gene expression profile of RP papillary tissue, imaging before treatment. which would contribute to developing molecular targeted therapies for idiopathic CaOx stones. Surgical Procedure For the PCNL, we accessed the renal collecting system from the lowerpoleinordertofragmentizeandremovethepelvicstone CONCISE METHODS with an 18 Fr mini-PCNL tract (Karl Storz, Tuttlingen, Germany) and lithotripter (Swiss LithoClast; Boston Scientific, Marlbor- Patients ough, MA). For the RIRS, ureteral access sheaths (12/14Fr Flexor; This study was approved by the Nagoya City University ethics board Cook Medical Inc., Bloomington, IN) were inserted for all (No. 929). All participants provided informed consent. We patients. We used a flexible ureteroscope ([Flex-X2; Karl Storz] obtained biopsies of renal papillary tissues from 23 idiopathic or [URF-V; Olympus, Tokyo, Japan]) and a holmium laser CaOx stone patients who had undergone percutaneous intrarenal lithotripsy system (VersaPulse; Boston Scientific). After the surgery (PCNL) or retrograde intrarenal surgery (RIRS) at our removal of fragments, we obtained renal papillary tissue institutions during November 2013 to April 2015. The age range of samples from the upper calyx using either BIGopsy (Cook Medical the stone-forming patients and controls was 20 to 80 years. Patients Inc.) or Piranha (Boston Scientific) biopsy forceps. Each group with active urinary tract infection, metabolic and autoimmune of samples was preserved in both 4% paraformaldehyde (PFA) disease, carcinoma, and severe hydronephrosis (grade 3 or 4 for immunohistochemical analysis and RNAlater (Qiagen, according to The Society for Fetal Urology guidelines) were Hilden, Germany) for microarray and qPCR analyses. Laser excluded. coagulation was performed if there was uncontrollable bleeding Samples were individually collected from two different regions in in the biopsied region by irrigation. A ureteral catheter was each patient: renal papillary tissue with RP (P group) and normal inserted at the end ofsurgery forallpatients who underwentPCNL papillary tissue without RP (N group). We also collected normal renal or RIRS. papillary tissue as a control (C group) from seven patients who underwent ureteroscopy or nephrectomy either for screening for Microscopic Analysis of RP Papillae urothelial tumor and hemorrhage or for adhesion of adrenal tumor Slices of 4% PFA-preserved renal sections from the RP group were without urolithiasis. For the nephrectomy, we cut the papillary examined by hematoxylin-eosin, Pizzolato, and von Kossa staining, as mucosa tissue with a scalpel. described previously.48,49

expression of each gene investigated was determined using qPCR performed using TaqMan assays. The data are presented as means6 SEMs. Control values are the average of the data for the C group. *P,0.05 for comparisons between the N and P groups; #P,0.05 compared with the C groups. C8orf4, chromosome 8 open reading frame 4; GDPD1, glycerophosphodiester phosphodiesterase domain containing 1; KCNJ1, potassium channel inwardly rectifying subfamily J member 1; MUC4, mucin 4; NAV2, neuron navigator 2.

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Figure 8. Validation of proinflammatory and apoptotic assay in RP and normal papillary tissues from CaOx SFs and normal papillary tissues from control patients. (A) mRNA expression. The data are presented as the means6SEMs. Control values are the average values of the data for the C group. #P,0.05 compared with the C groups. NOS2, nitric oxide synthase 2. (B) Immunohistochemical staining. Cells stained positive for CD68, CD138, and neutrophil elastase (each arrow) are shownineachrepresentativemicrophotograph.Magnification, 3400.

EDXanalysiswasperformedtomeasurethecomponentsofinorganic blocking solution. The secondary antibody was goat anti-rabbit IgG calcification of RP. The paraffin-embedded sections were dewaxed and gold colloidal particles (10 nm; BBI Solutions, Cardiff, UK). The spec- washed with phosphoric acid buffer. The sections were refixed first with imens were stained with 2% uranyl acetate for 5 minutes and modified 2.5% glutaraldehyde, and then with 2% osmium tetroxide. Dehydration Sato’s lead solution for 1 minute.50,51 was performed using a 50%–100% ethanol series. The samples were embedded in epoxy resin, coated with platinum, and then photo- Microarray Analysis graphed with a scanning electron microscope (S-4800; Hitachi, Tokyo, Total RNA was extracted from the tissues in RNAlater using an RNeasy Japan). The elemental spectra of the RPs in the specimens were deter- Micro Kit (Qiagen). cDNA amplified using the Ovation Pico System mined by performing energy-dispersive x-ray analysis using a Horiba (Nugen, San Carlos, CA) was subjected to transcriptome analysis using EMAX-5770 system (Horiba, Kyoto, Japan). Agilent SurePrint G3 microarrays. Microarray data were analyzed using The microstructure of the RP and surrounding tissue was examined the GeneSpring 13.1 program (Agilent Technologies, Santa Clara, CA). using TEM. The 4% PFA sections were perfusion-fixed in 0.1 mol/L Greater than twofold changes in gene expression between groups were phosphoric acid buffer (20 ml) and 2.5% glutaraldehyde (20 ml), deemed to be significantly different (P,0.01). All microarray data were extracted, washed with phosphoric acid buffer, and fixed with 2% deposited in Gene Expression Omnibus (Acc. No: GSE 73680). osmium tetroxide for 2 hours. The tissues were dehydrated using a Data were analyzed through the use of IPA (Qiagen Redwood City graded ethanol series (50%–100%), embedded in epoxy resin, and po- Inc., Redwood City, CA). Functional analysis was used to identify the lymerized at 60°C for 48 hours. Super slices (99 nm) were double stained biologic functions and/or diseases that were most significant for the with uranium and lead and examined under a JEM-1011 TEM micro- data set. The right-tailed Fisher exact test was used to calculate the P scope (JEOL, Tokyo, Japan). For immunohistochemical staining for value determining the probability that each biologic function and/or TEM, the tissues were incubated overnight with polyclonal anti-human disease assigned to that data set was assigned due to chance alone. A OPN (O-17) rabbit IgG (IBL Co. Ltd., Gunma, Japan) at 4°C in the same network is a graphical representation of the molecular relationships

344 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 333–347, 2017 www.jasn.org CLINICAL RESEARCH

