The aim of this project was to devise a simple and efficient method to identify cDNAs expressed specifically in the mouse fetal kidney which might be important in kidney development. A 14.5 day post coitum fetal kidney cDNA library was made which has a depth of 1.2 x 10<SUP>6</SUP> original clones and an average insert size of 1.5 kilobases. Using a novel subtractive hybridisation technique which identifies genes expressed in the fetal kidney but not in the adult liver, a subtracted 14.5 day post coitum fetal kidney cDNA pool was obtained. By screening the library with the subtracted fetal kidney cDNA, several clones were identified. Six, when analysed by sequencing and northern blot analysis, were shown to contain B1 embryonic repeats and were not analysed further. Three, when looked at by northern blot analysis and sequence homology, proved to be the most interesting. Further characterisation identified the clones as three independent isolates of a cDNA encoding the ε member of the 14-3-3 gene family, implicated in the regulation of protein kinase C activity and of exocytosis. Northern blot analysis of RNA identified a 2.0 kilobase transcript present in many tissues including the fetal kidney, but not in adult liver. More detailed <I>in situ</I> hybridisation analysis showed that, although the gene is expressed in most 12.5 day post coitum embryonic mouse tissues, the level of expression varies. In general, it is low in epithelial tissues and high in mesenchymal cells. As the mesenchyme in bone, kidney, gut and lung differentiates, the level of expression drops. This observation argues that the 14-3-3 ε isoform protein plays a role in mesenchyme differentiation including acting as the kidney mesenchyme epithelialises.

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Aim: Previous work carried out at the Institute of Child Health revealed that in the fetal sheep, combined urethral and urachal occlusion initiated at 75 days gestation (full term = 145 days) and maintained for 30 days resulted in dilated, hypocontractile and hypercompliant bladders, associated with uniformly disrupted kidney development. The aim of this project was to create a less severe model of fetal bladder outlet obstruction, and to define the role of the urachus. This model would then be utilised to investigate the prenatal onset of obstructive bladder dysfunction by means of in-utero radiotelemetered urodynamics. We hypothesised that short-term obstruction would result in a thick-walled bladder with preserved contractility and compliance, and that urachal ligation alone would result in similar features.;Methods: Male fetal lambs were assigned to urachal ligation and partial urethral occlusion, urachal ligation only, or sham, groups. Histological analyses, filling cystometry and contractility studies were performed following nine days of obstruction. Natural-fill radiotelemetered urodynamics were performed on the urachal and urethral occlusion group.;Results: Nine days urachal and urethral occlusion from mid-gestation caused hydronephrosis and increased bladder weight, protein and DNA content. Detrusor smooth muscle architecture was maintained but urothelia were thickened and showed basal apoptosis. Bladder compliance, wall stress and contractility were not significantly deranged. The thickest, most compliant bladders were found to be associated with kidneys exhibiting glomerular cysts. Urachal obstruction alone also resulted in similar changes, suggesting that the male fetal lamb urethra is a high-resistance conduit at this gestation. Radiotelemetered urodynamics were only feasible in the obstructed group from 94 days gestation, and revealed the presence of early-onset hypercontractility. Fetal voids became increasingly frequent and prolonged, occurring at higher voiding pressures baseline filling pressures did not vary significantly over the nine-day period of observation.;Conclusion: Short-term fetal bladder outflow obstruction from mid-gestation generated thick-walled bladders without evidence of contractile failure these were associated with cystic kidneys. Detrusor hypercontractility and raised voiding pressures were observed within hours of obstruction, although filling pressures remained stable. Future work will investigate the effect of vesico-amniotic shunting on bladder function.

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Pyrophosphate (PPi) has been known since the 1960s as an inhibitor of calcium renal stone formation. Naturally occurring mutations in a putative PPi transporter, ANK, causes renal calcification in mice. We hypothesised that the human homologue, ANKH, plays a role in PPi transport in the human kidney. Immunocytochemical localisation of ANKH in human kidney showed greater abundance in the cortical collecting duct than elsewhere in the nephron. The transport function of ANKH was investigated by heterologous expression of ANKH in Xenopus oocytes. Despite confirmation of ANKH expression at the oocyte plasma membrane, neither ANKH-mediated PPi efflux (the physiological mode of operation) nor influx was detectable compared to water-injected oocytes. Pyrophosphatase activity was detected at the surface of oocytes, suggesting hydrolysis of PPi to inorganic phosphate. Screening using a yeast two-hybrid method of the N-terminal of ANKH against a mouse renal library identified a possible protein-protein interaction with the fatty acid transporter SLC27A2, whose acyl-CoA synthetase activity yields PPi as an end product. This suggests that ANKH and protein partners such as SLC27A2 may form a biochemical couple whereby PPi is sequestered by transmembrane transport rather than by hydrolysis. Since there is no pyrophosphatase activity in peroxisomes, we suggest that the ANKH/SLC27A2 complex is a candidate protein for the peroxisomal membrane PPi transporter. AVP mediates increased expression and localisation of ANK to the apical membrane of a collecting duct model (mpkCCDcl4) in vitro, suggesting physiologically appropriate regulation, analogous to that of aquaporin-2. These findings offer insights into the cellular homeostasis of PPi. Instead of cytosolic hydrolysis, coupling of PPi generation and ANKH-mediated transport as part of a protein complex may allow PPi to be compartmentalised, preserving it for use within vesicular structures elsewhere in the cell or allowing export to the extracellular medium to assist in the regulation of apatite deposition.

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Accompanying the arterial hypertension in the dog following the partial constriction of one of a pair of renal arteries, there are rises in plasma renin activity, plasma volume and renal venous prostaglandins E and F of the contralateral kidney, reaching maxima on days 2, k and 5 respectively. By the tenth day all parameters were normal, with the exception of blood pressure. It is suggested that the contralateral kidney responds to the fluid retention by natriuresis and diuresis mediated by an increase in prostaglandin synthesis. It is also concluded that PGE is unlikely to play a role in the antihypertensive action of this kidney. A transient elevation of circulating PGA was observed in some experiments. In the two-kidney Goldblatt rat a transient decrease in renal plasma flow of the clipped kidney was seen whilst the contralateral kidney showed no change. Renal venous PC-E2 and PGF2CK concentrations changed inversely with renal plasma flow and calculated secretion rate of the clipped kidney fell, although it was not possible to establish the significance of this fall. Such a fall could lead to a reduction in intrarenal vasoconstrictor potentiation produced by prostaglandins in this species. The absence of a rise in secretion rate of PGE2 in either kidney raises further doubt concerning renal prostaglandins as mediators of the antihypertensive action of the kidney in the rat. In the pump-perfused dog kidney calculated secretion rate of PGE fell as perfusion pressure fell below the autoregulation range, whilst PGF secretion did not change. Meclofenamate failed to abolish renal blood flow autoregulation, despite a 70% fall in prostaglandin synthesis and it is therefore concluded that autoregulation is not dependent on prostaglandins.