Biomedical subjects

Prem Puri

Publications and source records attributed to Prem Puri.

At least 73 records · Page 4Linked to original sources

PubMed2003

Expression of heme oxygenase-1 and endothelial nitric oxide synthase in the lung of newborns with congenital diaphragmatic hernia and persistent pulmonary hypertension.

BACKGROUND/PURPOSE: Heme oxygenase (HO-1), an inducible isoform of HO is a regulator of vascular tone and cell proliferation through the production of endogenous carbon monoxide (CO). Endothelium-derived nitric oxide (NO) occurs in the endothelial layers of blood vessels and mediates vasorelaxation. Both CO and NO have similar properties and are potent vasodilators. The aim of this study was to examine the expression of HO-1 and endothelial nitric oxide synthase (eNOS) in the Congenital diaphragmatic hernia (CDI) lung. METHODS: RNA was extracted from archival formalin-fixed paraffin-embedded lung tissue from 11 patients with CDH complicated by persistent pulmonary hypertension (PPH). Five age-matched newborns served as controls. Reverse transcription polymerase chain reaction (RT-PCR) was performed using specific primers for human HO-1 and eNOS. Immunohistochemistry using HO-1 and eNOS antibodies was performed and examined using laser scanning microscope. RESULTS: HO-1 and eNOS mRNA expression was significantly decreased in CDH lung compared with controls (P <.05). HO-1 and eNOS immunoreactivity was reduced markedly reduced in the endothelium and arterial wall in the CDH samples compared with normal lung. CONCLUSIONS: Decreased expression of HO-1 and eNOS in the CDH lung suggests deficiency of endogenous NO and CO, which may contribute to altered vascular tone causing PPH.

Analysis of Variance↗

PubMed2003

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 Hirschsprung's 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.

Anastomosis, Surgical↗

PubMed2003

Gene expression of fibroblast growth factors 10 and 7 is downregulated in the lung of nitrofen-induced diaphragmatic hernia in rats.

BACKGROUND/PURPOSE: Newborns with congenital diaphragmatic hernia (CDH) still have a high mortality rate, which has been attributed to pulmonary hypoplasia and pulmonary hypertension. Fibroblast growth factors (FGFs) are essential components of the gene network that regulates lung development. Recent studies suggest that the new member of FGF family, FGF-10, plays a fundamental role in branching morphogenesis and is essential for lung formation. FGF-10-deficient mice exhibit complete absence of lungs. FGF-7 promotes epithelial proliferation and expansion leading to the formation of cystlike structures. The aim of this study was to determine the gene level expression of FGF-10 and FGF-7 in the lung of nitrofen-induced CDH. METHODS: Congenital diaphragmatic hernia (CDH) was induced in pregnant rats after administration of 100 mg of nitrofen on day 9.5 of gestation (term, 22 days). In control animals, the same dose of olive oil was given without nitrofen. Cesarean section was performed on day 21 of gestation. The fetuses were divided into 3 groups: normal controls (n = 16), nitrofen induced without CDH (n = 16), and nitrofen-induced CDH (n = 16). Total RNA and DNA were extracted from the lung in each group and measured. mRNA was extracted from total RNA. Reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate mRNA expressions of FGF-10 and FGF-7. Levels of mRNA were expressed as a ratio of the band density divided by that of beta-actin, a house-keeping gene. RESULTS: FGF-10 mRNA expression was decreased significantly in CDH lung (2.914 +/- 0.320) compared with controls (4.062 +/- 0.307; P <.05) and nitrofen induced without CDH lung (3.923 +/- 0.250; P <.01). FGF-7 mRNA expression was decreased significantly in CDH lung (0.777 +/- 0.097) compared with controls (1.028 +/- 0.093; P <.01). CONCLUSIONS: Decreased gene expression of FGF-10 and FGF-7 in the hypoplastic lung suggests that pulmonary hypoplasia in nitrofen-induced CDH rat may be caused by reduced synthesis of FGF-10 and FGF-7 during lung morphogenesis.