Figure 9. Schema of hypothetical theory of RP formation, based on this study’s results. (A) Anatomy of renal parenchyma. Renal papilla including loop of Henle, interstitial space, and urothelial cells was focused on in the following schemas. (B) Normal status of renal papillary tissue between the loop of Henle and renal calyx. Aligned urothelial cells, interstitial cells, and tubular cells can be seen. (C) Accumulation of immune cells (macrophages and plasma cells), collagen fibers, and OPN; cellular apoptosis occurs due to inflammation and oxidative stress due to activated molecules, such as LCN2, IL11, PTGS1, GPX3, and MMD. The disorder of SLC12A1 and NALCN causes apoptosis of interstitial cells. (D) Aggregation and precipitation of apatite crystals with collagen fibers and OPN result in the replacement from apoptotic interstitial and urothelial cells to RP. (E) A CaOx stone eventually develops and is attached to the RP. between molecules that are supported by at least one reference from Analysis System, version 9.1 (SAS Institute Inc., Cary, NC). Values of the literature, from a textbook, or from canonical information stored P,0.05 were considered statistically significant. in the Ingenuity Knowledge Base. qPCR ACKNOWLEDGMENTS For the qPCR, we used the amplified cDNA that was used for microarray analysis. To assess the gene expression results obtained by We thank Hiroshi Takase from the Medical Sciences Core Laboratory microarray analysis, validation experiments were performed using at Nagoya City University Graduate School for the assistance with TaqMan Gene Expression Assays (Life Technologies, Grand Island, TEM and EDX analyses, and Dr. Takeshi Sakakura from the De- NY) foreach complete mRNA sequence. The primers used are listed in partment of Urology, Konan Kosei Hospital and Dr. Yutaka Iwase Supplemental Table 6. qPCRs were performed using a TaqMan Fast from the Department of Urology, Toyota Kosei Hospital for obtaining Universal PCR Master Mix (4352042; Applied Biosystems) with a approval from the ethical committees of their hospitals. This work ’ 7500 Fast RT-PCR System (Applied Biosystems). Each gene s expres- was supported in part by Grants-in-Aid for Scientific Research from sion was normalized to that of the internal control, glyceraldehyde-3- the Ministry of Education, Culture, Sports, Science and Technology, phosphate dehydrogenase. Japan (Nos. 15H04976, 15K10627, and 25861443), the first research grant of the Japanese Society on Urolithiasis Research, the eighth Immunohistochemical Staining Young Researcher Promotion Grant of the Japanese Urological Immunohistochemistry for LCN2, IL11, SLPI, PTGS1, GPX3, MMD, Association, a medical research grant of the Takeda Science Foun- SCGB1D2, SLC12A1, NALCN, CD68, CD138, and neutrophil elastase dation, and the Medical Research Encouragement Prize of the Japan was performed on 4-mm-thick sliced biopsy sections. The antibodies Medical Association. listed in Supplemental Table 7 were used as primary antibodies. The reactions were analyzed using a Histofine Simple Stain Kit for mouse, rat, or rabbit IgG (Nichirei Biosciences Inc., Tokyo, Japan) according DISCLOSURES ’ to the manufacturer s instructions. None.

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J Am Soc Nephrol 28: 333–347, 2017 Gene Profiling of Randall’s Plaque 347 1 Supplementary Figure 1. Immunohistochemical distribution of urothelial cells, renal tubular cells, and interstitial cells stained

2 by uroplakin III, kidney specific protein, and vimentin, respectively. Magnification, ×100 (inset x400). Representative

3 micrographs were obtained from normal papillary tissues of CaOx stone formers.

