A HPLC method was developed for determination of cimetidine in human plasma and urine. Plasma samples were alkalinized followed by liquid extraction with water-saturated ethyl acetate then evaporated under nitrogen. The extracts were reconstituted in mobile phase and injected onto a C18 reversed-phase column; UV detection was set at 228nm. Urine samples were diluted with an internal standard/mobile phase mixture (1:9) prior to injection. The lower limit of quantification in plasma and urine were 100ng/ml and 10μg/ml, respectively; intra- and inter-day coefficients of variation were ≤4.2%. Advantages of this validated assay include a readily available internal standard, simplified plasma extraction and urine dilution methods, and applicability to clinical studies investigating the renal handling of cimetidine.

Cyclosporine A (CsA) is the most effective and widely used immunosuppressant drug in heart, lung and kidney transplantation. However, the effect of CsA is limited by the significant toxicity. The mechanism of CsA-induced toxicity is remaining controversial. Cellular apoptosis is being suggested as a possible mediator of CsA toxicity. To date, regarding the effects of CsA on apoptosis and apoptosis-related gene regulation in cardiomyocytes remain unclear. Therefore, the current study was designed to investigate the effect of CsA on apoptosis and apoptosis-related gene p53 expression in human cardiomyocytes. We hypothesized that CsA induces apoptosis in human cardiomyocytes through p53-dependent pathway.Human cardiac atrial tissue was obtained from open-heart surgery (n=5). The cardiac tissue was minced and incubated in the special tissue culture system for 24 hours in the absence or presence of CsA (10-7 M). To detect the DNA fragmentation, in situ terminal deoxymucleotidyl transferase dUTP nick end labeling (TUNEL) was performed.

C-type natriuretic peptide (CNP) is endothelial cell origin and has potent vasodilatory and antimitogenic actions. We reported that CNP was markedly increased in human cardiac tissue with severe congestive heart failure. To date, the effects of CNP on cardiomyocyte growth and death remain unclear. Therefore, the present study was designed to investigate the actions of CNP on apoptosis and apoptosis-related gene p53 expression in human cardiomyocytes. Human cardiac atrial tissue was obtained from open-heart surgery (n=6). The cardiac tissue was minced and incubated in the special tissue culture system in the absence or presence of CNP (10 -7 M) for 24 hours. These studies were repeated with HS-142-1 (HS, 10 -6 M), a natriuretic peptide biological receptor antagonist. To detect the DNA fragmentation, in situ terminal deoxymucleotidyl transferase dUTP nick end labeling (TUNEL) was performed. The p53 expression was determined by immunohistochemical staining (IHCS). An average of 1000 nuclei was analyzed for TUNEL and p53 staining studies.

920 Transforming growth factor-beta (TGF-β) is produced by a variety of cells including several renal cell types. TGF-β is a growth-regulating protein that has been shown to enhance collagen production in cultured cells. High cyclosporine-A (CsA) levels might lead to nephrotoxicity with increasing TGF-β and collagen synthesis in renal transplant recipients. We hypothesized that local renal TGF-β expression may increase with high circulating CsA levels. Therefore, the current study was designed to determine the TGF-β1 isoform expression in human renal biopsy tissue with high and low CsA levels by immunohistochemical staining (IHCS). Kidney biopsies from nine transplant recipients with chronic allograft nephropathy were obtained before (CsA level: 321±106 ng/ml) and after (CsA level: 141±40 ng/ml) reduction in CsA dosage (time between biopsies: 15.8±0.8 months). TGF-β1 IHCS score (S) (0-4) and positive staining area (%) were significantly decreased in patients with reduction in CsA levels. TGF-β1 was localized in the glomeruli and tubular cells, and was minimally evident in control biopsy specimens (IHCS score 0.2-0.4). Pathological evaluation included percentage expression (%) of interstitial fibrosis and tubular atrophy (FIB), vascular sclerosis (VS), transplant glomerulopathy (TG), and vascular hyalinosis (VH). Serum creatinine (CR, mg/dl) and BUN (mg/dl) levels were also investigated. Mean±SE, *p<.05 vs High CsA group. (Table)TableThese data indicate that chronic reduction in CsA may diminish production of renal TGF-β expression, and results in decreased renal fibrosis and improvement of renal function in patients with chronic allograft nephropathy.

178 Increasing renal cell apoptosis may contribute to tubular atrophy and fibrosis in cyclosporine A (CsA)-induced nephropathy. The mechanisms of apoptosis include p53-dependent and p53-independent pathways. To date, the relationships among circulating CsA level and renal cell apoptosis and p53 gene expression as well as renal pathological changes and renal functions remain unclear. Therefore, the current study was designed to determine the amount of apoptosis and p53 gene expression in human renal biopsies from ten patients with chronic allograft nephropathy and no acute rejection before and after a chronic reduction in CsA blood levels (356±94 to 95±13 ng/ml). Interval between biopsies was 14.1±0.7 months. Apoptosis was determined by TUNEL staining. The p53 protein expression was determined by immunohistochemical staining (IHCS). Positive TUNEL (%) and p53 (%) staining was evaluated in glomeruli and tubulointerstitium. Pathological evaluation included percentage expression (%) of interstitial fibrosis and tubular atrophy (FIB), vascular sclerosis (VS), transplant glomerulopathy (TG), and vascular hyalinosis (VH). Serum creatinine (CR, mg/dl) and BUN (mg/dl) levels were also investigated. (Table)TableThese data demonstrate that chronic reduction of CsA blood levels is associated with decreased renal cell apoptosis and p53 expression with improvement of renal pathological score and renal function. These studies suggest that renal cell apoptosis and apoptosis-related gene activation may play an important pathophysiological role in chronic allograft nephropathy, and that chronic CsA reduction may reduce the progression of this disease.