Valeria Solari

The role of nitric oxide in bladder urothelial injury after bladder outlet obstruction

To investigate the role of nitric oxide (NO) in the pathogenesis of injury to the bladder mucosa after bladder outlet obstruction (BOO).

Histopathological differences between recto-sigmoid Hirschsprung's disease and total colonic aganglionosis.

Total colonic aganglionosis (TCA) is a severe form of ultra long Hirschsprungs disease with an incidence of 2 to 14% among all forms of intestinal aganglionosis. C-kit positive interstitial cells of Cajal (ICCs) are pacemaker cells that play a key role in the motility function of the bowel. The aim of this study was to compare the innervation and ICCs distribution in total colonic and recto-sigmoid HD. Full thickness colonic specimens were obtained from four children with TCA, ten with recto-sigmoid HD and four controls. Single immunohistochemistry using peripherin, neuronal nitric oxide synthase (nNOS) and c-kit antibody was performed and analysed in light microscopy. Additionally, whole-mount preparations were stained using anti c-kit immunohistochemistry and NADPH-diaphorase. In the ganglionic bowel of TCA, recto-sigmoid HD and control patients there was a strong nNOS and peripherin immunoreactivity (IR) in ganglia of myenteric and submucous plexus and in thin nerve fibres in the muscle layers. In the TCA there was weak or lack of nNOS IR in the sparse, short nerve trunks of the myenteric and submucous plexuses and muscle layers, whereas nNOS weakly positive nerve trunks were observed in the recto-sigmoid HD bowel. Peripherin IR was markedly reduced in the TCA specimens compared to recto-sigmoid HD. In the TCA specimens there was a lack of ICCs-MY in the smooth muscle layer in all the specimens, whereas in the recto-sigmoid aganglionic bowel ICCs-MY were markedly reduced. Whole-mount preparations showed lack of ICCs-MY and a markedly reduced number of NADPH-positive nerve trunks in TCA. Our findings demonstrate clear histopathological differences between rectosigmoid Hirschsprungs disease and total colonic aganglionosis.

Distribution of Ca2+-activated K channels, SK2 and SK3, in the normal and Hirschsprung's disease bowel.

PURPOSE The aim of this study was to investigate the expression and distribution of SK2 and SK3 channels in the normal and Hirschsprungs disease (HD) bowel. METHODS Full-thickness colonic specimens were collected at pull-through operation from 10 patients with HD and from 6 patients during bladder augmentation. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for SK2 and SK3 channels and double immunostaining using anti SK2/c-kit, SK3/c-kit, SK2/alpha-SMA, and SK2/PGP 9,5 antibodies was performed. Immunolocalization was detected using laser scanning microscopy. RESULTS RT-PCR analysis showed strong expression of SK2 and SK3 mRNA in the normal human bowel and significantly reduced SK3 expression in the aganglionic bowel (P <.05). In the normal colon, double labeling immunohistochemistry showed strong SK3 immunoreactivity (IR) colocalizing in the c-kit-positive ICCs. In the aganglionic bowel, SK3 IR was reduced markedly in the sparsely found ICCs. There was strong SK2 IR mainly in smooth muscles in the normal and aganglionic bowel. CONCLUSIONS The results of this study provide the first evidence for the presence of SK2 and SK3 channels and for the immunocolocalization of SK3 channels in the ICCs in the normal human colon. Decreased expression SK3 channels in the aganglionic bowel may contribute to motility dysfunction in HD.

Up-regulation of angiotensin-converting enzyme (ACE) gene expression induces tubulointerstitial injury in reflux nephropathy.

