822 © 2 0 0 4 B J U I N T E R N A T I O N A L | 9 3 , 8 2 2 – 8 2 6 | doi:10.1111/j.1464-410X.2004.04751.x Original Article ANTI-CD75 REACTIVE PROTEINS IN G. KRAMER et al. Over-expression of anti-CD75 reactive proteins on distal and collecting renal tubular epithelial cells in calcium-oxalate stone-forming kidneys in Egypt G. KRAMER, G.E. STEINER, C. NEUMAYER, M. PRINZ-KASHANI, M. HOHENFELLNER*, M. GOMHA*, M. GHONEIM*, M. NEWMAN and M. MARBERGER Departments of Urology, Vienna, Austria and *Urology and Nephrology Centre, Mansoura University, Mansoura, Egypt Accepted for publication 6 November 2003 expression of CD75 were analysed using Western blotting, immunohistology and semi-quantitative confocal laser scanning microscopy (cLSM). Production was investigated by a2,6-sialyltransferase specific reverse transcription-polymerase chain reaction. RESULTS Western blotting showed one strong band at ª 43 kDa that reacted with anti-CD75 when renal epithelial and CAKI-1 tumour cell extracts were analysed. However, in renal tissue extracts of CaOx stone formers there were additional bands at 120 and 205 kDa. Image processing after cLSM showed that anti-CD75 reactivity was significantly greater on E-cadherin-positive distal and collecting tubular cells from CaOx stone-forming kidneys, at a mean (SD) intensity of 87 (7), than on those from normal kidneys, at 41 (5) (P = 0.005). CONCLUSION CD75 expression in human kidney was primarily on the luminal surface of distal tubules and collecting ducts. Whether increased epithelial CD75 expression in CaOx stone disease is a cause or result of the disease remains to be clarified. KEYWORDS CD75, calcium oxalate, stones, semi- quantitative confocal laser microscopy, sialic acid OBJECTIVE To assess the nature, distribution and expression pattern of CD75, a neuraminidase- sensitive lymphocyte cell surface differentiation antigen, in calcium oxalate (CaOx) stone disease, as cell-surface sialic acid might be involved CaOx crystal binding, and lectin-binding assays suggest that sialic acid in the a2,6 position is upregulated in stone- forming kidneys. MATERIALS AND METHODS Human CaOx stone-forming and normal kidneys (13 each) and primary kidney epithelial cells (CAKI-1, three samples) were analysed. The protein pattern, distribution and INTRODUCTION Idiopathic calcium oxalate (CaOx) nephrolithiasis appears to be a multifactorial disorder. The underlying molecular and cellular mechanisms are not completely understood. To clarify the situation in vivo, more attention has recently focused on human tissue studies, analysing primary and secondary effects of stone formation in situ [1,2]. Sialylation of cell-surface antigens is important for regulating cell-to-cell contact [3], and differential sialylation is involved in the adaptation of surface molecules for the specific functional requirements of T and B cells [4]. An increased a2,6-specific linkage of sialic acids is known to be involved in several cell-adhesion processes. Furthermore, cell surface sialic acid molecules in general have been proposed as candidate receptors for the adhesion of CaOx monohydrate crystals to renal tubular epithelial cells [2,5]. Other authors claim that sialic acid is not a crystal- binding molecule, but is involved in the exposure of a true crystal-binding molecule [6]. This directed our interest to CD75, a possible ligand for sialoglycan-specific lectins [7]. Originally it was thought that antibodies directed against CD75 specifically recognize a2,6 sialyltransferase [8]. Thus we investigated whether CD75 was expressed in human kidney [8]. However, recent transfection experiments using a2,6 sialyltransferase encoding cDNA of COS cells showed that both anti-CD75 antibodies (MB-2 and LN-1) recognize neuraminidase- sensitive a2,6 sialyltransferase-generated sugar antigens [10–14]. In the present study we investigated whether CD75 is expressed, which proteins and how many proteins are recognized by anti-CD75, whether the enzyme a2,6 sialyltransferase is actively synthesized in human kidney and if so, whether CD75 expression is up-regulated in human CaOx stone-forming kidneys. MATERIALS AND METHODS The CAKI-1 RCC cell line was purchased from the American Type Culture Collection. Normal renal tissue specimens were obtained during open surgery for renal tumours suspicious of malignancy, seven of which were benign and six localized RCC. The latter were obtained by open surgery in Mansoura, Egypt. Primary normal renal epithelial cells were prepared from normal renal tissue obtained during open surgery for a renal tumour suspicious of malignancy, which was later confirmed to be of benign origin. In brief, small fragments of renal tissue were digested using 200 U/mL type 1 collagenase and 100 mg/mL DNAse (Sigma Chemical Company, St. Louis, MO) overnight at 37 ∞C in RPMI-1640 (Gibco BRL, Paisley, UK) plus 10% fetal calf serum (Gibco). The resulting cell suspensions were washed three times and cultured at high densities for 48 h. Adherent cells consisted of > 95% cytokeratin-18-expressing cells (Becton Dickinson, CA, data not shown) and are herein