1

Supplementary Table 1. Top 100 upregulated genes in papillary tissue of both Randall’s Plaque and normal mucosa with calcium oxalate stone formers compared to those with control patients. Fold Agilent ID Gene Symbol Description change A_23_P128868 OR11H12 Homo sapiens olfactory receptor, family 11, subfamily H, member 12 (OR11H12), mRNA [NM_001013354] 26.613 Homo sapiens killer cell immunoglobulin-like receptor, two domains, short cytoplasmic tail, 2 (KIR2DS2), mRNA A_23_P130815 KIR2DS2 26.224 [NM_012312] A_24_P402855 PROL1 Homo sapiens proline rich, lacrimal 1 (PROL1), mRNA [NM_021225] 23.733 A_24_P917306 ZNF385D zinc finger protein 385D [Source:HGNC Symbol;Acc:26191] [ENST00000494108] 23.050 A_33_P3260667 OR2T34 Homo sapiens olfactory receptor, family 2, subfamily T, member 34 (OR2T34), mRNA [NM_001001821] 20.948 A_33_P3259440 GOLGA6A Homo sapiens golgin A6 family, member A (GOLGA6A), mRNA [NM_001038640] 20.628 A_33_P3417281 MUC4 Homo sapiens mucin 4, cell surface associated (MUC4), transcript variant 1, mRNA [NM_018406] 20.610 A_24_P239176 MUC4 Homo sapiens mucin 4, cell surface associated (MUC4), transcript variant 1, mRNA [NM_018406] 19.965 A_21_P0006968 SFTA1P Homo sapiens surfactant associated 1, pseudogene (SFTA1P), non-coding RNA [NR_027082] 19.344 A_23_P144512 GK2 Homo sapiens glycerol kinase 2 (GK2), mRNA [NM_033214] 19.311 A_33_P3370001 OR2L8 Homo sapiens olfactory receptor, family 2, subfamily L, member 8 (OR2L8), mRNA [NM_001001963] 18.255 A_24_P239177 MUC4 Homo sapiens mucin 4, cell surface associated (MUC4), transcript variant 1, mRNA [NM_018406] 17.474 A_23_P145159 DNAH8 Homo sapiens dynein, axonemal, heavy chain 8 (DNAH8), mRNA [NM_001206927] 16.324 A_33_P3369975 OR2L3 Homo sapiens olfactory receptor, family 2, subfamily L, member 3 (OR2L3), mRNA [NM_001004687] 15.979 A_33_P3216008 SKA3 Homo sapiens spindle and kinetochore associated complex subunit 3 (SKA3), transcript variant 1, mRNA [NM_145061] 15.735 A_33_P3372217 PSG5 pregnancy specific beta-1-glycoprotein 5 [Source:HGNC Symbol;Acc:9522] [ENST00000401992] 15.619 A_33_P3411682 TRIM49C tripartite motif containing 49C [Source:HGNC Symbol;Acc:38877] [ENST00000432771] 15.554 A_33_P3271276 PSG5 Homo sapiens pregnancy specific beta-1-glycoprotein 5 (PSG5), transcript variant 2, mRNA [NM_001130014] 14.939 A_21_P0013761 SSX4B Homo sapiens synovial sarcoma, X breakpoint 4B (SSX4B), transcript variant 1, mRNA [NM_001034832] 14.693 A_23_P45579 HSFY2 Homo sapiens heat shock transcription factor, Y linked 2 (HSFY2), transcript variant 1, mRNA [NM_153716] 14.631 A_24_P626932 MUC3 Homo sapiens SIB 276 intestinal mucin (MUC3) mRNA, partial cds. [AF007191] 14.574 A_23_P11397 TTTY13 Homo sapiens testis-specific transcript, Y-linked 13 (non-protein coding) (TTTY13), long non-coding RNA [NR_001537] 14.513 A_23_P66180 CACNG3 Homo sapiens calcium channel, voltage-dependent, gamma subunit 3 (CACNG3), mRNA [NM_006539] 14.484 A_33_P3405680 ARHGEF3-AS1 Homo sapiens ARHGEF3 antisense RNA 1 (ARHGEF3-AS1), long non-coding RNA [NR_046572] 14.480 A_33_P3384133 C17orf99 Homo sapiens chromosome 17 open reading frame 99 (C17orf99), mRNA [NM_001163075] 14.017 A_24_P167377 OR10P1 Homo sapiens olfactory receptor, family 10, subfamily P, member 1 (OR10P1), mRNA [NM_206899] 13.627 A_33_P3212764 FLJ40039 Homo sapiens cDNA FLJ40039 fis, clone SYNOV2000397. [AK097358] 13.562 A_33_P3346448 PCDH11X Homo sapiens protocadherin 11 X-linked (PCDH11X), transcript variant c, mRNA [NM_032968] 13.213 Homo sapiens killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 2, transcript variant 1, mRNA A_21_P0014000 KIR3DL2 13.198 [NM_006737] A_24_P375322 GGT8P Homo sapiens gamma-glutamyltransferase 8 pseudogene (GGT8P), non-coding RNA [NR_003503] 13.154 A_33_P3424122 TCHHL1 Homo sapiens trichohyalin-like 1 (TCHHL1), mRNA [NM_001008536] 13.146 A_33_P3381777 TREML1 Homo sapiens triggering receptor expressed on myeloid cells-like 1 (TREML1), transcript variant 1, mRNA [NM_178174] 13.097 A_33_P3292886 KRT6A Homo sapiens keratin 6A (KRT6A), mRNA [NM_005554] 12.978 A_23_P54736 GNG13 Homo sapiens guanine nucleotide binding protein (G protein), gamma 13 (GNG13), mRNA [NM_016541] 12.929 A_33_P3278279 TXNDC8 Homo sapiens thioredoxin domain containing 8 (spermatozoa) (TXNDC8), transcript variant 3, mRNA [NM_001286947] 12.755 A_33_P3395581 TRIM53AP Homo sapiens tripartite motif containing 53A, pseudogene (TRIM53AP), non-coding RNA [NR_028346] 12.684 A_24_P16353 TRIM53AP Homo sapiens tripartite motif containing 53A, pseudogene (TRIM53AP), non-coding RNA [NR_028346] 12.583 A_23_P150467 OR5I1 Homo sapiens olfactory receptor, family 5, subfamily I, member 1 (OR5I1), mRNA [NM_006637] 12.507 A_33_P3364488 FCAMR Homo sapiens Fc receptor, IgA, IgM, high affinity (FCAMR), transcript variant 3, mRNA [NM_001170631] 12.445 A_33_P3405479 OR4C45 Homo sapiens olfactory receptor, family 4, subfamily C, member 45 (OR4C45), mRNA [NM_001005513] 12.445 A_24_P623782 FRG2 Homo sapiens FSHD region gene 2 (FRG2), transcript variant 2, mRNA [NM_001005217] 12.288 A_23_P76234 RPH3A Homo sapiens rabphilin 3A homolog (mouse) (RPH3A), transcript variant 1, mRNA [NM_001143854] 12.194 A_32_P720220 C12orf36 Homo sapiens chromosome 12 open reading frame 36 (C12orf36), long non-coding RNA [NR_036555] 12.117 A_24_P400604 RBMY1B Homo sapiens RNA binding motif protein, Y-linked, family 1, member B (RBMY1B), mRNA [NM_001006121] 12.099 A_33_P3424507 OR51F1 Homo sapiens olfactory receptor, family 51, subfamily F, member 1 (OR51F1), mRNA [NM_001004752] 11.966 A_33_P3358143 C10orf71 Homo sapiens chromosome 10 open reading frame 71 (C10orf71), mRNA [NM_001135196] 11.840 A_23_P81018 PDHA2 Homo sapiens pyruvate dehydrogenase (lipoamide) alpha 2 (PDHA2), mRNA [NM_005390] 11.814 A_23_P409502 NLRP9 Homo sapiens NLR family, pyrin domain containing 9 (NLRP9), mRNA [NM_176820] 11.811 A_23_P94647 OR1L3 Homo sapiens