CD133+ endothelial progenitor cells as a potential cell source for a bioartificial glomerulus.

Development of a bioartificial glomerulus, a hemofilter in which the inner surface of hollow fibers is endothelialized, requires expandable, nonimmunogenic, antithrombogenic, and highly permeable endothelial cells. We used human umbilical cord blood CD133(+) endothelial progenitor cells (EPCs) to evaluate the feasibility of application of EPCs for bioartificial glomerulus. Numbers of adhered CD133(+) EPCs (adhered EPCs) was approximately 25 to 30 times as great in the expansion culture group as in the non-expansion group. Adhered EPCs had endothelial cell features, including the expression of CD31, Kinase domain region, von Willebrand factor, vascular endothelial-cadherin, positive for Ulex europeus agglutinin I staining, and up-take of acetylated low-density lipoprotein. Adhered EPCs secreted 6-keto-prostaglandin F(1alpha) identically to that secreted by human umbilical vein endothelial cells (HUVECs). The cells also expressed messenger RNA for phospholipase A(2), cyclooxygenase (COX)-1, COX-2, prostaglandin I(2) synthase, tissue plasminogen activator, and thrombomodulin (TM). TM protein in adhered EPCs properly activated protein C. Scanning electron microscopy revealed the suppression of platelet adhesion and aggregation on the surface of cell monolayer. Adhered EPCs treated with 50 microg/mL of cytochalasin B induced a larger diameter and a greater number of fenestrae, subsequently producing significantly more ultrafiltration than the non-treated cell. These results suggest that CD133(+) EPCs would potentially be applicable in bioartificial glomerulus.

Suppression of parathyroid hormone production in vitro and in vivo by RNA interference.

We used RNA interference, which causes sequence-specific degradation of target mRNAs to suppress the production of parathyroid hormone by cells of patients with secondary hyperparathyroidism in vitro and in vivo. Transfection of small interfering RNA (siRNA) against human parathyroid hormone into monolayers of parathyroid cells cultured from these patients caused a dose-dependent decrease of secretion and mRNA levels with 80% or more suppression using 40 nM siRNA. Parathyroid cells cultured on non-adherent plastic produced spheroid cell aggregates which secreted parathyroid hormone for more than 150 days. Transfection of these spheroids with 50 nM targeted siRNA decreased parathyroid hormone production to 20% of the control level, with half of them being suppressed for 50 days. When parathyroid cells were transplanted into the livers of athymic nude mice, plasma human parathyroid hormone rose to 100-300 pg/ml within one month and remained at about this level for at least 39 days. Systemic delivery of hormone-targeted siRNA into these mice caused a dose-dependent suppression of circulating human parathyroid hormone for at least one month, with a maximum 80% suppression achieved by 80 microg of siRNA. Our study shows that hormone secretion by parathyroid cells of patients with secondary hyperparathyriodism can be suppressed both in vitro and in vivo by targeted siRNAs.

Enhancement of permeability in endothelial cells for the development of an antithrombogenic bioartificial hemofilter.

For the development of an antithrombogenic bioartificial hemofilter, in which the inner surface of hollow fibers is lined by endothelial cells, it is essential to increase the permeability of the cells in order to achieve a sufficient ultrafiltrate. We tried to increase it by using an actin microfilament polymerization inhibitor, cytochalasin B (CyB). Fifty microg/mL CyB was added for 2 h to the culture medium of confluent rat glomerular endothelial cells (RGEC) and human umbilical vein endothelial cells (HUVEC). Under the 130 mmHg hydrostatic pressure, the CyB-treated group produced significantly more ultrafiltration than the non-treated control group and this increase was maintained for at least 7 days. Horseradish peroxidase (HRP) permeability acutely and reversibly increased in the CyB-treated group compared with the non-treated control group. Scanning electron microscopy revealed a larger average diameter and increased number of fenestrae on the CyB-treated endothelial cells, compared with the non-treated cells. This phenomenon also lasted for at least 7 days. The platelet adherence test showed that CyB did not deteriorate the antithrombogenic property of endothelial cells. These results indicate that CyB is potentially applicable for the enhancement of endothelial cell permeability in an antithrombogenic bioartificial hemofilter.

Prevention of LLC-PK(1) cell overgrowth in a bioartificial renal tubule device using a MEK inhibitor, U0126.

A common approach to construct a bioartificial renal tubule system is to utilize renal tubular cells seeded in porous polymer membrane hollow fibers. We have reported that overgrowth of renal tubular cells was not beneficial for the transport and reabsorption functions of bioartificial tubules. Therefore, long-term maintenance of a confluent monolayer of cells in hollow fibers is essential and technically challenging. In this study, we examined whether MEK inhibitor, U0126, could maintain the monolayer of Lewis-lung cancer porcine kidney 1 (LLC-PK(1)) cells on polystyrene plates and in a dialysis module housing hollow fibers made of ethylene vinyl alcohol (EVAL). We also evaluated the leakage of urea nitrogen (UN) and creatinine (Cr) through the cell-lined hollow fibers, and reabsorption of glucose and sodium by the cells, comparing the U0126-treated cells with nontreated cells in the module. Treatment with 50micromol l(-1) U0126 prevented the overgrowth of cells cultured on polystyrene plates. Moreover, U0126-treatment reduced the leakage of UN, and increased the reabsorption of electrolytes in 65cm(2) modules. Scanning electron microscopy revealed that it also prevented the overconfluence of cells in modules. Therefore, application of U0126 is a potentially effective method to improve the performance of the device.