Calcium oxalate (COM) crystals are the commonest component of kidney stones. These arise mainly in the distal tubules and collecting ducts. To gain further insight for the cellular damage in terms of oxidative stress caused by COM deposition, in vitro and in vivo model studies were performed. In vitro In renal distal tubule cells, COM and free oxalate treatment caused a 3- and 2-fold increase respectively in superoxide (O2*") formation, originating from mitochondria. This was measured by lucigenin chemiluminescence in digitonin permeabilised cells. However, hydroxyapatite produced a much lower but significant enhancement of 02*", whilst other micro-particles, uric acid crystals, brushite, zymosan, and latex beads had no effect. When EDTA was omitted during O2*" monitoring, COM induced mitochondrial 02*" was ablated indicating a requirement for the release of free oxalate. Mitochondrial oxalate uptake was studied by employing different oxalate transport inhibitors. Omitting phosphate from the media or using mersalyl both of which block dicarboxylate transport, caused a significant decrease in the 02*" formation evoked by COM treatments. Using the membrane potential sensitive-probe tetramethylrhodamine methyl ester (TMRM) together with confocal microscopy, evidence is presented that in cells where COM binding had occurred a marked change in the mitochondrial membrane potential (Aij/m) occurred. COM also modulated intracellular Ca2+ signalling as demonstrated using the Ca2- sensitive dye Fura-2 AM, and this was via a non-mitochondrial mechanism. In Vivo Using a rat model of crystalluria and renal stones initiated by treatment with ethylene glycol (EG) and 1, 25-dihydroxycholecalciferol (DHC), nephrolithiasis arose in kidneys and this was linked to oxidative stress. In the EG + DHC treated animals where crystalluria was evident, this oxidative insult was manifest by a decrease in total and mitochondrial glutathione concentration, as well as an increased activity of glucose-6-phosphate dehydrogenase. Severe kidney damage at the mitochondria level was a further observation, indicated by the diminished O2 consumption resulting in a lowered O2 production. In addition, histopathological analysis revealed increased renal tubular pathology characterised by obstruction, distension and interstitial inflammation. The above findings were not observed in hyperoxaluria (EG) or calciuria (DHC) and are therefore a direct effect of crystal formation in kidney distal tubules that have implications in kidney stone disease which are discussed.

Background: The interest in living donor transplantation has been driven by the continuing fall in available cadaveric organs for transplantation. During the last five years there has been a substantial growth in living donor kidney transplantation in the UK but there is still considerable room for expansion in comparison with activity in Scandinavia and the USA. Traditionally kidneys have been harvested from donors via a loin incision with partial resection of the twelfth rib, which placed a considerable burden on the donors in terms of post-operative pain, absence from work, and morbidity. Laparoscopic live donor nephrectomy developed in 1995, promised to reduce these burdens on the donors and reduce some of the disincentives to kidney donation. Several comparative studies have shown this new technique to hold promise in terms of less pain and faster inpatient and outpatient recovery. However there were some concerns in procuring the kidneys with this technique, namely, increase in warm ischaemia times and the quality of graft. Methods: This was addressed in the setting of a prospective randomised controlled trial of laparoscopic versus limited incision live donor nephrectomy. Live kidney donors were randomly assigned in a 2:1 ratio to laparoscopic (LDN n=56) or short incision open donor nephrectomy (ODN n=28). Quality of life was assessed using the Short-Form 36 questionnaire. Postoperative analgesia was by morphine PCAS. Pain scores were recorded using visual analogue and verbal response scales. Donor convalescence was self-reported using a prospective diary system. Our study was the first randomised control trial to present live donor transplant recipient data at a minimum follow-up of four years. There were no differences in renal function or allograft survival for kidneys removed by LDN (laparoscopic donor nephrectomy) or ODN (open donor nephrectomy) at this point. The other aspect of this study is that this is the first study to compare respiratory function after LDN and ODN. During the evolution of LDN, the vessels are secured with various methods (endoclips, polymer clips and stapling device). These methods were compared with respect to complications and maximum length of vessels obtained. Technical modifications and improvement of techniques especially when comparing right and left donor nephrectomy are described. Results: Postoperative morphine requirement was lower in the LDN group [median (range) 59 (6-136) vs ODN 90 (35-312)mg; p=0.01]. Donors in the LDN group returned to normal activities more quickly compared to the ODN group [median (range) days to: driving 21 (7- 70) vs 28 (7-70); p=0.05), exercise 28 (7-77) vs 42 (14-84); p=0.001, return to work 42 (14- 84) vs 66.5 (14-112); p=0.001]. When compared to the pre-operative baseline, norm adjusted physical component scores (PCS) fell significantly at 6 weeks in both the LDN (mean±SD 46.3±8.9 vs 55±6.9; p=0.001) and ODN groups (44.0±7.9 vs 52.7±9.0; p=0.008). Nonetheless, the bodily pain domain score of PCS was significantly better in the LDN group (57.5 to 49.5; p=0.0001). The mental component score also fell in the ODN group (48±10.2 vs 53.5±7.6; p=0.02). In contrast, there was no fall in the mental component score after LDN (mean±SD 51.9±7.2 vs 53.8±6.4; p=0.29). Conclusions: In conclusion, our trial has shown that laparoscopic donor nephrectomy removes some of the disincentives to live kidney donation. This can be achieved without any additional morbidity in the recipient. This study provides high-level evidence to show that laparoscopic donor nephrectomy improves recovery back to the normal activities of daily life, is less painful than open surgery and improves the mental component of quality of life.

