Immunohistochemical localization and mRNA quantification of osteopontin and Tamm-Horsfall protein in canine renal tissue after potassium oxalate injection

Mohamaden et al. BMC Veterinary Research 2014, 10:70 http://www.biomedcentral.com/1746-6148/10/70 RESEARCH ARTICLE Open Access Immunohistochemical localization and mRNA quantification of osteopontin and Tamm-Horsfall protein in canine renal tissue after potassium oxalate injection Walaa Mohamaden1,2, Heng Wang1,2, Huawei Guan1,2, Xia Meng1,2 and Jianji Li1,2* Abstract Background: Urinary macromolecules contribute to promoting or inhibiting crystal retention in renal tissue and stone formation. Osteopontin (OPN) and Tamm-Horsfall protein (THP) are the most important proteins involved in this process. Although these two proteins were discovered a long time ago, their role in setting kidney stone formation has not yet been fully investigated. We conducted a study to explore the role of OPN and THP in canine renal oxalosis. Ten dogs were carefully examined prior to the study. Six dogs were assigned to the treatment group and were injected intravenously with 0.5 M potassium oxalate (KOx). The other four dogs were assigned to a control group and were injected intravenously with 0.9% NaCl three times a day (tid) for 7 consecutive days. Then kidneys were harvested for pathological, immunohistochemical examination and OPN and THP mRNA expression levels were quantified by quantitative real-time PCR. Results: Calcium oxalate crystals deposition was observed in both renal cortex and medulla. Immunohistochemistry examination revealed increased tissue expression of OPN in the renal tissue while THP was significantly decreased. OPN mRNA expression level significantly increased in treated dogs compared to that in the controls, while THP mRNA level significantly decreased. Conclusion: Together, these results suggest that THP and OPN are both involved in the pathogenesis and response to oxalate exposure. Keywords: Canine, Osteopontin, Tamm-Horsfall, mRNA, IHC Background In the past two decades, calcium oxalate urolithiasis (COU) has been a frustrating problem in dogs and cats worldwide [1]. Usually the presence of oxalate crystals in urine is a result from urinary supersaturation of oxalate salts. A problem that might be established in kidneys first and subsequently leads to retention of oxalate crystals within the renal tissue, inducing injury to renal tubules [2] due to adhesion between crystals and renal epithelial tissue [3]. Kidneys normally produce macromolecules such * Correspondence: 1 Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, China 2 Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu 225009, China as osteopontin (OPN), nephrocalcin, fibronectin, TammHorsfall protein (THP) and other macromolecules that bind to calcium salts in order to prevent or promote crystal growth and aggregation [4]. OPN is a negatively-charged aspartic acid-rich protein expressed in tissues, such as bone, liver, kidney, lung, bladder, and pancreas, as well as in macrophages [5]. It inhibits the formation of calcium oxalate (CaOx) crystals and regulates physiologic and pathologic mineralization [6]. The role of OPN in nephrolithiasis formation is unclear. In vitro, studies suggested that OPN inhibits nucleation, and CaOx crystal growth and aggregation in cultured epithelial cells. It also enhances the formation of calcium oxalate dihydrate (COD) crystals that are less adherent to renal tissue than calcium oxalate monohydrate (COM) crystals © 2014 Mohamaden et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mohamaden et al. BMC Veterinary Research 2014, 10:70 http://www.biomedcentral.com/1746-6148/10/70 [7]. Conversely, other studies found OPN associated with nephrolithiasis through the mineralization and dystrophic calcification of the urolithiasis matrix [8]. OPN is also an important mediator of tissue injury. Increased expression of OPN in renal tubule cells is related to an accumulation of macrophages in the damaged tissue, since OPN is involved in the recruitment and retention of macrophages in the inflamed site [9-11]. Another urinary macromolecule involved in renal tissue crystals deposition is uromodulin or THP, a glycosylphosphatidylinositol protein, the most abundant urinary protein and the main constituent of hyaline urinary casts [12]. THP forms a gel like matrix that traps bacteria and prevents their adhesion to plasma membranes. It also acts as an inhibitor of stone formation in healthy individuals by trapping crystals in the same manner [13]. However, this function may be subverted under some circumstances and THP may facilitate crystal aggregation and then promote stone formation [14]. In humans, studies investigating the association between THP expression and urinary excretion were inconsistent in stone forming patients [15]. THP was expressed in the thick ascending limbs of Henle’s loops in normal renal tissue of dogs [16], and it was chosen as a new biomarker of canine renal toxicity [17], indicating that THP is involved in the development of renal diseases in dogs. Despite numerous proteins that engaged in the retention of CaOx crystals in the urinary tract, understanding the precise mechanisms by which OPN and THP influence the several aspects of crystallization and urinary stone formation is only beginning to emerge. For instance, THP expression decreased in ethylene glycol-administered rats [18], while in other studies it increased [19] or remained unchanged in others [20]. Some studies dealt only with one of the two proteins, this does not provide a logical understanding of how these two proteins interact. Most of the studies that focused on both proteins were conducted in either laboratory animals [18,21] or in Madin Darby canine kidney cell culture (MDCK) [22], which may not be representative of the real situation in canine renal tissue in vivo. However the conflicting roles of THP and OPN makes the study of their reaction toward kidney stone formation also valuable for the explanation of the pathogenesis of renal oxalosis in dogs. We hypothesized that THP and OPN expression in the renal tissue will be changed after exposure to oxalates. The aim of current study is to record the canine renal tissue injury upon oxalates injection in vivo and to quantify THP and OPN gene expressions of renal tubular cells in order to investigate their role of pathogenesis in canine renal oxalosis. Methods Chemicals 0.5 mol/l KOx (K2C2O4 · H2O) (MW = 184 g) was prepared KOx solution was filtered (...truncated)