Cepharanthine induces the proliferation of human dermal papilla cells and stimulates vascular endothelial growth factor expression through increased intracellular calcium mobilization and hypoxia-inducible factor activation.

BACKGROUND: Cepharanthine (CEP), a compound extracted from the vine Stephania cephalantha, is commonly prescribed to treat alopecia areata; however, the scientific evidence for its efficacy is limited. AIM: To investigate the effect of CEP and its structural analogues on human hair growth in vitro. METHODS: The effects of CEP and three of its structural analogues on the proliferation of human dermal papilla cells (hDPCs) and human outer root sheath cells (hORSCs) were investigated. Their effects on vascular endothelial growth factor (VEGF) expression were also assessed by real-time PCR. Activation of pathways leading to VEGF expression, such as intracellular Ca2+ mobilization and hypoxia-inducible factor (HIF) expression, was also characterized. RESULTS: CEP and two of its structural analogues significantly stimulated the growth of hDPCs but not hORSCs. Moreover, CEP and all three structural analogues significantly induced the expression of VEGF in hDPCs. CEP increased the intracellular Ca2+ concentration in hDPCs. CEP also increased the expression of HIF-1α and HIF-2α and induced the expression of HIF-responsive genes in hDPCs, even under normoxia. CONCLUSIONS: These results suggest that CEP and its structural analogues have the potential to restore hair growth by promoting the proliferation of hDPCs and increasing their expression of VEGF.

Combined treatment with nafamostat mesilate and aspirin prevents heparin-induced thrombocytopenia in a hemodialysis patient.

We report on the management of a 36-year-old hemodialysis patient with heparin-induced thrombocytopenia (HIT, type II) and clot formation in extracorporeal circulation. Platelet aggregation test and measurement of anti-platelet factor 4/heparin complex antibody by enzyme-linked immunosorbent assay revealed to us that our patient had developed HIT. Instead of heparin, we used nafamostat mesilate (NM) as an anticoagulant during hemodialysis, but could not completely prevent HIT-induced thrombocytopenia or clot formation in the extracorporeal circuit. Combined use of NM and aspirin completely inhibited platelet aggregation, decrease in platelet count and clot formation in the extracorporeal circuit.

Cellular component of vascular calcification. Fibroblasts are essential for calcium deposition in cultured cells.

It has been reported that calcium deposition and calcium content in cultured human aortic smooth muscle cells (SMC) increased when the cells are incubated in a medium with a high phosphate concentration (2 mM). To determine the cellular components or soluble factors contributing to the deposition, we cultured commercially available SMC, fibroblasts (Fb) and endothelial cells (Ed). These cells and their mixtures were incubated for 10 days in normal or high-phosphate media. Calcium crystals were stained by the von-Kossa staining and counted in the defined area. Calcium content was measured by a colorimetric assay. SMC were incubated in high-phosphate media (up to 2 mM) or beta-glycerophosphate (beta-GP) media, resulting in no obvious deposition of calcium crystals, irrespective of the coating of type I collagen on the dish. Next, various combinations of cells were cultured, and a significant number of depositions were observed only when Fb were included in the combination. The calcium content was significantly higher in cultures of SMC and Fb. The calcium deposition on single or mixture of the cells did not increase compared with control when cells were incubated in a high concentration of phosphate, cultured in the existence of beta-GP or uremic serum. We therefore conclude that Fb, rather than SMC or Ed, are essential for calcium deposition and calcium accumulation in culture. Phosphate concentration in the medium and uremic serum did not influence the deposition of calcium.

[Prospective evaluation of renal function by serum cystatin-C: comparison with three other parameters of glomerular filtration rate].