Animals↗

PubMed2003

Epidermal growth factor and monocyte chemotactic peptide-1 expression in reflux nephropathy.

OBJECTIVES: Reflux nephropathy (RN) is recognised as a major cause of end stage renal failure in children and young adults. The histological findings of RN are tubular atrophy and interstitial monocyte infiltration. Epidermal growth factor (EGF) produced by tubular cells playing a pivotal role in the modulation of tubular cell growth, while monocyte chemotactic peptide-1 (MCP-1) is a powerful and specific chemotactic and activating factor for monocytes. It has been suggested that the modulation of local EGF production is directly involved in the pathogenesis of tubular damage. We designed this study to investigate the expression of EGF and MCP-1 in severe reflux nephropathy in order to further understand the pathogenesis of reflux nephropathy. METHODS: The kidney specimens from 12 children with severe reflux nephropathy were obtained at the time of nephrectomy. Control material included normal kidney specimens obtained from three adult patients during partial nephrectomy for an incidentaloma. Single-label immunofluorescence histochemistry was carried out using polyclonal antibodies to EGF and MCP-1 employing laser scanning confocal microscopy. EGF and MCP-1 gene expression were evaluated by in situ hybridization (ISH). TUNEL method was utilized to assess tubular apoptosis. RESULTS: In the normal kidney there was strong EGF immunoreactivity in the proximal tubules compared to the reflux nephropathy where there was lack of immunoreactivity in the proximal tubules. Normal kidney demonstrated lack of MCP-1 immunoreactivity, whereas reflux nephropathy kidney showed strong MCP-1 immunoreactivity in the proximal tubules and tubulointerstitial space. In the normal kidney there was marked EGF mRNA expression in the proximal tubules whereas EGF mRNA expression was undetectable in reflux nephropathy kidney. MCP-1 mRNA expression was undetectable in normal kidney, whereas there was strong MCP-1 mRNA expression at the tubulointerstitial level in reflux nephropathy kidney. Decreased EGF expression and increased MCP-1 expression at the tubulointerstitial levels in reflux nephropathy strongly correlated with severity of apoptosis in reflux nephropathy compared with controls. CONCLUSIONS: Our data suggests that the downregulation of EGF with simultaneous upregulation of MCP-1 may be involved in the pathogenesis of tubulointerstitial damage in reflux nephropathy.

Adolescent↗

PubMed2003

Intestinal neuronal dysplasia.

Intestinal neuronal dysplasia (IND) is a clinical condition that resembles Hirschsprung's disease. In the past many years investigators have raised doubts about the existence of IND as a distinct histopathologic entity. One strong piece of evidence that IND is a real entity stems from animal models. Recently, two different HOX11L1 knockout mouse models and a heterozygous endothelin B receptor-deficient rat demonstrated abnormalities of the submucous plexus similar to that observed in human IND. This review describes in detail the diagnostic criteria of IND, staining techniques, correlation between histological findings and clinical symptoms, and management of IND.

Biopsy↗

PubMed2003

Immunocolocalization of the heme oxygenase-2 and interstitial cells of Cajal in normal and aganglionic colon.

