Cadmium is more toxic to LLC-PK1 cells than to MDCK cells acting on the cadherin-catenin complex.

Cadmium toxicity to renal cells was investigated in Madin-Darby canine kidney (MDCK) and LLC-PK1 cells as models of the distal tubule/collecting duct and proximal tubule, respectively. Cells were grown on two-compartment filters and exposed to 0.1-50 microM Cd2+. In MDCK cells, Cd2+ was more toxic from the basolateral than from the apical side and dependent on the extracellular Ca2+ concentration. Toxicity was evident within 24 h, as shown by a decrease in transepithelial resistance (TER), reduced proliferation (bromodeoxyuridine incorporation), reduction in ATP concentration, and morphological changes. On confocal microscopy, E-cadherin and alpha-catenin staining patterns indicated interference with the cadherin-catenin complex. LLC-PK1 cells showed a similar toxicity pattern, which was evident at lower Cd2+ concentrations. An increase of E-cadherin and alpha-catenin molecules in the Triton X-100-insoluble fraction was detectable at high Cd2+ concentrations in LLC-PK1 cells but not in MDCK cells. Lactate dehydrogenase release indicated membrane leakage in LLC-PK1 cells. Rhodamine-phalloidin staining, a probe for F-actin filaments, demonstrated alterations of the actin cytoskeleton in both cell lines. In conclusion, cadmium caused ATP depletion and interfered with the cadherin-catenin complex and probably the tight junctions changing renal cell morphology and function.

Apoptosis and necrosis during ischaemia in renal tubular cells (LLC-PK1 and MDCK).

BACKGROUND: Ischaemia is the most frequent cause of acute renal failure. It has been previously demonstrated that ischaemia is connected with signs of necrosis and apoptosis. Apoptosis is an energy-dependent process. During ischaemia intercellular energy levels decline rapidly. Therefore, the goal of the investigation was to reveal the time dependency of cell death mechanisms during ischaemia leading to irreversibility of tissue damage. METHODS AND RESULTS: A model of renal ischaemia induced by ATP depletion was used in LLC-PK1 and MDCK-cells. Cell proliferation, determined by 3H-thymidine and BrdU incorporation and by the Ki67-labelling index was affected already after 1-2 h of ATP depletion in both cell lines. Cell viability and membrane leakage, estimated by trypan blue and propidium iodide exclusion and LDH release, was profoundly increased after 8-16 h. Evaluation of mechanisms of necrotic or apoptotic cell death was calculated from fraction of cells with pyknotic nuclei, investigation of DNA fragmentation and, by translocation of phosphatidylserine (PS) from the inner membrane face to the surface. In both cell lines increased numbers of cells with condensed nuclei was not a major sign of apoptosis. Only in MDCK cells were the numbers of cells with condensed nuclei significantly increased after 1 h compared to controls. As a hallmark of apoptosis, ATP depletion resulted in intranucleosomal DNA fragmentation after 1 h. After 8-16 h this pattern changed to a smear pattern, as a sign for necrosis. PS staining was detectable at the cell surface after 1 h. CONCLUSIONS: Ischaemia is associated with a rapid loss of proliferation and signs of apoptosis at early stages in a small proportion of cells. Most cells undergo the necrotic pathway of cell death after prolonged ATP depletion (8 h). There was no difference in behaviour comparing proximal (LLC-PK1) with more distal (MDCK) cell culture models. These results may help to explain the findings that apoptosis and necrosis have both been described after renal ischaemia.

Tubular toxicity of cyclosporine A and the influence of endothelin-1 in renal cell culture models (LLC-PK1 and MDCK).

To evaluate the effect of cyclosporine A (CyA) at high concentrations (10(-4) and 10(-5) M) and the influence of endothelin-1 (ET-1) at physiological and pharmacological concentrations (10(-14) to 10(-6) M) on epithelial cell function, LLC-PK1 cells were studied as a model of the proximal tubule and MDCK cells as a model of the distal tubule/collecting duct. CyA caused time- and concentration-dependent acute toxicity. In LLC-PK1 cells, CyA caused a decrease in transepithelial resistance, indicating a loss of cell contacts, a release of lactate dehydrogenase (LDH) and villin into the supernatant, suggesting destruction of the apical membrane with loss of brush border, and finally release of uvomorulin, suggesting a disruption of the cell-cell adhesion, the zonula adherens. DNA synthesis, as evaluated by bromodeoxyuridine (BrdU) incorporation, was significantly affected at > or = 10(-5) M CyA. The toxicity of CyA was higher when given from the apical rather than the basolateral compartment. ET-1 alone was without effect, but in combination with CyA, ET-1 significantly enhanced toxicity. The ET-1 effect was partially inhibitable by an ET(B), but not an ET(A), antagonist. Immunofluorescence for alpha-catenin, another protein of the zonula adherens, demonstrated no change in polarity for this protein, and immunoprecipitation of the complex indicated relative stability of the zonula adherens despite loss of cadherin into the supernatant. In MDCK cells the effects were different. CyA was not associated with LDH release, but with an increase in transepithelial resistance, indicating increased paracellular resistance. Morphological alterations were significantly less, but BrdU incorporation was decreased. This pattern of toxicity is compatible with a direct toxic effect of CyA on cells of the proximal tubule, with predominant morphological destruction of the cells, with concomitant proximal tubular dysfunction, and a functional alteration in cells of the distal tubule associated with increased paracellular resistance, which may lead to solute and water loss.

Tamm-Horsfall protein as a marker of tubular maturation.

Tamm-Horsfall protein (THP), a glycoprotein with a molecular weight of 95 kilodaltons, is produced and secreted in the ascending loop of Henle. To evaluate the measurement of THP in the assessment of fetal renal development and function, we stained fetal kidney sections for THP and measured THP concentrations in 129 amniotic fluid samples from healthy pregnancies, together with other parameters such as transferrin, albumin, alpha 1- and beta 2-microglobulin. After the 16th week of gestation THP could be detected immunohistochemically in the distal tubular cells, but was not consistently detected by sandwich enzyme immunoassay until after the 20th week of gestation (detection limit 50 ng/ml). Between the 15th and 19th week of gestation THP was only detected occasionally, but after the 20th week of gestation the concentration increased significantly reaching levels of 0.4-4 mg/l at term. The THP concentration was lower in samples taken directly before birth than in the corresponding first urine after birth, indicating that THP is produced from the fetal kidney only and does not pass the placental barrier. This pattern was different from other proteins studied. Transferrin and albumin were significantly lower in the first urine voided, microglobulins remained unchanged, and the creatinine concentration increased. This indicates that maternal to fetal exchange or transport is likely for most of the other proteins. Measurement of THP concentrations, in addition to other proteins in the amniotic fluid, can improve fetal renal assessment, but because the range of THP concentrations is wide accurate predictions are still not possible.