Osteopontin (OPN) immunostaining of human kidney tissue from implantation biopsies, using a peroxidase-diaminobenzidine procedure. Tissue sections were counterstained with periodic acid-Schiff reagent. Distal tubular cells (DTC) exhibited pronounced apical OPN immunostaining (black arrows); however, some DTC also exhibited OPN signals in perinuclear vesicles (white arrow). Slightly damaged proximal tubular cells (PTC) demonstrated perinuclear OPN staining (arrowheads). Magnification, ϫ 400. 

Context in source publication

Context 1

... is present at the apical surface of cells in the distal nephron. After ischemic or toxic renal damage in rats, OPN is upregulated in distal tubular cells (DTC) and expressed de novo in perinuclear vesicles in proximal tubular cells (PTC). In the first phase of this study, OPN localization in ischemic human biopsies was compared with that in ischemic rat kidneys. In the second phase, cultures of PTC and DTC were used to investigate human renal OPN synthesis, secretion, and localization. OPN localization in human biopsies after renal ischemia was comparable to that in ischemic rat kidneys. Microscopic and flow cytometric detection of immunofluorescent OPN staining in tubular cell cultures demonstrated strong plasma membrane localization in DTC, whereas mainly perinuclear intracellular expression was observed in PTC. Northern blotting and reverse transcription-PCR demonstrated production of a single OPN Osteopontin (OPN) is a glycosylated phosphoprotein that was originally isolated from bone (1) but is expressed in a variety of tissues, including renal tubular epithelium (2– 4). In normal human and rat kidneys, OPN expression is mainly confined to the apical cell surface of a few cells in Henle’s loop and in the more distal parts of the nephron (2,3,5,6). Constitutive OPN expression is thought to play a role in the process of nephrolithiasis (7–9). In several experimental models of renal disease, highly upregulated OPN levels have been reported (5,6,10 –16). The precise role of OPN in renal pathophysiologic processes re- mains unclear, however. A protective role for OPN was de- duced from its ability to reduce inducible nitric oxide synthase levels (17,18). Through its interactions with several integrins, OPN has been reported to promote cell attachment and migra- tion (19,20) and to decrease tubular cast formation (21). The stimulation of transforming growth factor- ␤ activity, collagen deposition (22), and macrophage attraction (22,23) and the reduction of apoptotic cell death (22) suggest a role for OPN in renal fibrosis. mRNA in PTC and DTC. Detection of OPN by Western blotting and enzyme-linked immunosorbent assay demonstrated that PTC and DTC synthesized and secreted the same three molecular mass OPN forms, in comparable amounts. Finally, confocal microscopy demonstrated different staining patterns for endocytotic/lysosomal vesicles and perinuclear OPN; however, perinuclear OPN exhibited colocalization with the Golgi apparatus. In conclusion, human renal OPN localization in cell cultures demonstrated differences between PTC and DTC comparable to those observed after renal ischemia in vivo . Therefore, these cell cultures represented an excellent model for the study of human OPN synthesis, secretion, and localization in PTC versus DTC. It is reported for the first time that intracellular OPN is located in the Golgi apparatus of both PTC and DTC and that PTC and DTC are able to produce and secrete the same OPN isoforms, in comparable amounts. Upregulated OPN expression after acute renal injury in rats exhibits a specific pattern (5,6). Distal tubular cells (DTC) demonstrate pronounced OPN expression at their apical cell surfaces, whereas only a few DTC exhibit perinuclear staining. In proximal tubular cells (PTC), however, OPN is predominantly observed in perinuclear vesicles and apical cell surface expression is restricted to regenerating PTC (6). The same pattern of OPN expression, although less prominent, was observed by Hudkins et al. (2) in normal human kidney at sites of interstitial inflammation and fibrosis. An understanding of renal OPN function requires elucida- tion of this peculiar OPN expression pattern. OPN synthesis, secretion, and localization in primary cell cultures of human PTC and DTC were examined, to obtain better insight into the cellular OPN expression pattern and to learn more regarding human OPN expression in general. A cell culture system for mixed and pure human PTC and DTC was previously developed in our laboratory (24 –26). Flow cytometric immunodissection of PTC and DTC, on the basis of their expression of leucine aminopeptidase (LAP) and epithelial membrane antigen (EMA), respectively, has been described as an accurate method to obtain pure primary cultures of human PTC and DTC (24 –26). In the mixed cultures, PTC or DTC origins can be determined by means of LAP or EMA expression, respectively (24 –26). OPN localization was investigated by microscopic and flow cytometric analyses after immunofluorescent staining of cell cultures for OPN, LAP, and EMA. OPN transcription was studied by Northern blotting and reverse transcription (RT)- PCR, and OPN synthesis and secretion were investigated by Western blotting