PURPOSE: Interstitial cells of Cajal (ICCs) are pacemaker cells that play an important role in the control of gut motility. Carbon monoxide (CO) has been proposed as an endogenous messenger molecule between ICC and smooth muscle cells in the gastrointestinal tract (GIT). Heme oxygenase-2 (HO-2) is the main physiologic mechanism for generating CO in human cells. The aim of this study was to investigate the immunocolocalization of the HO-2 and ICCs in normal and aganglionic bowel of Hirschsprung's disease (HD). METHODS: Full-thickness specimens were obtained from aganglionic colon during pull-through operation from 10 patients diagnosed as having HD. Normal control large bowel specimens were collected from 4 patients during bladder augmentation procedures. Double immunostaining was carried out using c-kit and HO-2 antibodies. Immunolocalization was detected by means of confocal laser scanning microscopy. RESULTS: HO-2 immunoreactivity (IR) was found in many ICCs present around the myenteric plexus, within the longitudinal and circular muscle layers and at the innermost part of the circular muscle layer in normal colon. In the aganglionic colon there was absence of HO-2 IR in the sparsely found ICCs. In the transitional zone of HD bowel the colocalization of HO-2 IR and ICCs was much reduced compared with controls. CONCLUSIONS: The results of this study provide the first evidence for the presence of HO-2 immunoreactivity in the ICCs in normal human colon and absence of HO-2 immunoreactivity in sparsely appearing ICCs in the bowel of HD patients. The lack of HO-2 in the ICCs in the bowel of HD patients may result in impaired intracellular communication between ICCs and SMCs causing motility dysfunction.

Carbon Monoxide↗

PubMed2003

Is primary endoscopic puncture of ureterocele a long-term effective procedure?

BACKGROUND/PURPOSE: For more than a decade, endoscopic puncture of ureterocele has been recommended as an initial and, in the majority of the patients, as a definitive procedure. This study evaluates the long-term effectiveness of primary endoscopic puncture of ureterocele. METHODS: Over the last 18 years (1984 through 2001), 52 patients (median age 3 months) underwent primary endoscopic puncture of ureterocele. The median follow-up was 9 years (6 months to 18 years). Antenatal ultrasound scan detected hydronephrosis and led to the postnatal diagnosis of ureterocele in 12 (23%) children, whereas in the remaining 40 (77%) children the diagnosis was made on investigation for urinary tract infection (UTI). The ureterocele presented as a part of renal duplication in 48 (92%) patients and a single system in 4 (8%). Forty-four (92%) of the patients with duplication presented with non- or poorly functioning upper poles. Vesicoureteric reflux (VUR) was seen in the lower moiety of the ipsilateral kidney in 31 and in 18 of the contralateral kidney comprising 49 renal refluxing units (RRU). RESULTS: Complete decompression of the ureterocele was achieved in 48 (92%) patients after the first endoscopic puncture. Four (8%) patients required a second puncture of ureterocele. Nine (17%) of the 52 patients underwent nephrectomy for a nonfunctioning kidney. Ten (19%) patients required upper pole partial nephrectomy owing to nonfunctioning upper pole. Twenty-nine (59%) of the 49 RRU showed spontaneous resolution of VUR. Sixteen (33%) RRU underwent endoscopic correction of VUR. One required ureteric reimplantation. The remaining 4 (8%) are maintained on prophylactic antibiotics. Five (10%) patients had VUR in the upper pole moieties after ureterocele puncture. CONCLUSIONS: Our data suggest that primary endoscopic puncture of ureteroceles is a simple, long-term, effective, and safe procedure avoiding complete reconstruction in the majority of the patients.

Child↗

PubMed2003

Familial vesicoureteral reflux.

PURPOSE: Vesicoureteral reflux is known to occur in families. We summarized worldwide data on the incidence and nature of vesicoureteral reflux in siblings of children with vesicoureteral reflux. MATERIALS AND METHODS: We searched MEDLINE using the words siblings vesicoureteral reflux, familial vesicoureteral reflux, offspring vesicoureteral reflux and vesicoureteral reflux screening. All articles that we identified that were published from 1972 to 2002 were analyzed for the incidence of renal damage in siblings of patients with vesicoureteral reflux. RESULTS: Siblings of patients with vesicoureteral reflux have a much higher incidence of reflux than the normal population. There is a direct relationship of patient age to the incidence and severity of reflux. Most investigators advocate screening asymptomatic siblings of patients with vesicoureteral reflux. CONCLUSIONS: The incidence of sibling reflux is significant. When vesicoureteral reflux is discovered in symptomatic siblings, it is usually high grade and associated with a high incidence of reflux nephropathy. Randomized controlled studies are needed to compare renal damage in patients with reflux detected through screening to renal damage in those diagnosed after urinary tract infection to establish how much renal damage may be prevented by screening in asymptomatic siblings.