Abstract.Reflux nephropathy (RN) is the cause of end-stage renal failure in 3%–25% of children and 10%–15% of adults. Angiotensin-converting enzyme (ACE) converts the inactive decapeptide angiotensin I (Ang I) to the active octapeptide angiotensin II (Ang II), a potent vasoconstrictor. ACE is localized in highest concentrations on the luminal surface of endothelial cells, but is also found in several other cell types, including the epithelial cells of the proximal renal tubule. Recent studies have suggested that ACE increases production of the components of extracellular matrix (ECM) such as fibronectin (Fib) mediated through Ang II. Since RN is a primary tubulointerstitial disease, we hypothesized that local overexpression of ACE may induce renal fibrosis via up-regulation of Ang II. In this study, we investigated the expression of ACE in severely refluxing kidneys from eight patients (age range 6 months–14 years) with severe RN secondary to primary high-grade vesicoureteral reflux at nephrectomy. Control material included normal kidney specimens obtained from three adult patients during partial nephrectomy for an incidentaloma. Soluble enzyme immunohistochemistry was carried out using polyclonal antibodies to ACE and Fib. In-situ hybridization (ISH) was performed utilizing biotin-labelled antisense oligonucleotide probe. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to evaluate the relative amount of ACE mRNA. In the refluxing kidney, there was strong ACE immunoreactivity in the glomerulus and proximal tubules and moderate-to-strong immunoreactivity in the distal tubules accompanied by strong Fib immunoreactivity in the glomerulus, proximal tubule, and interstitial space. There was strong ACE mRNA expression in the glomerulus and proximal tubules and moderate expression in the distal tubules. In the control kidneys, homogeneous weak ACE immunoreactivity and mRNA expression was demonstrated only in the proximal tubules. RT-PCR showed strong ACE expression in the refluxing kidneys compared to controls. Up-regulation of ACE in RN accompanied by an increase in ECM in the tubulointerstitial space suggests that ACE is involved in the pathogenesis of the renal parenchymal damage in patients with RN. Pharmacologic blockade of ACE may be helpful in preventing the renal fibrosis associated with RN.

Increased expression of EGFR and TGF-α in segmental renal dysplasia in duplex kidney

Renal dysplasia (RD) is a disorganised development of renal parenchyma that results in a deficit of functional renal tissue. It is known that the epidermal growth factor (EGF) and the transforming growth factor-α (TGF-α) enhance renal cell proliferation, migration and differentiation during kidney development through binding to the same EGF receptor (EGFR). The aim of the study was to analyse the expression of TGF-α and EGFR in the dysplastic kidney. The specimens of dysplstic upper poles duplex kidneys were surgically resected from 19 patients. Indirect immunohistochemistry was performed using the ABC method employing antibodies against EGFR and TGF-α, and gene expression using primers specific to the human genes. There was absent or weak EGFR and TGF-α immunoreactivity in normal kidney tissue. In dysplastic kidneys, there was strong TGF-α and EGFR immunoreactivity in the epithelium of primitive tubules and strong EGFR immunoreactivity in the connective tissue around the primitive tubules. Our findings of markedly increased local expression of EGFR and TGF-α in primitive tubules suggest that EGFR and TGF-α may play an important role in altering renal morphogenesis resulting in renal dysplasia.

Increased c-Met tyrosine kinase expression in segmental renal dysplasia

Renal dysplasia (RD) is a disorganized development of renal parenchyma that results in a deficit of functional renal tissue. It has been suggested in the animal model that increased expression of HGF receptor, c-Met tyrosine kinase in the epithelial cells during kidney development may induce a growth of dysplastic epithelia and result in RD. The aim of this study was to investigate the immunoreactivity of c-Met tyrosine kinase in the dysplastic kidney in order to further understand the pathogenesis of RD. Specimens of dysplastic upper pole kidney were obtained from 19 patients during upper pole partial nephrectomy for non-functioning upper moiety of duplex kidney. In the dysplastic kidney, there was strong c-Met immunoreactivity in the epithelium of primitive tubules. In contrast, c-Met immunoreactivity was barely detectable in the normal kidney. Markedly increased expression of HGF receptor, c-Met tyrosine kinase in renal dysplasia suggests that HGF may be involved in the development of renal dysplasia.