Background Anaemia is common in patients with functioning kidney transplants. Mycophenolic acid (MPA) is anecdotally associated with anaemia, but the mechanism of action is not proven. The effect of recombinant human erythropoietin (rHuEPO) in this setting is poorly studied. Hypothesis 1) MPA causes decreased erythropoiesis in vitro. 2) rHuEPO treatment attenuates progression of renal failure in Post-Transplant Anaemia (PTA). 3) rHuEPO treatment improves Health Quality of Life (HRQOL) and cardiovascular biomarkers. Methods An in vitro model of erythropoiesis in UT-7 cells (human haemo-Ieukaemic cells responsive to EPO) and murine bone marrow cells was used to study the effects of mycophenolic acid on erythroid cell lines. An open labelled, randomised, controlled trial of the use of rHuEPO in anaemic kidney transplant recipients (KTRs) was performed. The primary end points were renal progression measured by rate of change of estimated Glomerular Filtration Rate (eGFR),; change in Protein: Creatinine Ratio (P: CR) and change in blood pressure (B~). Secondary outcomes were changes in SF-36® Health Quality-of-Life (HRQOL) scores and change in Left Ventricular Mass Index (LVMI). A subgroup of the trial was studied for changes in cardiovascular biomarkers using flow cytometry. Results MPA significantly decreased both the proliferation of UT-7 cells (P < 0.001) and erythropoiesis in murine bone marrow cells (P < 0.0001). This was associated with an increase in caspase-3 activity in UT-7 cells in a dose-dependent manner (P < 0.01). Inhibition was reversed in UT-7 cells and in murine bone marrow by guanosine, but not by caspase inhibitors. The apoptosis induced by MPA was also reversed by guanosine. UT-7 cells treated with MPA showed a decrease IMPDH activity. Epoetin beta (the rHuEPO used) treatment improved haemoglobin concentration resulting in a significant improvement of the Vitality Health Domain scales of the SF-36 QOL in the treatment group (P = 0.02). There was no significant difference in the primary outcomes. There was no difference in LVMI or in cardiovascular biomarkers. Conclusion Mycophenolic acid inhibits proliferation of UT-7 cells and inhibits erythropoiesis in murine bone marrow cells via direct inhibition of IMPDH. These in vitro findings offer an explanation to the clinical association of anaemia and MPA use. The treatment with rHuEPO of PTA was found to be associated with improved HRQOL in certain domains. There were no obvious safety concerns demonstrated. However it did not affect the rate of progression of renal failure. Similarly no effect was seen on biomarkers of cardiovascular morbidity.

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Colocystoplasty has an important role in bladder reconstructive surgery. However it can be affected by chronic inflammation and excessive mucous production which may lead to urinary tract infectioh, stone formation and occasionally malignant.transformation. We postulate that changes in the colonic segment within the augmentation are affected by a condition known as diversion colitis, which is found in bowel segnients diverted away from the faecal stream. The aetiology ofthis condition is a luminal deficiency ofshort chain fatty acids (SCFAs) which are produced by bacterial fermentation ofdietary fibre and include butyrate, propionate and acetate. Studies have shown that they are the colonocytes' preferred energy substrate and have an important role in colonic mucosal health andprevention of malignant transformation. The purpose ofthis study was to investigate the effect ofSCFAs on the bladder which is an .important consideration when contemplating intravesical therapy in colocystoplasty. Using monolayer cell cultures ofboth primary urothelial cells and urothelial cancer cell lines the effects of SCFAs were inves~igated. The MIT (3-[4,5-dimethyl thiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazolyl blue) cytotoxicity assay was employed to study cell growth. Fluorescence microscopy with acridine orange and flow cytometry were used to study apoptosis and the cell cycle. It was found that all three SCFAs inhibit cell growth, induce apoptosis and induce cell cycle arrest. Butyrate had the' most potent effect in vitro followed by propionate and then acetate. To assess the significance ofthese results in vivo, intravesical instillation of SCFAs was performed in a rodent model which demonstrated no significant adverse effects both histologically and on urothelial cell turnover.

The glomerulus is the site of ultrafiltration within in the nephron of the kidney. The Glomerular Filtration Barrier (GFB) restricts protein loss whilst allowing the passage of water and small solutes. There are two cell types that contribute to the formation of the GFB: the Glomerular Endothelial Cell (GEne) and the podocyte. Both cell types contribute to the formation of the Glomerular Basement Membrane (GBM). The podocytes are composed of three structurally distinct segments: the cell body, major processes and foot processes. The foot processes regularly interdigitate. The gap between interdigitating foot processes is bridged by the slit diaphragm. The slit diaphragm is a molecular sieve that enables the podocyte to restrict urinary protein loss. Podocyte injury leads to foot process effacement 'and the concomitant loss of the slit diaphragm. Therefore podocyte injury compromises the integrity of the GFB leading to the proteinuria and nephrotic syndrome. Foot process effacement requires increased podocyte motility. Indeed, podocyte motility in vitro is being increasingly used as a marker of foot process effacement in vivo. Whilst the pathogenesis of nephrotic syndrome is not completely understood, it is well established that the podocyte is the target cell. In this study, two sources of podocyte injury have been studied.

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