Child↗

PubMed2003

Endoscopic treatment of primary grades IV and V vesicoureteral reflux in children with subureteral injection of polytetrafluoroethylene.

PURPOSE: We evaluated our experience with endoscopic STING (subureteral polytetrafluoroethylene injection) for grades IV and V vesicoureteral reflux. MATERIALS AND METHODS: We reviewed the records of 221 boys and 305 girls who underwent STING for primary grades IV (430) and V (96) vesicoureteral reflux between 1984 and 2000. Median patient age at STING was 3 years (range 3 months to 14 years). Reflux was unilateral and bilateral in 335 and 191 children, respectively (717 refluxing units). Median followup was 11.6 years (range 1 to 17). RESULTS: Reflux was corrected in 420 of the 717 refluxing units (58%) after a single injection. Reflux resolved after a second and third injection in 185 ureters (26%). High grade reflux was converted to grades I and II in 112 ureters (15%) and did not require any further treatment. STING failed to correct reflux in 7 units (0.9%), which were managed by ureteral reimplantation (5) and nephrectomy (2) due to poor renal function. Voiding cystourethrography showed recurrent vesicoureteral reflux in 9 units (1.2%), including 2 with low grade reflux for which no treatment was given. Seven ureters required repeat injection due to grades III and IV reflux. No untoward effects were noted in any patients in whom polytetrafluoroethylene was used as the injected material. CONCLUSIONS: STING is a simple, safe and effective outpatient procedure for grades IV and V vesicoureteral reflux.

Adolescent↗

PubMed2003

Treatment of vesicoureteral reflux by endoscopic injection of dextranomer/hyaluronic Acid copolymer: preliminary results.

PURPOSE: Endoscopic subureteral injection of tissue augmenting substances has become an established alternative to long-term antibiotic prophylaxis and open surgery for the management of vesicoureteral reflux (VUR) in children. Recently, dextranomer/hyaluronic acid copolymer (Deflux, Q-Med AB, Uppsala, Sweden) a biodegradable injectable material has been reported to be a promising alternative to other tissue augmenting substances. However, the experience with dextranomer/hyaluronic acid copolymer is limited. We prospectively evaluate the effectiveness of dextranomer/hyaluronic acid copolymer in the endoscopic treatment of VUR. MATERIALS AND METHODS: A total of 32 males and 81 females with a median age of 1 year (range 3 months to 10 years) underwent endoscopic correction of primary VUR with dextranomer/hyaluronic acid copolymer. Reflux was unilateral in 58 cases and bilateral in 54, affecting 166 ureters. Reflux was grades II to V in 7 (4.2%) cases, 91 (54.8%), 63 (38%) and 5 (3%), respectively. All patients underwent endoscopic correction as a day procedure and have been followed for 3 months to 1 year. RESULTS: The reflux was corrected in 143 (86%) of the 166 ureters after a single injection, and resolved after a second and third injection in 22 (13%) and 1 (1%) ureter, respectively. No untoward effects were seen in any of these patients with the use of dextranomer/hyaluronic acid copolymer as an injectable material. CONCLUSIONS: Dextranomer/hyaluronic acid copolymer is an effective tissue augmenting substance in the endoscopic treatment of all grades of VUR.

Administration, Intravesical↗

PubMed2003

Cyclooxygenase-2 up-regulation in reflux nephropathy.