olfactory receptor, family 1, subfamily L, member 3 (OR1L3), mRNA [NM_001005234] 11.775 A_33_P3319900 PRG1 Homo sapiens p53-responsive gene 1 (PRG1), long non-coding RNA [NR_026881] 11.738 A_23_P96291 MAGEA1 Homo sapiens melanoma antigen family A, 1 (directs expression of antigen MZ2-E) (MAGEA1), mRNA [NM_004988] 11.698 A_33_P3363071 ITIH6 Homo sapiens inter-alpha-trypsin inhibitor heavy chain family, member 6 (ITIH6), mRNA [NM_198510] 11.618 A_23_P62099 MAGEC2 Homo sapiens melanoma antigen family C, 2 (MAGEC2), mRNA [NM_016249] 11.546 A_33_P3317225 OR2L5 Homo sapiens olfactory receptor, family 2, subfamily L, member 5 (OR2L5), mRNA [NM_001258284] 11.510 A_21_P0012072 REG1P Homo sapiens regenerating islet-derived 1 pseudogene (REG1P), non-coding RNA [NR_002714] 11.507 A_33_P3216570 MUC5AC PREDICTED: Homo sapiens mucin 5AC, oligomeric mucus/gel-forming (MUC5AC), partial mRNA [XM_003119481] 11.473 A_23_P15832 OR1D5 Homo sapiens olfactory receptor, family 1, subfamily D, member 5 (OR1D5), mRNA [NM_014566] 11.419 A_33_P3239278 OR4D10 Homo sapiens olfactory receptor, family 4, subfamily D, member 10 (OR4D10), mRNA [NM_001004705] 11.413 A_23_P409888 FAM83C Homo sapiens family with sequence similarity 83, member C (FAM83C), mRNA [NM_178468] 11.399 A_24_P794458 MOBP Homo sapiens myelin-associated oligodendrocyte basic protein (MOBP), transcript variant 3, mRNA [NM_182935] 11.388 A_24_P70906 PDILT Homo sapiens protein disulfide isomerase-like, testis expressed (PDILT), mRNA [NM_174924] 11.383 A_32_P196193 PAQR9 Homo sapiens progestin and adipoQ receptor family member IX (PAQR9), mRNA [NM_198504] 11.343 A_33_P3326075 CYP2C19 Homo sapiens cytochrome P450, family 2, subfamily C, polypeptide 19 (CYP2C19), mRNA [NM_000769] 11.256 A_21_P0012346 FAM230C family with sequence similarity 230, member C [Source:HGNC Symbol;Acc:24482] [ENST00000411581] 11.142 A_33_P3387781 OR2L1P Homo sapiens olfactory receptor, family 2, subfamily L, member 1 pseudogene (OR2L1P), non-coding RNA [NR_002145] 11.126 A_23_P65189 PDX1 Homo sapiens pancreatic and duodenal homeobox 1 (PDX1), mRNA [NM_000209] 11.056 A_24_P298716 FAM197Y2 Homo sapiens family with sequence similarity 197, Y-linked, member 2 (FAM197Y2), non-coding RNA [NR_001553] 11.055 A_33_P3300312 DMBT1 Homo sapiens deleted in malignant brain tumors 1 (DMBT1), transcript variant 2, mRNA [NM_007329] 11.018 A_23_P411157 WNT1 Homo sapiens wingless-type MMTV integration site family, member 1 (WNT1), mRNA [NM_005430] 11.008 A_23_P28023 OR7A17 Homo sapiens olfactory receptor, family 7, subfamily A, member 17 (OR7A17), mRNA [NM_030901] 10.994 A_23_P321892 HRG Homo sapiens histidine-rich glycoprotein (HRG), mRNA [NM_000412] 10.983 A_33_P3258206 OR6N2 Homo sapiens olfactory receptor, family 6, subfamily N, member 2 (OR6N2), mRNA [NM_001005278] 10.952 A_33_P3342153 USP17L8 Homo sapiens ubiquitin specific peptidase 17-like family member 8 (USP17L8), mRNA [NM_001256872] 10.950 A_23_P84000 CACNG2 Homo sapiens calcium channel, voltage-dependent, gamma subunit 2 (CACNG2), mRNA [NM_006078] 10.884 Homo sapiens myelin-associated oligodendrocyte basic protein (MOBP), transcript variant 6, non-coding RNA A_33_P3391439 MOBP 10.858 [NR_103505] A_21_P0001324 MGC27382 Homo sapiens cDNA FLJ34438 fis, clone HLUNG2001144. [AK091757] 10.853 A_24_P30557 TBX5 Homo sapiens T-box 5 (TBX5), transcript variant 1, mRNA [NM_000192] 10.808 A_32_P129120 FAM182A Homo sapiens family with sequence similarity 182, member A (FAM182A), long non-coding RNA [NR_026713] 10.792 A_33_P3270360 OR1S1 Homo sapiens olfactory receptor, family 1, subfamily S, member 1 (OR1S1), mRNA [NM_001004458] 10.741 Homo sapiens membrane-spanning 4-domains, subfamily A, member 14 (MS4A14), transcript variant 4, mRNA A_33_P3290577 MS4A14 10.725 [NM_001261828] A_33_P3525377 FLJ41130 Homo sapiens cDNA FLJ41130 fis, clone BRACE2022549. [AK123125] 10.676 A_23_P93169 LGSN Homo sapiens lengsin, lens protein with glutamine synthetase domain (LGSN), transcript variant 1, mRNA [NM_016571] 10.663 A_23_P97826 TECTB Homo sapiens tectorin beta (TECTB), mRNA [NM_058222] 10.650 A_33_P3213288 AIF1 Homo sapiens allograft inflammatory factor 1 (AIF1), transcript variant 2, mRNA [NM_004847] 10.645 A_24_P325146 KRTAP4-8 Homo sapiens keratin associated protein 4-8 (KRTAP4-8), mRNA [NM_031960] 10.621 A_23_P350396 CDSN Homo sapiens corneodesmosin (CDSN), mRNA [NM_001264] 10.614 A_23_P102694 DEFB129 Homo sapiens defensin, beta 129 (DEFB129), mRNA [NM_080831] 10.475 A_23_P353667 MIR7-3HG Homo sapiens MIR7-3 host gene (non-protein coding) (MIR7-3HG), long non-coding RNA [NR_027148] 10.464 A_24_P387875 KCNJ10 Homo sapiens potassium inwardly-rectifying channel, subfamily J, member 10 (KCNJ10), mRNA [NM_002241] 10.456 A_32_P88262 SLC7A14 Homo sapiens solute carrier family 7, member 14 (SLC7A14), mRNA [NM_020949] 10.455 A_33_P3286846 TP53TG3C Homo sapiens TP53 target 3C (TP53TG3C), transcript variant 1, mRNA [NM_001205259] 10.446 A_33_P3209406 RTBDN Homo sapiens retbindin (RTBDN), transcript variant 3, mRNA [NM_001270440] 10.437 A_32_P219660 SPANXA1 Homo sapiens sperm protein associated with the nucleus, X-linked, family member A1 (SPANXA1), mRNA [NM_013453] 10.419 A_24_P342484 CRP Homo sapiens C-reactive protein, pentraxin-related (CRP), mRNA [NM_000567] 10.414 A_23_P376124 RNASE8 Homo sapiens ribonuclease, RNase A family, 8 (RNASE8), mRNA [NM_138331] 10.399 A_23_P380526 DPPA4 Homo sapiens developmental pluripotency associated 4 (DPPA4), mRNA [NM_018189] 10.387 A_23_P1691 MMP1 Homo sapiens matrix metallopeptidase 1 (interstitial collagenase) (MMP1), transcript variant 1, mRNA [NM_002421] 10.378 A_21_P0000439 SNORD115-3 Homo sapiens small nucleolar RNA, C/D box 115-3 (SNORD115-3), small nucleolar RNA [NR_003295] 10.372 A_21_P0012347 FAM230C family with sequence similarity 230, member C [Source:HGNC Symbol;Acc:24482] [ENST00000411581] 10.366

