Simonne Dauwe

Chondrocyte rather than osteoblast conversion of vascular cells underlies medial calcification in uremic rats

Objective—To investigate cell biological changes in calcified aortas of rats that experienced chronic renal failure. Methods and Results—Vascular smooth muscle cells have the potential to transdifferentiate to either chondrocytes or osteoblasts, depending on the molecular pathways that are stimulated. Uremia-related medial calcification was induced by feeding rats an adenine low-protein diet for 4 weeks. Aortic calcification was evaluated biochemically and histochemically and with in vivo micro–computed tomographic scanning. Immunohistochemistry and RT-PCR were applied to analyze the time-dependent aortic expression of molecules involved in the segregation between the chondrocyte versus osteoblast differentiation pathway. After 4 weeks, 85% of the uremic rats had developed distinct aortic medial calcification, which increased to severely calcified lesions during further follow-up. The calcification process was accompanied by a significant time-dependent increase in the expression of the chondrocyte-specific markers sex determining region Y-box 9 (sox9), collagen II, and aggrecan and a nonsignificant trend toward enhanced core binding factor alpha 1 (cbfa1), and collagen I. The expression of the osteoblast marker osterix and both lipoprotein receptor–related protein 6 and &bgr;-catenin, molecules of the wingless-type MMTV integration site family member (Wnt)/&bgr;-catenin pathway induced during osteoblast differentiation, was suppressed. Conclusion—In the aorta of uremic rats, medial smooth muscle cells acquire a chondrocyte rather than osteoblast phenotype during the calcification process.

An active renal crystal clearance mechanism in rat and man

The kidney has several defense mechanisms to avert nephrocalcinosis by preventing intratubular crystal formation and adherence. Little is known about the fate of luminally adhered crystals. In order to study post-crystal adhesion defense mechanisms we quantified the number and morphology of crystal-containing tubules in rats at various time points following ethylene glycol administration as well as in renal biopsies of patients diagnosed with nephrocalcinosis of different etiology. In rats, nephrocalcinosis was completely cleared by epithelial overgrowth of adherent crystals, which were then translocated to the interstitium and subsequently disintegrated. These processes correlated with a low to moderate infiltration of inflammatory cells. Patients with nephrocalcinosis due either to acute phosphate nephropathy, primary hyperoxaluria, preterm birth, or transplantation also showed epithelial crystal overgrowth independent of the underlying disorder or the nature of the crystals. Our study found a quantitative association between changes in tubular and crystalline morphology and crystal clearance, demonstrating the presence of an important and active nephrocalcinosis-clearing mechanism in both rat and man.

Hepatocellular transport and gastrointestinal absorption of lanthanum in chronic renal failure

Lanthanum carbonate is a new phosphate binder that is poorly absorbed from the gastrointestinal tract and eliminated largely by the liver. After oral treatment, we and others had noticed 2-3 fold higher lanthanum levels in the livers of rats with chronic renal failure compared to rats with normal renal function. Here we studied the kinetics and tissue distribution, absorption, and subcellular localization of lanthanum in the liver using transmission electron microscopy, electron energy loss spectrometry, and X-ray fluorescence. We found that in the liver lanthanum was located in lysosomes and in the biliary canal but not in any other cellular organelles. This suggests that lanthanum is transported and eliminated by the liver via a transcellular, endosomal-lysosomal-biliary canicular transport route. Feeding rats with chronic renal failure orally with lanthanum resulted in a doubling of the liver levels compared to rats with normal renal function, but the serum levels were similar in both animal groups. These levels plateaued after 6 weeks at a concentration below 3 microg/g in both groups. When lanthanum was administered intravenously, thereby bypassing the gastrointestinal tract-portal vein pathway, no difference in liver levels was found between rats with and without renal failure. This suggests that there is an increased gastrointestinal permeability or absorption of oral lanthanum in uremia. Lanthanum levels in the brain and heart fluctuated near its detection limit with long-term treatment (20 weeks) having no effect on organ weight, liver enzyme activities, or liver histology. We suggest that the kinetics of lanthanum in the liver are consistent with a transcellular transport pathway, with higher levels in the liver of uremic rats due to higher intestinal absorption.

Flow cytometric immunodissection of the human nephron in vivo and in vitro.

In the present article, we show that flow cytometric immunodissection of cells immediately following their preparation from a tumor nephrectomy specimen is an accurate way of obtaining pure human primary cultures of proximal convoluted tubule origin, proximal straight tubule origin, distal tubular origin and/or collecting duct origin. By studying the expression of a panel of cell surface markers in these purified cultures, we could identify a number of markers that retain their lineage specificity in vitro. Using these appropriate stable markers, flow cytometry provides a simple yet accurate way of determining cell composition in previously unsorted (mixed type) tubular epithelial cultures in terms of proximal versus distal tubule/collecting duct subpopulations. Both subpopulations in mixed type cultures are shown to retain functional characteristics of their in vivo counterparts (glucose uptake, hormonal stimulation of adenylate cyclase) as well as cell type-specific response patterns (such as inducibility of cell adhesion and histocompatibility molecules), indicating the usefulness of studying cell responses in vitro in a cell-type-dependent way. Finally we illustrate that multi-parameter flow cytometry is a powerful tool for assessing constitutive characteristics of and/or responses by the distinct cell subpopulations present in mixed type cultures.

Detection and Immunohistochemical Localisation of Hplap and CA 125 in Sera and Tissues of Patients with Ovarian, Nonovarian Tumors, and Liver Disorders

Abstract Cancer Antigen 125 (CA 125) was compared to human placental alkaline phosphatase (hPLAP) and CEA in sera of patients with different types of ovarian tumors, non ovarian tumors and patients with liver disorders. In ovarian tumor cases the sensitivity of hPLAP was 44%, 69% for CA 125 and only 13% for CEA. 45 nonovarian tumor patients were examined on hPLAP, CA 125 and CEA. Serum hPLAP and CA 125 levels were almost equally increased (22% and 24% respectively), whereas CEA was increased in 44%. hPLAP was not increased in sera of patients with benign liver pathologies, in contrast to CA 125 and CEA, which were increased in 54% and 24% respectively. Cirrhotic icteric patients had the highest prevalence of CA 125 (89%) and CEA (44%) in their serum. hPLAP, CA 125 and CEA were localized immunhistochemically in paraffin sections of ovarian tumors. A positive reaction was observed in 90%, 99% and 45% respectively. Immunohistochemical staining of CA 125 in normal liver tissue of a patient with a metastatized pancreatic adenocarcinoma and a high CA 125 level in the serum was observed in organelles compatible with the lysosomes of hepatocytes. Normal fetal liver was immunohistochemically negative for hPLAP, CA 125 or CEA, and one fetal ovary studied was positive only for hPLAP.