Romina Graziotto

Perforin, Granzyme B, and fas ligand for molecular diagnosis of acute renal-allograft rejection: analyses on serial biopsies suggest methodological issues.

Background. The Perforin-Granzyme B and Fas/Fas Ligand apoptotic mechanisms are involved in the development of acute renal rejection (AR). We describe our experience of analyzing the expression of cytotoxic T-lymphotoxins (CTL) in biopsies and peripheral blood leukocytes (PBL) for the diagnosis of AR. Methods. We analyzed Perforin (P), Granzyme B (GB) and Fas Ligand (FL) expression in 68 renal biopsies and 64 PBL using comparative kinetic RT-PCR and, for GAPDH and FL, we also replicated with real-time RT-PCR. The levels of expression were measured in different groups, such as T0 (biopsies before reperfusion and PBL in recipient before the transplant [Tx]), Td (biopsies and PBL collected for clinical purposes) and Tp (biopsies and PBL two months after Tx). Results. A higher CTL expression was seen in nonrejecting (NR) biopsies in the first 2 months after Tx. P and FL were significantly more expressed during AR in all biopsies and in Td, while P remained upregulated in Tp. In PBL, there was no significant increase in CTL transcription during AR. A variable expression of CTL emerged in all T0 biopsies. Conclusions. Two lytic pathways are activated in biopsies when AR occurs shortly after Tx, whereas the P/GB mechanism prevails if it occurs later on. Only P and FL in biopsies might be able to predict AR diagnosis, but with a considerable variability in each sample, possibly due to the small portion of tissue core, which may be inadequate for molecular diagnosis. CTL expression in PBL does not correlate with histological AR.

Early activation of fibrogenesis in transplanted kidneys: A study on serial renal biopsies

BACKGROUND/AIMS In kidney transplants, the renin-angiotensin system (RAS) is involved in systemic and local changes that may induce fibrosis. Our aim was to use gene expression and immunohistochemical analysis to investigate the RAS and several factors involved in the fibrogenic cascade in allograft biopsies. METHODS We considered 43 donor biopsies (T0), 18 biopsies obtained for diagnostic purposes (Td) and 24 protocol biopsies (Tp) taken 2 months after transplantation in patients with stable renal function. Morphometric alpha SMA and TGF beta 1 analysis, and Massons Trichrome staining were performed. mRNA levels of angiotensinogen, renin, ACE, AT1-R, AT2-R, TGF beta 1, BMP-7, Coll III, fibronectin and alpha SMA were analyzed by real-time RT/PCR. MDRD a year after the transplant was also considered. RESULTS Significantly higher levels of AT1-R and alpha SMA transcripts were found in Tp than in T0. Regression analysis showed significant TGF beta 1-independent positive correlations between RAS and matrix components in T0 and Tp, but more evident in Tp, where a positive correlation between TGF beta 1 and Massons Trichrome stained areas was also seen. CONCLUSION Our results suggest that RAS and TGF beta 1-related fibrogenic loops are activated as early as 2 months after kidney transplantation.

Dent’s disease and prevalence of renal stones in dialysis patients in Northeastern Italy

AbstractDents disease (DD) involves nephrocalcinosis, urolithiasis, hypercalciuria, LMW proteinuria, and renal failure in various combinations. Males are affected. It is caused by mutations in the chloride channel CLCN5 gene. It has been suggested that DD is underdiagnosed, occurring in less overt forms, apparently without family history. A possible approach to this problem is to search for CLCN5 mutations in patients who may have a high prevalence of mutations: end-stage renal disease (ESRD) patients with previous calcium, struvite, or radio-opaque (CSR) stones. We looked for CLCN5 mutations in 25 males with ESRD-CSR stones selected from all of the patients (1,901 individuals, of which 1,179 were males) of 15 dialysis units in the Veneto region. One DD patient had a new DD mutation (1070 G>T) in exon 7. The new polymorphism IVS11-67 C>T was detected in intron 11 in one patient and one control. We also found 28 females with ESRD and stone history, and seven more males with ESRD and non-CSR stones. The prevalence of stone formers among dialysis patients in our region was 3.2%, much lower than the prevalence observed in older studies. Struvite stones continue to play a major role in causing stone-associated ESRD .

A novel gene (PD1) with a potential role on rat spermatogenesis

PD1 is a novel protein particularly expressed at the testicular level. The relative cDNA sequences were cloned from human and rat testis libraries revealing an open reading frame for a protein of 520 and 511 amino acids respectively. The human PD1 amino acid sequence shows 85% identity with rat sequence suggesting that PD1 gene has been highly conserved during mammalian evolution. Immunohistochemical analysis showed that this protein is detected in the tubular compartment of the testis and, in particular, in the cytoplasm of the Sertoli cells. PD1 expression is not constitutive but seems to be under the influence of neighboring spermatogenic cells as demonstrated by its reduction in hypospermatogenesis with respect to normal spermatogenesis and a further reduction in Sertoli cell-only syndrome. During testicular development in the rat (from 2 to 45 days of age) the PD1 mRNA level became detectable at 14 days and then increased steadly with an advancement of age. These findings suggest that PD1 may play a role in the regulation of spermatogenesis and may be a potential candidate gene for defects of male fertility.