Supplementary Table 2. Top 100 downregulated genes in papillary tissue of both Randall’s Plaque and normal mucosa with calcium oxalate stone formers compared to those with control patients.

Gene Fold Agilent ID Description Symbol change

A_23_P58082 CCDC80 Homo sapiens coiled-coil domain containing 80 (CCDC80), transcript variant 1, mRNA [NM_199511] -13.184 A_23_P150053 ACTA2 Homo sapiens actin, alpha 2, smooth muscle, aorta (ACTA2), transcript variant 2, mRNA [NM_001613] -12.840 A_24_P277934 COL1A2 Homo sapiens collagen, type I, alpha 2 (COL1A2), mRNA [NM_000089] -12.467 A_23_P216361 COL14A1 Homo sapiens collagen, type XIV, alpha 1 (COL14A1), mRNA [NM_021110] -12.049 A_33_P3281191 NID1 Homo sapiens nidogen 1 (NID1), mRNA [NM_002508] -11.939 A_23_P33196 COL5A2 Homo sapiens collagen, type V, alpha 2 (COL5A2), mRNA [NM_000393] -11.736 A_23_P350001 GUCY1A2 Homo sapiens guanylate cyclase 1, soluble, alpha 2 (GUCY1A2), transcript variant 2, mRNA [NM_000855] -11.347 A_33_P3382924 SPARC Homo sapiens secreted protein, acidic, cysteine-rich (osteonectin) (SPARC), mRNA [NM_003118] -11.297 A_33_P3238166 PXDN Homo sapiens peroxidasin homolog (Drosophila) (PXDN), mRNA [NM_012293] -11.209 A_24_P935491 COL3A1 Homo sapiens collagen, type III, alpha 1 (COL3A1), mRNA [NM_000090] -11.107 A_23_P143817 MYLK Homo sapiens myosin light chain kinase (MYLK), transcript variant 1, mRNA [NM_053025] -11.035 A_24_P921366 CALD1 Homo sapiens caldesmon 1 (CALD1), transcript variant 1, mRNA [NM_033138] -10.885 A_33_P3210488 COL6A3 Homo sapiens collagen, type VI, alpha 3 (COL6A3), transcript variant 1, mRNA [NM_004369] -10.735 A_23_P152305 CDH11 Homo sapiens cadherin 11, type 2, OB-cadherin (osteoblast) (CDH11), mRNA [NM_001797] -10.719 A_23_P99063 LUM Homo sapiens lumican (LUM), mRNA [NM_002345] -10.322 A_23_P31064 MOXD1 Homo sapiens monooxygenase, DBH-like 1 (MOXD1), transcript variant 2, mRNA [NM_015529] -10.303 A_33_P3304668 COL1A1 Homo sapiens collagen, type I, alpha 1 (COL1A1), mRNA [NM_000088] -10.261 A_24_P168925 CHRDL1 Homo sapiens chordin-like 1 (CHRDL1), transcript variant 1, mRNA [NM_001143981] -10.190 A_33_P3382856 DCN Homo sapiens decorin (DCN), transcript variant E, mRNA [NM_133507] -10.138 A_23_P21618 PDZRN3 Homo sapiens PDZ domain containing ring finger 3 (PDZRN3), mRNA [NM_015009] -9.845 A_24_P373152 CFL2 Homo sapiens cofilin 2 (muscle) (CFL2), transcript variant 1, mRNA [NM_021914] -9.827 A_23_P151805 FBLN5 Homo sapiens fibulin 5 (FBLN5), mRNA [NM_006329] -9.626 A_23_P2492 C1S Homo sapiens complement component 1, s subcomponent (C1S), transcript variant 2, mRNA [NM_001734] -9.518 A_23_P88069 LHFP Homo sapiens lipoma HMGIC fusion partner (LHFP), mRNA [NM_005780] -9.487 A_23_P415791 KIAA1210 Homo sapiens KIAA1210 (KIAA1210), mRNA [NM_020721] -9.485 A_23_P214168 COL12A1 Homo sapiens collagen, type XII, alpha 1 (COL12A1), transcript variant long, mRNA [NM_004370] -9.454 A_33_P3271455 PXDN Homo sapiens peroxidasin homolog (Drosophila) (PXDN), mRNA [NM_012293] -9.297 A_32_P80850 COL14A1 Homo sapiens collagen, type XIV, alpha 1 (COL14A1), mRNA [NM_021110] -9.180 A_23_P132956 UCHL1 Homo sapiens ubiquitin carboxyl-terminal esterase L1 (ubiquitin thiolesterase) (UCHL1), mRNA [NM_004181] -9.083 A_33_P3351416 SMOC2 Homo sapiens SPARC related modular calcium binding 2 (SMOC2), transcript variant 1, mRNA [NM_022138] -9.041 A_21_P0004312 MIR143HG Homo sapiens MIR143 host gene (non-protein coding) (MIR143HG), long non-coding RNA [NR_027180] -9.036 A_23_P64873 DCN Homo sapiens decorin (DCN), transcript variant A1, mRNA [NM_001920] -8.994 A_21_P0000096 CPXM1 Homo sapiens carboxypeptidase X (M14 family), member 1 (CPXM1), transcript variant 1, mRNA [NM_019609] -8.896 A_32_P32254 COL6A1 Homo sapiens collagen, type VI, alpha 1 (COL6A1), mRNA [NM_001848] -8.858 A_23_P88404 TGFB3 Homo sapiens transforming growth factor, beta 3 (TGFB3), mRNA [NM_003239] -8.841 A_33_P3373358 GJC1 Homo sapiens gap junction protein, gamma 1, 45kDa (GJC1), transcript variant 1, mRNA [NM_005497] -8.834 A_23_P85015 MAOB Homo sapiens monoamine oxidase B (MAOB), mRNA [NM_000898] -8.795 A_23_P50786 CLIP3 Homo sapiens CAP-GLY domain containing linker protein 3 (CLIP3), transcript variant 2, mRNA [NM_015526] -8.544 Homo sapiens solute carrier family 8 (sodium/calcium exchanger), member 1 (SLC8A1), transcript variant A, mRNA A_24_P136866 SLC8A1 -8.541 [NM_021097] A_32_P192376 ENPP1 Homo sapiens ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), mRNA [NM_006208] -8.457 A_33_P3348239 FBN1 Homo sapiens fibrillin 1 (FBN1), mRNA [NM_000138] -8.441 A_23_P101093 COPZ2 Homo sapiens coatomer protein complex, subunit zeta 2 (COPZ2), mRNA [NM_016429] -8.434 A_24_P334130 FN1 Homo sapiens fibronectin 1 (FN1), transcript variant 7, mRNA [NM_054034] -8.398 A_23_P58251 CPZ Homo sapiens carboxypeptidase Z (CPZ), transcript variant 3, mRNA [NM_001014448] -8.391 A_23_P34744 CTSK Homo sapiens cathepsin K (CTSK), mRNA [NM_000396] -8.355 A_23_P205031 COL4A2 Homo sapiens collagen, type IV, alpha 2 (COL4A2), mRNA [NM_001846] -8.336 A_24_P12626 CAV1 Homo sapiens caveolin 1, caveolae protein, 22kDa (CAV1), transcript variant 1, mRNA [NM_001753] -8.321 A_33_P3212257 MMRN1 Homo sapiens multimerin 1 (MMRN1), mRNA [NM_007351] -8.315 A_23_P67661 COX7A1 Homo sapiens cytochrome c oxidase subunit VIIa polypeptide 1 (muscle) (COX7A1), mRNA [NM_001864] -8.296 A_32_P152437 AKAP12 Homo sapiens A kinase (PRKA) anchor protein 12 (AKAP12), transcript variant 1, mRNA [NM_005100] -8.245 A_32_P133072 SPON1 Homo sapiens spondin 1, extracellular matrix protein (SPON1), mRNA [NM_006108] -8.229 A_23_P157299 AEBP1 Homo sapiens AE binding protein 1 (AEBP1), mRNA [NM_001129] -8.214 A_23_P157865 TNC Homo sapiens tenascin C (TNC), mRNA [NM_002160] -8.136 A_19_P00324470 MIR143HG Homo sapiens MIR143 host gene (non-protein coding) (MIR143HG), long non-coding RNA [NR_027180] -8.096 A_23_P83028 RECK Homo sapiens reversion-inducing-cysteine-rich protein with kazal motifs (RECK), mRNA [NM_021111] -8.088 A_33_P3254096 PI15 Homo sapiens peptidase inhibitor 15 (PI15), mRNA [NM_015886] -8.032 A_33_P3278313 MSRB3 Homo sapiens methionine sulfoxide reductase B3 (MSRB3), transcript variant 3, mRNA [NM_001193460] -7.980 A_23_P157914 MAMDC2 Homo sapiens MAM domain containing 2 (MAMDC2), mRNA [NM_153267] -7.957 A_33_P3269539 COL6A2 Homo sapiens collagen, type VI, alpha 2 (COL6A2), transcript variant 2C2a, mRNA [NM_058174] -7.950 A_23_P50426 KANK2 Homo sapiens KN motif and ankyrin repeat domains 2 (KANK2), transcript variant 1, mRNA [NM_015493] -7.930 A_23_P64879 KCNJ8 Homo sapiens potassium inwardly-rectifying channel, subfamily J, member 8 (KCNJ8), mRNA [NM_004982] -7.928 A_23_P41476 SHISA3 Homo sapiens shisa family member 3 (SHISA3), mRNA [NM_001080505] -7.910 Homo sapiens sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 1 (SPOCK1), mRNA A_24_P354689 SPOCK1 -7.857 [NM_004598] A_33_P3382177 TIMP2 Homo sapiens TIMP metallopeptidase inhibitor 2 (TIMP2), mRNA [NM_003255] -7.781 A_21_P0003931 MIR143HG Homo sapiens MIR143 host gene (non-protein coding) (MIR143HG), long non-coding RNA [NR_027180] -7.717 A_23_P361014 TSHZ3 Homo sapiens teashirt zinc finger homeobox 3 (TSHZ3), mRNA [NM_020856] -7.717 A_33_P3229617 SGCE Homo sapiens sarcoglycan, epsilon (SGCE), transcript variant 1, mRNA [NM_001099401] -7.663 A_23_P213857 C7 Homo sapiens complement component 7 (C7), mRNA [NM_000587] -7.650 Homo sapiens sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 (SVEP1), mRNA A_23_P216596 SVEP1 -7.649 [NM_153366] A_24_P879740 MAP1B Homo sapiens microtubule-associated protein 1B (MAP1B), mRNA [NM_005909] -7.603 A_33_P3363799 NCAM1 Homo sapiens neural cell adhesion molecule 1 (NCAM1), transcript variant 5, mRNA [NM_001242607] -7.556 A_23_P356554 BAG2 Homo sapiens BCL2-associated athanogene 2 (BAG2), mRNA [NM_004282] -7.543 A_23_P144959 VCAN Homo sapiens versican (VCAN), transcript variant 1, mRNA [NM_004385] -7.522 A_23_P100711 PMP22 Homo sapiens peripheral myelin protein 22 (PMP22), transcript variant 1, mRNA [NM_000304] -7.508 A_33_P3321432 FAM198B Homo sapiens family with sequence similarity 198, member B (FAM198B), transcript variant 2, mRNA [NM_016613] -7.467 A_23_P82990 OGN Homo sapiens osteoglycin (OGN), transcript variant 1, mRNA [NM_033014] -7.426 A_33_P3230478 C1S Homo sapiens complement component 1, s subcomponent (C1S), transcript variant 1, mRNA [NM_201442] -7.389 A_23_P210425 MYL9 Homo sapiens myosin, light chain 9, regulatory (MYL9), transcript variant 2, mRNA [NM_181526] -7.367 A_33_P3344477 FERMT2 Homo sapiens fermitin family member 2 (FERMT2), transcript variant 2, mRNA [NM_001134999] -7.341 A_23_P257649 RBP1 Homo sapiens retinol binding protein 1, cellular (RBP1), transcript variant 1, mRNA [NM_002899] -7.333 A_24_P261417 DKK3 Homo sapiens dickkopf WNT signaling pathway inhibitor 3 (DKK3), transcript variant 1, mRNA [NM_015881] -7.298 A_23_P113351 SPARCL1 Homo sapiens SPARC-like 1 (hevin) (SPARCL1), transcript variant 2, mRNA [NM_004684] -7.279 A_23_P432573 MRGPRF Homo sapiens MAS-related GPR, member F (MRGPRF), transcript variant 2, mRNA [NM_145015] -7.246 A_24_P131522 ANTXR1 Homo sapiens anthrax toxin receptor 1 (ANTXR1), transcript variant 1, mRNA [NM_032208] -7.239 A_24_P55496 OSR2 Homo sapiens odd-skipped related transciption factor 2 (OSR2), transcript variant 2, mRNA [NM_053001] -7.235 A_33_P3231953 COL12A1 Homo sapiens collagen, type XII, alpha 1 (COL12A1), transcript variant long, mRNA [NM_004370] -7.216 A_23_P97990 HTRA1 Homo sapiens HtrA serine peptidase 1 (HTRA1), mRNA [NM_002775] -7.184 A_23_P98645 DCHS1 Homo sapiens dachsous cadherin-related 1 (DCHS1), mRNA [NM_003737] -7.147 A_24_P925062 MXRA7 Homo sapiens matrix-remodelling associated 7 (MXRA7), transcript variant 1, mRNA [NM_001008528] -7.105 Homo sapiens extracellular matrix protein 2, female organ and adipocyte specific (ECM2), transcript variant 1, mRNA A_23_P303671 ECM2 -7.092 [NM_001393] A_33_P3364741 MRC2 Homo sapiens mannose receptor, C type 2 (MRC2), mRNA [NM_006039] -7.072 A_23_P369899 TMEM158 Homo sapiens transmembrane protein 158 (gene/pseudogene) (TMEM158), mRNA [NM_015444] -7.038 A_24_P937405 PRSS23 Homo sapiens protease, serine, 23 (PRSS23), mRNA [NM_007173] -7.002 A_19_P00807411 RNF180 Homo sapiens ring finger protein 180 (RNF180), transcript variant 1, mRNA [NM_001113561] -6.993 A_32_P36694 JAZF1 Homo sapiens JAZF zinc finger 1 (JAZF1), mRNA [NM_175061] -6.980 A_33_P3270599 TPM2 Homo sapiens tropomyosin 2 (beta) (TPM2), transcript variant 2, mRNA [NM_213674] -6.974 Homo sapiens metastasis associated lung adenocarcinoma transcript 1 (non-protein coding) (MALAT1), long A_19_P00324839 MALAT1 -6.965 non-coding RNA [NR_002819] A_33_P3236102 IER5L Homo sapiens immediate early response 5-like (IER5L), mRNA [NM_203434] -6.866 A_33_P3366120 FLNA Homo sapiens filamin A, alpha (FLNA), transcript variant 2, mRNA [NM_001110556] -6.822