Cystatin-C is a low-molecular-weight basic protein produced at a stable rate by all nucleated cells. It is freely filtered through the renal glomeruli and primarily catabolized in the proximal tubule cells. Since the serum cystatin-C concentration is not affected by muscle mass nor inflammation, it has been postulated to be an improved marker of glomerular filtration rate (GFR) compared with the serum creatinine level. To evaluate the clinical usefulness in terms of estimation of the glomerular filtration rate(GFR), we compared the serum cystatin-C concentration with other markers of GFR, such as serum levels of creatinine(SCr), alpha 1-microglobulin(alpha 1MG), and beta 2-microglobulin(beta 2MG). Their variations were analyzed based on 2-hour creatinine clearance (2hCCr) as a standard marker of GFR. The logarithmic value of serum cystatin-C level showed a stronger negative correlation(-0.959) with the logarithmic value of 2hCCr than that of other markers(-0.924, -0.942, -0.888; SCr, alpha 1MG, beta 2MG, respectively). Although beta 2MG showed the next strongest correlation with 2hCCr, it had a significantly lower sensitivity when detecting mild reduction of GFR. In addition, serum cystatin-C showed the greatest area under the curve of receiver-operating characteristic(ROC) of all GFR markers at both higher(90 ml/min.) and lower(70 ml/min.) cut-off value of 2hCCr. These data suggest that serum cystatin-C is useful for estimating GFR, even if the reduction of GFR is very mild.

Cloning and functional studies of splice variants of the alpha-subunit of the amiloride-sensitive Na+ channel.

The alpha-subunit of the amiloride-sensitive epithelial Na+ channel (alpha ENaC) is critical in forming an ion conductive pore in the membrane. We have identified the wild-type and three splice variants of the human alpha ENaC (h alpha ENaC) from the human lung cell line H441, using RT-PCR. These splice variants contain various structures in the extracellular domain, resulting in premature truncation (h alpha ENaCx), 19-amino acid deletion (h alpha ENaC-19), and 22-amino acid insertion (h alpha ENaC + 22). Wild-type h alpha ENaC and splice variants were functionally characterized in Xenopus oocytes by coexpression with hENaC beta- and gamma-subunits. Unlike wild-type h alpha ENaC, undetectable or substantially reduced amiloride-sensitive currents were observed in oocytes expressing these splice variants. Wild-type h alpha ENaC was the most abundantly expressed h alpha ENaC mRNA species in all tissues in which its expression was detected. These findings indicate that the extracellular domain is important to generate structural and functional diversity of h alpha ENaC and that alternative splicing may play a role in regulating hENaC activity.

Transcriptional activation of RACTK1 K+ channel gene by apical alkalization in renal cortical collecting duct cells.

We have previously demonstrated that RACTK1 cDNA encodes a pH sensitive K+ channel expressed in the apical side of renal collecting tubule cells. To determine whether extracellular pH induces the RACTK1 gene expression in the renal cortical collecting duct (CCD) cells, we measured mRNA of the RACTK1 using cultured rabbit CCD cells. Alkalization of incubation medium activated the transcription of the RACKTK1 gene in a time- and dose-dependent manner after 1 h, and reached a maximal level after 12 h. To examine whether the stimulation of mRNA by alkalization of body fluid occurs also in vivo, mRNA levels were measured in mice loaded with acid or alkali. The RACTK1 mRNA was increased in association with the rise in urinary pH. To examine side face of the effect of pH on stimulation of mRNA, we observed the effect of pH in the apical or the basolateral side in the preparation where CCD cells were cultured on filter membrane supports. Alkalization of the apical side but not of the basolateral side, was shown to be a determinant in inducting the RACTK1 mRNA. These findings suggest that, in addition to rapid direct regulation of RACTK1 K+ channel conductance by intracellular pH, this channel is also regulated by the changes in luminal pH through synthesis of channel protein by transcriptional activation.

HTLV-I-associated myelopathy in a patient with chronic renal failure.

A case of human T-cell lymphotrophic virus type I (HTLV-I)-associated myelopathy (HAM) occurring in a 62-year-old Japanese male with a history of hemodialysis and repeated blood transfusions due to chronic renal failure (CRF) is reported. The patient was a lifelong resident of Chiba Prefecture, a nonendemic area for HTLV-I infections. The clinical course was characterized by abrupt onset and rapid progression of neurological signs and symptoms, which appeared to have responded quite well to prednisolone. Although patients with a history of CRF, hemodialysis, and transfusion of blood units which were not screened for anti-HTLV-I antibodies appear to belong to the high risk group for HTLV-1 infections and subsequent development of HAM, reports on such cases have been scanty. Only 3 cases have been reported to date. It appears quite possible that many patients with CRF and HAM remain misdiagnosed.