PURPOSE: Reflux nephropathy (RN) is a major cause of end stage renal failure in children and hypertension is a frequent complication. Cyclooxygenase-2 (COX-2) is an enzyme responsible for the prostaglandin synthesis. It has been shown that COX-2 up-regulates renin production leading to renovascular hypertension. We investigate COX-2 expression in the kidneys of children with RN. MATERIALS AND METHODS: Kidney specimens from 12 patients 2 to 13 years old with severe RN secondary to primary high grade vesicoureteral reflux obtained at the time of nephrectomy and 5 controls were examined. Single labeled immunohistochemistry was performed with 2 COX-2 antibodies using light and confocal microscopy. Quantification of COX-2 was determined by Western blotting analysis. COX-2 gene expression was evaluated by real-time quantitative reverse transcription polymerase chain reaction. RESULTS: There was a strong COX-2 immunoreactivity in the proximal tubules and tubulointerstitial space in the RN samples compared to controls. Immunoreactive COX-2 protein expression was markedly increased in RN samples compared to controls. Real-time reverse transcription polymerase chain reaction showed a significant increase in COX-2 mRNA expression in the RN samples compared to controls (p <0.05). CONCLUSIONS: Over expression of COX-2 in reflux nephropathy suggests that COX-2 may be involved in the pathogenesis of tubulointerstitial damage associated with severe reflux nephropathy.

Blotting, Western↗

PubMed2003

Altered expression of interstitial cells of Cajal in congenital ureteropelvic junction obstruction.

PURPOSE: Peristaltic contractions in the upper urinary tract serve to move urine from the kidney through the ureter to the bladder. Ureteropelvic junction (UPJ) obstruction is the most common cause of congenital hydronephrosis in children. To our knowledge the pathophysiology of UPJ obstruction is unknown. C-kit positive interstitial cells of Cajal (ICCs) are pacemaker cells that facilitate active propagation of electrical events and mediate neurotransmission. We investigated the expression of c-kit positive cells in the muscle layer of normal and obstructed UPJ specimens. MATERIALS AND METHODS: A total of 19 human formalin fixed, paraffin embedded specimens of intrinsic UPJ obstruction from children with a mean age of 2.3 years (range 2 months to 12 years) and 7 control samples from children with a mean age of 4.5 years (range 11 months to 9 years) were investigated immunohistochemically for the expression of c-Kit oncoprotein and peripherin by light and laser scanning microscopy. Quantification of immunolabeled structures was quantified using computerized image analysis. RESULTS: Peripherin immunoreactivity was strong in the muscle layer of normal UPJ specimens, while in UPJ obstructed specimens there was a decrease in peripherin positive nerve fibers. In normal UPJ specimens there were many c-Kit positive ICCs between the muscle bundles. The density of ICCs was markedly decreased in the muscle layers of UPJ obstructed specimens. CONCLUSIONS: To our knowledge this study shows for the first time the immuno-expression of c-Kit positive ICCs in the proximal part of the normal human upper ureter. The altered density of c-Kit positive cells in UPJ obstruction may have a role in the failure of transmission of peristaltic waves across the UPJ.

Child↗

PubMed2003

Nitrergic hyperinnervation in appendicitis and in appendices histologically classified as normal.

CONTEXT: The pathogenesis of appendicitis remains poorly understood. Despite new diagnostic techniques, appendices removed from patients with suspected appendicitis often appear histologically normal on conventional examination. There is increasing evidence of involvement of the enteric nervous system in immune regulation and in inflammatory responses in the gastrointestinal system. OBJECTIVE: To investigate the nitrergic innervation of (a) acutely inflamed appendices, (b) appendices classified as histologically normal from patients with a clinical diagnosis of appendicitis, and (c) normal control appendix specimens, using the whole-mount preparation technique. PATIENTS AND DESIGN: Full-thickness specimens were collected from 28 acutely inflamed appendices (age range, 3.2-13.4 years), 31 histologically normal appendices removed from patients (age range, 5.7-13.6 years) with suspected appendicitis, and 23 histologically normal appendices from patients (age range, newborn to 12.1 years) undergoing elective abdominal surgery (controls). Whole-mount preparation using nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry and neuronal nitric oxide synthase immunohistochemistry were performed. The density of myenteric plexus was measured with a computerized analysis system. RESULTS: The density of myenteric plexus in normal appendix specimens was similar to that of large bowel from the newborn period up to 3 years of age; this density decreased significantly thereafter. The myenteric plexus of normal appendix specimens from patients older than 4 years demonstrated smaller ganglia connected by thin nerve bundles, compared to larger ganglia and nerve bundles in large bowel. Significant neuronal hypertrophy was found in 55% of acutely inflamed and 41% of histologically classified normal appendix specimens. The myenteric plexus of these appendix specimens had even thicker nerve bundles connecting an increased number of ganglion cells. CONCLUSIONS: Differences in the architecture of the myenteric plexus in patients older than 3 years suggest an altered function and motility of appendix in the early years of life. The significant increase in neuronal components of the myenteric plexus in a high proportion of acutely inflamed and histologically normal appendix specimens is unlikely to have developed during a single acute inflammatory episode. This suggests an underlying chronic abnormality as a secondary response to chronic luminal obstruction or repeated inflammatory episodes in the histologically normal appendix.

