CLINICAL RESEARCH www.jasn.org 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 J Am Soc Nephrol 28: 333–347, 2017 ISSN : 1046-6673/2801-333 333 CLINICAL RESEARCH www.jasn.org 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.