and enzyme-linked immunosorbent assays (ELISA) of both cell culture conditioned media and cell lysates. Intracellular OPN localization was investigated by means of confocal microscopy, using antibodies raised against OPN and specific organelle markers. As presented in Figure 1, pronounced OPN immunoreactivity was present at the apical cell surface of distal tubules. Some DTC also exhibited OPN signals in perinuclear vesicles. In PTC, however, OPN immunostaining was restricted to perinuclear vesicles in slightly damaged tubules. All biopsies were obtained from the cortex; therefore, the proximal tubules are S1/S2 segments. Flow cytometric immunodissection resulted in 97% pure cultures of PTC and DTC. Both mixed and pure tubular cells grew to 90% confluence within approximately 6 d. As noted earlier by Helbert et al. (25), cultures retained exclusive LAP or EMA expression during that period. OPN expression was therefore investigated in primary mixed and pure cultures of PTC and DTC. Because flow cytometric immunodissection resulted into low cell yields, mixed cultures were used when possible. Immunocytochemical analyses clearly demonstrated different OPN expression patterns in DTC and PTC (Figures 2 and 3). In both mixed (Figure 2, A, A', B, and B') and pure (Figure 2, C and D) tubular cultures, virtually all DTC (EMA-positive) demonstrated strong OPN immunoreactivity (Figure 2, A and A'), whereas PTC (LAP-positive) were weakly OPN positive (Figure 2, B and B'). OPN staining of EMA-positive DTC was intense and covered the entire cell surface (consistent with plasma membrane expression) (Figure 2C). Much less plasma membrane staining was observed in LAP-positive PTC (Figure 2D), where OPN staining appeared mainly intracellular and perinuclear. This perinuclear intracellular OPN staining in PTC was clearly demonstrated when cells were permeabilized before immunostain- ing and were observed with confocal laser scanning microscopy (Figure 3). Intracellular perinuclear OPN staining in DTC could be observed only when plasma membrane OPN staining was less prominent (Figure 3). Flow cytometric detection of OPN on living, unfixed, nonpermeable cells confirmed the presence of OPN at the extracellular cell surface (plasma membrane OPN expression). To obtain quantitative data on this plasma membrane expression, we performed a flow cytometric analysis with OPN/LAP/EMA triple-staining. EMA- and LAP-positive populations were de- fined (Figure 4, A and B). Plasma membrane OPN expression was mainly concentrated on EMA-positive DTC (Figure 4C), with much less expression on LAP-positive PTC (Figure 4D). LAP/EMA/OPN staining of three different kidney specimens indicated that 93.3 Ϯ 5.6% of DTC were OPN-positive, whereas only 17.5 Ϯ 15.5% of PTC were OPN-positive; OPN expression on PTC varied considerably among different kidney specimens, whereas OPN expression on DTC was rather con- stant. Furthermore, it could be demonstrated that the OPN fluorescence signal was much stronger in EMA-positive cells. Indeed, the OPN signal of DTC was clearly shifted to the right, compared with the negative control signal (Figure 4C), whereas the OPN fluorescence of PTC was difficult to distin- guish from the negative control signal (Figure 4D). Consequently, the results of microscopic and flow cytometric OPN analyses were in close agreement with each other. Northern blot analysis revealed that tubular cell cultures produced a single OPN mRNA of 1.6 kb (Figure 5). Comparable amounts of this transcript were present in PTC, DTC, and mixed tubular cell cultures. The size of the OPN mRNA produced in human tubular cell cultures agreed with the size of the OPN mRNA in human kidney tissue (data not shown) and with the reported size of the complete OPN cDNA sequence (GenBank accession number AF052124). RT-PCR resulted in one product, with the expected size of 960 bp, in PTC and DTC (data not shown). Sequence analysis revealed that the coding sequences for OPN were identical in PTC and DTC. Therefore, no alternative splicing occurred in PTC or DTC. Western blot analysis demonstrated full-length human recombinant OPN (produced as described in Materials and Methods) as a 58-kD protein (Figure 6). Analysis of urinary OPN revealed 66- and 72-kD protein bands (Figure 6). In conditioned media and cell lysates of mixed PTC and DTC, OPN forms of 66 and 72 kD (equivalent to urinary OPN) and a 58-kD form (equivalent to human recombinant OPN) were identified (Figure 6). Some lower-molecular mass forms were also detected in cell lysates and conditioned media, probably because of protein degradation. No differences were observed between PTC and DTC or between cell lysates and conditioned media. ELISA allowed quantification of OPN production and secretion. Comparable results were observed for PTC and DTC, which produced 7.3 and 6.4 mg OPN/g protein, respectively, of which 7.1 and 5.9 mg OPN/g protein were secreted into the medium. Therefore, no important quantitative differences in the levels of OPN production were observed between PTC and DTC. Uptake of FITC-labeled dextran resulted in a punctuate fluorescence pattern throughout the cell cytoplasm (Figure 7). Lysosomal staining was also scattered throughout the cytoplasm (Figure ...