Internalization of calcium oxalate crystals by renal tubular cells: A nephron segment-specific process? Background. Crystal retention in the kidney is caused by the interaction between crystals and the cells lining the renal tubules. These interactions involve crystal attachment, followed by internalization or not. Here, we studied the ability of various renal tubular cell lines to internalize calcium oxalate monohydrate (COM) crystals. Methods. Crystal-cell interactions are studied by light-, electron-, and confocal microscopy with cells resembling the renal proximal tubule [porcine kidney (LLC-PK 1)], proximal/distal tubule [Madin-Darby canine kidney II (MDCK-II)], and distal tubule and/or collecting ducts [(Madin-Darby canine kidney I (MDCK-I), rat cortical collecting duct 1 (RCCD 1)]. Crystalbinding strength and internalization are characterized and quantified with radiolabeled COM. Results. Microscopy studies showed that crystals were firmly embedded in the membranes of LLC-PK 1 and MDCK-II cells to be subsequently internalized. On the other hand, crystals bound only loosely to MDCK-I and RCCD 1 and were not taken up by these cells. Crystal uptake by LLC-PK 1 and MDCK-II, expressed in g/10 6 cells, is temperature-dependent and gradually increases from 0.88 and 0.15 in 30 minutes, respectively, to 4.70 and 3.85, respectively, after five hours, whereas these values never exceeded background levels in MDCK-I and RCCD 1 cells. Conclusion. The adherence of COM crystals to renal cells with properties of the proximal tubule is inevitable and actively followed by their uptake, whereas crystals attached to cells resembling the distal tubule and/or collecting duct are not internalized. Since crystal formation usually occurs in segments beyond the renal proximal tubule, crystal uptake may be of less importance in the etiology of idiopathic calcium oxalate stone disease. Kidney stones are composed of myriad microliths pasted with organic material. Although most stones are found in the renal pelvis, the stone-forming process actu

Background/Aim: Retention of crystals in the kidney ultimately leads to renal stone formation. Hyaluronan (HA) has been identified as binding molecule for calcium oxalate monohydrate crystals. The association of high molecular mass (M_r) HA with cell surface receptors such as CD44 gives rise to pericellular matrix (PCM) formation by many eukaryotic cells in culture. Here, we study the ability of several renal tubular cell lines to assemble PCMs and to synthesize high-M_r HA during proliferation in relation to crystal retention. Methods: PCM assembly by MDCK-I, MDCK-II, and LLC-PK₁ cells was visualized by particle exclusion assay. Metabolic labeling studies were performed to estimate the cellular production of HA. The expression of CD44 and HA was studied using fluorescent probes, and crystal binding was quantified with radiolabeled calcium oxalate monohydrate. Results: PCMs were formed, and HA was expressed by most MDCK-I and some MDCK-II, but not by LLC-PK₁ cells. All cell types expressed CD44 at their apical surface. MDCK-I and MDCK-II cells secreted, respectively, 14.7 ± 1.6 and 0.5 ± 0.2 pmol [³H]glucosamine incorporated in high-M_r HA, whereas LLC-PK₁ cells did not secrete HA. Streptomyces hyaluronidase treatment significantly decreased crystal binding (µg/cm²) to MDCK-I cells (from 8.6 ± 0.4 to 3.9 ± 0.9), but hardly to MDCK-II cells (from 10.2 ± 0.2 to 9.6 ± 0.1) or LLC-PK₁ cells (from 10.2 ± 0.8 to 9.9 ± 0.3). Conclusions: There are various forms of crystal binding to renal tubular cells in culture. Crystal attachment to MDCK-I and some MDCK-II cells involves PCM assembly that requires high-M_r HA synthesis. HA production and PCM formation do not play a role in crystal binding to LLC-PK₁ and the majority of MDCK-II cells. It remains to be determined which form of binding is involved in renal stone disease.

<p>Supplementary Figures S1-S4; Supplementary Tables S1-S5.</p>

<div>Abstract<p><b>Purpose:</b> High-grade osteosarcoma is a malignant primary bone tumor with a peak incidence in adolescence. Overall survival (OS) of patients with resectable metastatic disease is approximately 20%. The exact mechanisms of development of metastases in osteosarcoma remain unclear. Most studies focus on tumor cells, but it is increasingly evident that stroma plays an important role in tumorigenesis and metastasis. We investigated the development of metastasis by studying tumor cells and their stromal context.</p><p><b>Experimental Design:</b> To identify gene signatures playing a role in metastasis, we carried out genome-wide gene expression profiling on prechemotherapy biopsies of patients who did (<i>n</i> = 34) and patients who did not (<i>n</i> = 19) develop metastases within 5 years. Immunohistochemistry (IHC) was performed on pretreatment biopsies from 2 additional cohorts (<i>n</i> = 63 and <i>n</i> = 16) and corresponding postchemotherapy resections and metastases.</p><p><b>Results:</b> A total of 118/132 differentially expressed genes were upregulated in patients without metastases. Remarkably, almost half of these upregulated genes had immunological functions, particularly related to macrophages. Macrophage-associated genes were expressed by infiltrating cells and not by osteosarcoma cells. Tumor-associated macrophages (TAM) were quantified with IHC and associated with significantly better overall survival (OS) in the additional patient cohorts. Osteosarcoma samples contained both M1- (CD14/HLA-DRα positive) and M2-type TAMs (CD14/CD163 positive and association with angiogenesis).</p><p><b>Conclusions:</b> In contrast to most other tumor types, TAMs are associated with reduced metastasis and improved survival in high-grade osteosarcoma. This study provides a biological rationale for the adjuvant treatment of high-grade osteosarcoma patients with macrophage activating agents, such as muramyl tripeptide. <i>Clin Cancer Res; 17(8); 2110–9. ©2011 AACR</i>.</p></div>