Adolescent↗

PubMed2003

Distribution of heme oxygenase-2 in nerves and interstitial cells of Cajal in the normal pylorus and in infantile hypertrophic pyloric stenosis.

CONTEXT: Interstitial cells of Cajal (ICCs) are pacemaker cells, which are of fundamental importance in regulating gastrointestinal motility. Recent evidence suggests that carbon monoxide is a neurotransmitter involved in neurotransmission between ICC and smooth muscle cells. Heme oxygenase-2 (HO-2) is the major physiological mechanism for the generation of carbon monoxide in the enteric nervous system. OBJECTIVE: To investigate the immunocolocalization of HO-2 and ICCs in the normal pylorus and in infantile hypertrophic pyloric stenosis (IHPS). DESIGN: Specimens from 18 infants with IHPS and 8 control specimens were examined using double-immunostaining with c-Kit and HO-2 antibodies. The immunolocalization was detected with the help of confocal laser scanning microscopy. RESULTS: Abundant HO-2 immunoreactivity was found in ICCs in the smooth muscle layer of normal pylorus. There was a decrease in the number of ICCs and lack of HO-2 immunoreactivity in ICCs in IHPS. CONCLUSIONS: The results of the present study provide the first evidence for the presence of HO-2 in ICCs in the normal human pylorus. The lack of ICCs and HO-2 in IHPS suggests impaired intracellular communication between ICCs and smooth muscle cells, contributing to motility dysfunction in IHPS.

Enteric Nervous System↗

PubMed2003

Distribution of interstitial cells of Cajal in the internal anal sphincter of patients with internal anal sphincter achalasia and Hirschsprung disease.

CONTEXT: Interstitial cells of Cajal (ICCs) are pacemaker cells in the smooth muscles of the gut. The internal anal sphincter (IAS) is the most caudal part of gastrointestinal tract. It has the important function of maintaining fecal continence. It has been proposed that ICCs in the IAS mediate the inhibitory innervation of the recto-anal reflexes. OBJECTIVE: To investigate the distribution of ICCs in the normal IAS and in the IAS of children diagnosed with internal anal sphincter achalasia (IASA) and Hirschsprung disease (HD). METHODS: At the time of IAS myectomy, specimens of the IAS were taken from 8 patients with IASA, 4 patients with HD, and 4 normal controls. All specimens were examined using anti-c-Kit and antiperipherin antibodies; immunolocalization was detected with light microscopy. Density of the ICCs was graded by computerized image analysis. RESULTS: There was strong peripherin immunoreactivity in the ganglia cells and nerve fibers in the normal IAS. The number of peripherin-positive nerve fibers was markedly reduced in the IAS in patients with IASA. In HD patients, there was lack of peripherin immunoreactivity in the IAS, but hypertrophic nerve trunks stained strongly. Many c-Kit-positive ICCs were present among the muscle fibers and between the muscle bundles in the normal IAS. In HD and IASA patients, ICCs were absent or markedly reduced. CONCLUSION: Altered distribution of ICCs in the internal sphincter in IASA and HD may contribute to motility dysfunction in these patients.