Supplementary Table 3. Top 15 networks in both Randall’s Plaque and normal papillary tissue gene expressions are differed from control mucosa by 2 folds

Network Focus Molecules in Network Score Top Diseases and Functions ID Molecules ABO,ATP10A,C11orf54,CDHR1,CRY1,CSTF2T,DNAAF3,DPF3,DPM2,DTWD1,FAM53C,FNDC3B,F OXRED1,GOLIM4,HAAO,HHLA2,HIST1H2BG,HNF4A,KDELC1,LINC00574,LINC01558,PRODH2,R Developmental Disorder, Hereditary 1 41 35 EG1A,RNF113A,SGCB,SGCE,SPATA5L1,STXBP5L,TCEAL3,TMEM79,TMEM242,TMIGD1,TTC22, Disorder, Respiratory Disease VAC14,ZNHIT6 ABCC9,C1QTNF6,CDC42SE2,CENPM,CENPN,CFL2,DCBLD2,DDX54,DNAH2,DNAH3,DNAH8,DN Cardiac Arteriopathy, Cardiovascular 2 AH9,DNAH10,DNAH12,DNAH17,DNAI1,EML1,ESR1,GAL,GULP1,Kcnj,KCNJ8,KCTD6,KLHL24,NP 38 34 Disease, Organismal Injury and W,ONECUT2,PCDHB5,PCDHB17P,PGC,PRDM2,RBM23,RTN4RL1,TMEM72,UMODL1,VEZF1 Abnormalities

