A laparoscopic approach to allograft nephrectomy and bilateral native nephrectomy: a case report.

INTRODUCTION: Laparoscopic surgery is rapidly emerging as the standard of care for a variety of urological conditions, even among patients who have undergone prior renal transplantation. We describe the technique of bilateral native nephrectomy and allograft nephrectomy by laparoscopy. CASE REPORT: A 32-year-old man with end-stage renal disease who had undergone a cadaveric renal transplant presented with chronic graft dysfunction. He had received a living donor kidney transplant with a postoperative course complicated by persistent proteinuria and refractory hypertension. Our nephrology service indicated the need for bilateral native nephrectomy and allograft nephrectomy for better blood pressure control following a second transplant. Bilateral native nephrectomy was performed following the previous reported techniques for pure laparoscopic nephrectomy. Allograft nephrectomy started by dissection of the iliac vessels to identify the vascular anastomosis. The hilum of the transplanted kidney was accessed. The renal vessels were clipped and transected. The ureter was identified and clipped. All three kidneys were removed from the abdominal cavity through a 3-cm skin incision. RESULTS: The left nephrectomy took 25 minutes and the right nephrectomy, 40 minutes. The estimated blood loss was 300 mL and the total operative time was 210 minutes. The patient had an uneventful postoperative course and was discharged on the third postoperative day. CONCLUSIONS: We demonstrate the feasibility of laparoscopic allograft nephrectomy and bilateral native nephrectomy in a transplant recipient.

A comparison of kinetic and regulatory properties of the tetrameric and dimeric forms of wild-type and Thr427-->Pro mutant human phenylalanine hydroxylase: contribution of the flexible hinge region Asp425-Gln429 to the tetramerization and cooperative substrate binding.

Recombinant human phenylalanine hydroxylase (hPAH, phenylalanine 4-monooxygenase EC 1.14.16.1) is catalytically active both as a tetramer and a dimer [Knappskog, P.M., Flatmark, T., Aarden, J.M., Haavik, J. and Martínez, A. (1996) Eur. J. Biochem. 242, 813-821]. In the present study we have further characterized the differences in kinetic and regulatory properties of the two oligomeric forms when expressed in Escherichia coli. The positive cooperativity of L-Phe binding to the tetrameric form both in enzyme kinetic studies (h = 1.6) and intrinsic tryptophan fluorescence measurements (h = 2.3) was abolished in the dimer, which also revealed a catalytic efficiency (Vmax/[S]0.5) of only 35% of the tetramer. Whereas the catalytic activity of the tetramer was activated fivefold to sixfold by preincubation with L-Phe, the dimer revealed only a 1.6-fold activation. The crystal structure has identified a five-residue flexible hinge region (Asp425-Gln429) that links the beta-strand Tbeta2 (Ile421-Leu424) and the 24 residue amphipathic alpha-helix Talpha1 (Gln428-Lys452) at the C-terminus which forms an antiparallel coiled-coil structure in the center of the tetramer [Fusetti, F., Erlandsen, H., Flatmark, T. & Stevens, R.C. (1998) J. Biol. Chem. 273, 16962-16967]. The potential role of this flexible hinge in the tetramerization and the conformational transition of wt-hPAH on the cooperative binding of L-Phe was examined by site-specific mutagenesis. Substitution of Thr427 by a Pro (as in tyrosine hydroxylase) resulted in a mutant protein which was isolated mainly (about 95%) as a dimer. The isolated tetramer of T427P revealed no kinetic cooperativity of L-Phe binding, the catalytic efficiency (Vmax/[S]0.5) was decreased to about 39% of the wild-type tetramer and it was not activated by L-Phe preincubation. The dimeric forms of T427P and wt-hPAH revealed rather similar kinetic properties. The lack of kinetic cooperativity of the T427P tetramer was associated with a corresponding change in the binding isotherm for L-Phe as studied by intrinsic tryptophan fluorescence measurements. Protein stability was also reduced both for the E. coli expressed and the in vitro synthesized mutant enzyme. Collectively, these results indicate that the positive cooperativity of L-Phe binding to wt-hPAH requires a tetrameric enzyme with a C-terminal flexible hinge region (Asp425-Gln429) which has a structural role in the formation of the enzyme tetramer. Furthermore, this hinge region represents a motif in the PAH structure that is involved in the conformational change transmitted through the protein on the cooperative binding of L-Phe to tetrameric wt-hPAH. This conclusion is further supported by studies on two disease (phenylketonuria)-associated mutant forms.