Adolescent↗

PubMed2003

The intramural innervation of the rabbit upper urinary tract.

The role of the autonomic innervation of the upper urinary tract for pyeloureteral motility is not completely understood. It is still debatable if the autonomic nervous system might play a modulating role on the ureteral peristalsis. The aim of this study was to investigate the distribution and regional variation of the intramural innervation using whole-mount preparations of the rabbit upper urinary tract. Whole-mount preparation was performed at upper urinary tracts of healthy rabbits. Immunohistochemistry was employed using Neurofilament (NF), Tyrosine Hydroxylase (TH), Choline Acetyltransferase (ChAT) and Substance P (SP) antibodies. NADPH-diaphorase and Acetylcholinesterase (AChE) histochemistry was carried out at the specimens. The stains were evaluated using brightfield, fluorescence and laser confocal microscopy. NF-, TH-, ChAT- and SP-immunoreactive (-IR) nerves formed distinct neuronal plexuses at the submucosal and muscle layers. Perivascular TH-, ChAT- and SP-IR fibres were demonstrated. AChE positive nerves were revealed in all layers, but only moderate NADPH-diaphorase positive innervation was found. Renal pelvis, upper and lower ureter showed enriched intrinsic innervation. Ganglia were found at the ureteropelvic border and the distal ureter. Whole-mount preparation technique revealed detailed informations about morphology and regional variation of the intramural innervation of the rabbit upper urinary tract.

Acetylcholinesterase↗

PubMed2002

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

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.

Adolescent↗

PubMed2002

The role of nitric oxide in reflux nephropathy.

Reflux nephropathy (RN) is recognized as a major cause of end-stage renal failure in children and young adults. Inhibition of nitric oxide (NO) exacerbates and enhanced production ameliorates tubulointerstitial fibrosis (TIF) in experimental obstructive uropathy. NO is synthesised by NO synthase (NOS), three distinct isoforms of which have been identified: inducible (iNOS), endothelial (eNOS), and neuronal (nNOS). It has been reported that iNOS induces immunologic injury to glomerular cells and enhances accumulation of extracellular matrix in the glomerulus and tubulointerstitial space. Furthermore, it has been suggested that nNOS and eNOS have beneficial effects in ameliorating TIF. We investigated the expression of different isoforms of NOS in severe refluxing kidneys in order to further understand the pathogenesis of RN in kidney specimens from nine children with severe RN obtained at nephrectomy. Control material included normal kidney specimens from three adult patients undergoing partial nephrectomy for small kidney tumours. Histochemistry for NO was performed using nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase. Single-label immunofluorescence histochemistry was carried out using polyclonal antibodies to nNOS, iNOS, eNOS, and transforming growth factor (TGF)-beta 1 employing laser-scanning confocal microscopy. The TUNEL method was used to assess tubular apoptosis. Strong NADPH staining was observed in the proximal tubules of RN kidneys compared to controls, where there was weak staining. Control kidneys demonstrated weak immunoreactivity for iNOS in the proximal tubules and a lack of immunoreactivity for nNOS and eNOS. RN kidneys demonstrated strong immunoreactivity for nNOS in the tubulointerstitial space, for eNOS in the glomerulus, and for iNOS in the glomerulus and proximal tubules. Strong immunoreactivity for TGF beta 1 was seen in the glomerulus and proximal tubules identical to iNOS. Increased immunoreactivity for iNOS and TGF-beta 1 strongly correlated with the severity of apoptosis in RN. Our data demonstrate that NO derived from nNOS, iNOS, and eNOS is strongly expressed in RN. The selective shunting of NO via iNOS may induce renal fibrosis in RN. The upregulation of nNOS and eNOS in RN appears to be a compensatory mechanism of ameliorating TIF.

Adolescent↗