ADGRA1,ADGRG4,ADGRG5,ADGRG7,Gpcr,GPR12,GPR35,GPR55,GPR61,GPR62,GPR88,GPR1 Cell-To-Cell Signaling and Interaction, 3 01,GPR139,GPR148,GPR155,GPR157,GPR182,GPR137C,HTR6,HTR5A,MC3R,MC5R,MCHR1,N 38 34 Cell Signaling, Nucleic Acid MBR,NPBWR2,NTSR1,NTSR2,OPRL1,RGR,RRH,RXFP2,SLC52A1,TAAR5,TAAR8,XCR1 Metabolism BNIP3,CCDC57,CCDC155,CD302,CDC20B,FAM90A1,FATE1,FRA10AC1,Gsk3,H2AFB3 (includes Cardiovascular System Development others),KRTAP10-1,KRTAP10-5,KRTAP2-3/KRTAP2-4,KRTAP3-2,KRTAP4-2,LCE3E,LCE4A,LDLR and Function, Auditory and Vestibular 4 36 33 AD1,LNPEP,MOBP,OTX1,PAQR9,PLAGL2,PRM2,S6K1,SPATA8,STRIP2,SUN3,TMCC2,TMEM190 System Development and Function, ,TRAF3IP3,TRIM36,TSGA10IP,TSHZ3,ZFC3H1 Embryonic Development ABI2,ACTN2,ADAM29,APBA1,Caspase Cancer, Connective Tissue Disorders, 3/7,CFAP61,CTAGE1,DDN,DENND2A,DLG4,DLGAP2,DLGAP3,ENPP1,FAM81A,GAS2L2,GRM5,H 5 36 33 Dermatological Diseases and UNK,KCNJ5,KCNJ6,KCNJ10,MAGI1,MARCH10,NEFL,NLGN1,NMDA Receptor,PRAMEF4 Conditions (includes others),ROBO2,RTKN2,SDK2,SLIT1,SNX22,SPATA16,SYNPO2L,ZDHHC8,ZIC5 alpha2-adrenergic receptor,ALX4,B4GAT1,DLX1,ELAVL2,Focal adhesion kinase,GUCY,GUCY1A2,HOXC13,HOXD12,IRX4,LHX2,LHX9,LMX1B,LPP,NEUROD1,NHLH1,NR2 Embryonic Development, Organismal 6 34 32 E1,NR2E3,OLFM1,OR8B8,OSR2,PALLD,PAX9,POLK,POU4F1,POU4F2,PRRX1,RCVRN,RUNDC3 Development, Gene Expression A,SHPRH,SSBP3,SSX4/SSX4B,TBL3,ZFHX4 AHSG,ASIC2,ATP13A4,AZGP1,BOLL,BPIFA1,BRIP1,C1orf94,CD177,COL1A2,COL6A1,DMRT1,E Amino Acid Metabolism, NaC,ERCC2,FGF18,GJA3,HIST1H2BC,HNRNPLL,IFN alpha 7 32 31 Post-Translational Modification, Small receptor,KLK14,NR2F2,P4HA1,P4HA2,p85 (pik3r),PPP3R1,PSG1,QKI,RBMY1A1 (includes Molecule Biochemistry others),RFX2,SEMG2,SHBG,STRA8,TFIIH,UVSSA,ZNF385C Organismal Injury and Abnormalities, AKR1D1,ALPP,CDYL2,CHURC1,CRABP2,FBXL7,FHL1,FLRT2,GFI1,H3F3A/H3F3B,HOXA10,HSD Dermatological Diseases and 8 3B1,KDELC2,KDM4B,KLK12,KMT2D,LYZL1,MMRN1,MYOM3,N4BP2L2,NAA38,NDUFS7,Neurotro 32 31 Conditions, Cell-mediated Immune phin,NFIL3,NKTR,PAX7,PTGR1,RELL1,Shc,SLC38A6,Sos,TFAP2D,TPP1,Trk Receptor,XKRX Response ACKR3,Adaptor protein 2,Beta Cardiovascular Disease, Congenital Arrestin,C1orf74,CALCR,CFC1/CFC1B,Clathrin,DAB2,DPPA2,DPPA4,EDNRA,ESPL1,F2RL2,Fgf,F 9 32 31 Heart Anomaly, Developmental GF13,FGF16,FOXJ3,FZD1,FZD2,HK3,KIFC1,KIT,LGALS1,MICAL1,MITF,MUC16,PAX3,PITX2,RA Disorder MP1,SLC19A1,SLC45A2,SPC24,SYT6,TBX4,TFAP2A ABCC2,BCAN,chymotrypsin,Collagen type XVIII,CTRC,DKK3,EFEMP2,FBLN1,FBN1,FN1,FXR ligand-FXR-Retinoic Post-Translational Modification, 10 acid-RXRα,GYPA,GYPB,HAPLN4,HBE1,HBG1,HBQ1,HBZ,hemoglobin,HIST1H2BD,HIST1H4C,HI 30 30 Protein Synthesis, Cellular ST1H4E,HIST1H4F,HIST1H4H,HIST1H4K,IL22RA1,KIF21B,KLF1,KMT2E,MDM4,SLC4A1,TMPRS Development S6,TRIM10,Trypsinogen,ZAK ANKRA2,ARNT,CARTPT,CCKBR,CPS1,Creb,CRHR2,DAPK3,DRD3,FFAR4,FSH,GHRHR,GIPR,G Cell Signaling, Nucleic Acid 11 NPAT,GNRH,GPR119,HNF1A,HRH3,INS,KIAA1324,LEP,Lh,NDN,P2RY14,PDX1,Proinsulin,PTGER 30 30 Metabolism, Small Molecule 3,QRFP,REG1B,REG1P,SLC2A2,SST,SSTR3,XKR4,ZNF331 Biochemistry

ARRDC3,CAGE1,CCDC33,CHRDL1,CT55,Ctnna,CTNNA3,DYDC1,Filamin,FLNA,HAS1,HEXB,HO Inflammatory Disease, Respiratory 12 XD10,Integrinβ,KLHL38,LGALS14,MAGEC2,MAOB,MBIP,NCS1,NREP,OR4D6,PAK1,PARP2,POL 28 29 Disease, Hereditary Disorder R1C,PRSS21,Rnr,Sapk,SAXO1,STARD6,TRIM42,TXLNB,UBIQUITIN LIGASE,USHBP1,ZFYVE26 26s Proteasome,ADAMTS17,ADARB2,AHNAK2,APCDD1,ASB10,CDKN1B,CNOT6L,DCLK3, Cell Cycle, Dermatological Diseases DNAJC5B,DNAJC5G,GLI3,GPT2,GRPR,HISTONE,HSP,Hsp70,Hsp90,HSPA12A,HSPB9,KLK3,LO 13 28 29 and Conditions, Developmental XL2,MGEA5,NAP1L1,NEAT1,ODF1,PRDM9,PUM1,SPATA2L,SSX3,SSX5,TSACC,TSSK6,Ubiquitin Disorder ,ZSCAN1 AKAP,AKAP4,AKAP12,AKAP14,CABYR,cldn,CLDN9,CLDN17,CTPS1,DCD,DCT,Dlg,EMX1,ESX1, Nervous System Development and 14 FAM107A,FLVCR2,HOXB1,LHX5,MEIS1,MITF-p300/CBP,MPDZ,OR1D2,OR2AG2,OR5D16,OTX2, 26 28 Function, Organ Morphology, Pka,RAD51D,ROPN1,SLC39A12,SLC6A20,SOX2-OCT4-NANOG,sPla2,TPH1,TPH2,XKR3 Organismal Development Ampa Receptor,CABP1,CABP2,cacn,CACNA1S,Cacng,CACNG2,CACNG3,CACNG7,CALML5, Cell-To-Cell Signaling and Interaction, Calmodulin,CaMKII,CNTN2,CPLX2,CPLX4,EEA1,GPHA2,N-type Calcium Nervous System Development and 15 26 28 Channel,NEUROD2,PCP4,PHOSPHO1,RAB22A,RIMBP2,RIMS1,RPH3A,SCN2A, Function, Cellular Assembly and SEZ6,Snare,SRY,SYN3,SYT1,SYT3,TRPM3,TSHB,WNK4 Organization

