LYSOSOMAL IRON ACCUMULATION AND TUBULAR DAMAGE IN RAT PUROMYCIN NEPHROSIS AND AGEING

1. Energy dispersive X-ray spectrometry was used to examine the relationship between proteinuria and increased urinary iron excretion, and structural and functional damage in puromycin nephrosis. 2. After 11–12 days rats treated with puromycin (10 mg/100g, i.v.i.) had greater proteinuria (211.6 ± 35.7 mg/day, mean ± s.e.m.) and urinary iron excretion (15.4 ± 2.2 μg/day) than salinetreated controls (14.5 ± 1.4 mg/day and 1.1 ± 0.2 μg/day, respectively, both P<0.001). 3. On day 13, mean lysosomal iron concentration of proximal tubular cells (306.6 ± 64.5 vs 11.9 ± 8.6 mg%, P<0.001), and proximal tubular cell damage assessed semi-quantitively (1.17 ± 0.10 vs 0.62 ± 0.10, P<0.001) were higher and creatinine clearance (0.15 ± 0.01 vs 0.29 ± 0.02 mL/min perg kidney weight, P<0.001) lower than in control rats. 4. At days 35, 60 and 360 there were no differences in any of the measured parameters between rats treated with puromycin or saline, and in both groups proteinuria, tissue damage and lysosomal iron concentration increased with time. 5. Lysosomal iron accumulation was the only independent predictor of both functional and structural damage. 6. In conclusion, the apparent association between proteinuria and tubulo-interstitial damage in puromycin nephrosis, and with ageing, is best explained by factors associated with accumulation of iron within lysosomes of proximal tubule cells.     

Mycophenolate Mofetil Is Associated with Altered Expression of Chronic Renal Transplant Histology

Mycophenolate mofetil (MMF) reduces acute rejection in controlled trials of kidney transplantation and is associated with better registry graft survival. Recent experimental studies have demonstrated additional antifibrotic properties of MMF, however, human histological data are lacking. We evaluated sequential prospective protocol kidney biopsies from two historical cohorts treated with cyclosporine (CSA)-based triple therapy including prednisolone and either MMF (n = 25) or azathioprine (AZA, n = 25). Biopsies (n = 360) were taken from euglycemic kidney-pancreas transplant recipients. Histology was independently assessed by the Banff schema and electron microscopic morphometry. MMF reduced acute rejection and OKT3 use (p < 0.05) compared with AZA. MMF therapy was associated with limited chronic interstitial fibrosis, striped fibrosis and periglomerular fibrosis (p < 0.05-0.001), mesangial matrix accumulation (p < 0.01), chronic glomerulopathy scores (p < 0.05) and glomerulosclerosis (p < 0.05). MMF was associated with delayed expression of CSA nephrotoxicity, reduced arteriolar hyalinosis, striped fibrosis and tubular microcalcification (p < 0.05-0.001). The beneficial effects of MMF remained in recipients without acute rejection. Retrospective analysis shows that MMF therapy was associated with substantially reduced fibrosis in the glomerular, microvascular and interstitial compartments, and a delayed expression of CSA nephrotoxicity. These outcomes may be due to a limitation of immune-mediated injury and suggest a direct effect of reduced fibrogenesis.     

Androgen receptor YAC transgenic mice carrying CAG 45 alleles show trinucleotide repeat instability

X-linked spinal and bulbar muscular atrophy (SBMA) is caused by a CAG repeat expansion in the first exon of the androgen receptor (AR) gene. Disease-associated alleles (37-66 CAGs) change in length when transmitted from parents to offspring, with a significantly greater tendency to shift size when inherited paternally. As transgenic mice carrying human AR cDNAs with 45 and 66 CAG repeats do not display repeat instability, we attempted to model trinucleotide repeat instability by generating transgenic mice with yeast artificial chromosomes (YACs) carrying AR CAG repeat expansions in their genomic context. Studies of independent lines of AR YAC transgenic mice with CAG 45 alleles reveal intergenerational instability at an overall rate of approximately 10%. We also find that the 45 CAG repeat tracts are significantly more unstable with maternal transmission and as the transmitting mother ages. Of all the CAG/CTG repeat transgenic mice produced to date the AR YAC CAG 45 mice are unstable with the smallest trinucleotide repeat mutations, suggesting that the length threshold for repeat instability in the mouse may be lowered by including the appropriate flanking human DNA sequences. By sequence-tagged site content analysis and long range mapping we determined that one unstable transgenic line has integrated an approximately 70 kb segment of the AR locus due to fragmentation of the AR YAC. Identification of the cis - acting elements that permit CAG tract instability and the trans -acting factors that modulate repeat instability in the AR YAC CAG 45 mice may provide insights into the molecular basis of trinucleotide repeat instability in humans.      

Cryopreservation of human oocytes: a review of current problems and perspectives

It is well recognized that the ability to cryopreserve unfertilizedhuman oocytes would make a significant contribution to infertilitytreatment. However, despite considerable interest, very fewsuccessful pregnancies have arisen from cryopreserved oocytesafter thawing, insemination and transfer of the subsequent embryo.The reasons for this lack of progress may well result from adearth of information on how the various biophysical changesduring a cryopreservation regimen affect human oocyte function.Recently, fundamental studies on the effects of cooling, membranepermeability, cryoprotectant addition and ice formation havebeen performed on human oocytes by a number of groups, and theseform the basis of the current review. It is likely that successfulhuman oocyte cryopreservation will only follow once these factorsare fully understood, but the existing base of knowledge shouldprovide a platform for further improvements in the techniquescurrently employed.