Supplementary Table 4. All networks in Randall’s Plaque tissue gene expression is differed from normal papillary tissue by 2 folds

Network Focus Top Diseases and Molecules in Network Score ID Molecules Functions

ADAMTS12,Akt,C8orf4,CAPG,CCL20,CCL22,cyclooxygenase,ERK1/2,FAM3B,gelati Inflammatory Response, nase,GHRH,IL1,IL11,IL17C,IRF6,Kcnj,KCNJ1,LCN2,MEOX2,MMD,MUC4,NALCN,N 1 25 10 Endocrine System Disorders, FAT5,PI3K (complex),Pka,POU3F3,Pro-inflammatory Cytokine, Hereditary Disorder PROK1,PRTN3,PTGS1,S100a7a,SFTPB,SLC12A1,SLPI,Tgf beta ADAT2,ADRB2,ANLN,ATF5,CAPG,CCL17,CKAP2,CORO1C,DHRS9,DRD4,GABBR Molecular Transport, Cellular 2,GDPD1,Girk,GPX3,KCNJ1,KCNJ5,KCNJ6,KCNJ10,KIF23,NAV2,OXSR1,PGRMC1 2 19 8 Development, Cellular ,PPRC1,PRDM16,PTGFR,RBM15B,RGS9,SCGB1D2,SEPHS1,SREBF1,TAGLN2,TP Growth and Proliferation 53,TRIO,UBC,ZNF705G

Supplementary Table 5. Upstream regulators for genes which showed over/lower 2 folds between Randall’s Plaque and normal papillary tissue in CaOx stone formers. Predicted Activation p-value of Upstream Regulator Molecule Type Activation Target molecules in dataset z-score overlap State IL1B cytokine Activated 2.400 0.000028 IL11,KCNJ1,LCN2,MUC4,PTGS1,SLC12A1 lipopolysaccharide chemical drug Activated 2.360 0.000141 AQP1,IL11,KCNJ1,LCN2,PTGS1,SLC12A1,SLPI

TNF cytokine Activated 2.214 0.000001 AQP1,IL11,KCNJ1,LCN2,MMD,MUC4,PTGS1,SLC12A1,SLPI phorbol myristate acetate chemical drug Activated 2.169 0.000696 IL11,LCN2,MUC4,PTGS1,SLPI

TGFB1 growth factor 1.972 0.006700 AQP1,IL11,LCN2,MUC4,PTGS1

TGFA growth factor 1.969 0.000001 LCN2,MUC4,PTGS1,SLPI

IFNG cytokine 1.424 0.000025 AQP1,IL11,KCNJ1,LCN2,MUC4,SLC12A1,SLPI chemical - endogenous tretinoin 1.102 0.000490 AQP1,DHRS9,MUC4,NAV2,PTGS1,SLPI mammalian HOXA10 transcription regulator 0.000 0.000010 AQP1,DHRS9,IL11,LCN2 chemical - endogenous beta-estradiol -0.302 0.000020 AQP1,C8orf4,DHRS9,GPX3,LCN2,MMD,PTGS1,SLPI mammalian dexamethasone chemical drug -0.358 0.000011 AQP1,C8orf4,IL11,KCNJ1,LCN2,PTGS1,SLC12A1,SLPI ligand-dependent NR3C1 -1.000 0.001410 AQP1,C8orf4,GPX3,IL11 nuclear receptor

Supplementary Table 6. TaqMan primer information which were used for Quantitative Reverse Transcription polymerase chain reaction. Probe Gene Name TaqMan Assay ID

LCN2 lipocalin 2 Hs01008571_m1 IL11 interleukin 11 Hs01055413_g1 SLPI secretory leukocyte peptidase inhibitor Hs00268204_m1 MUC4 mucin 4, cell surface associated Hs00366414_m1 PTGS1 prostaglandin-endoperoxide synthase 1 Hs00377726_m1 GPX3 glutathione peroxidase 3 Hs01078668_m1 MMD monocyte to macrophage differentiation-associated Hs00948031_m1 C8orf4 chromosome 8 open reading frame 4 Hs00535539_s1 SCGB1D2 secretoglobin, family 1D, member 2 Hs00255208_m1 KCNJ1 potassium channel, inwardly rectifying subfamily J, member 1 Hs00165012_m1 NAV2 neuron navigator 2 Hs01093707_m1 GDPD1 glycerophosphodiester phosphodiesterase domain containing 1 Hs00402246_m1 SLC12A1 solute carrier family 12 (sodium/potassium/chloride transporter), memberHs00165731_m1 1 NALCN sodium leak channel, non selective Hs00291166_m1 AQP1 aquaporin 1 Hs01028916_m1 IL1B interleukin 1β Hs00174097_m1 IL6 interleukin 6 Hs00985639_m1 NOS2 nitric oxide synthase 2 Hs01075529_m1 TNF tumor necrosis factor Hs01113624_g1 GAPDH glyceraldehyde-3-phosphate dehydrogenase Hs02758991_g1 Supplementary Table 7. Primary antibodies for immunohistochemical stain.

Protein Name Antibodies Product Codes

LCN2 anti-human lipocalin-2 / NGAL rat monoclonal IgG (R&D Systems) MAB1757 IL11 anti-human interleukin 11 rabbit polyclonal IgG (Novus Biologicals) NBP1-19815 SLPI anti-human SLPI mouse monoclonal IgG (abcam) ab17157 PTGS1 anti-human COX1 / Cyclooxygenase 1 rabbit monoclonal IgG (abcam) ab109025 GPX3 anti-human Glutathione Peroxidase 3 rabbit polyclonal IgG (abcam) ab170313 MMD anti-human MMD (N-14) goat polyclonal IgG (Santa Cruz) sc-243494 SCGB1D2 anti-human SCGB1D2 / Lipophilin B (T-14) goat polyclonal IgG (Santa sc-48327 SLC12A1 anti-human SLC12A1 rabbit polyclonal IgG (abcam) ab60301 NALCN anti-human NALCN (N-20) rabbit polyclonal IgG (Santa Cruz) sc-84520 CD68 anti-human CD68 (KP1) mouse monoclonal IgG (Dako) M0814 CD138 anti-human CD138 (MI15) mouse monoclonal IgG (Dako) M7228 Neutrophil Elastaseanti-human Neutrophil Elastase (NP57) mouse monoclonal IgG (Dako) M0752 uroplakin III anti-human uroplakin III (SFI-1) mouse monoclonal IgG (abcam) ab78196 KSP anti-human KSP (aa1-30) rabit polyclonal IgG (LSBio) LS-C164201 vimentin anti-human vimentin (V9) mouse monoclonal IgG (Dako) M0725