Evaluation of a rapid real-time RT-PCR assay for detection of enterovirus RNA in cerebrospinal fluid specimens.

BACKGROUND: We previously described the characteristics of a single-tube real-time enterovirus reverse transcriptase polymerase chain reaction (RT-PCR) assay based on a fluorogenic probe and primers directed to highly conserved sequences in the 5'-untranslated region (UTR) of the enterovirus genome. OBJECTIVES: To evaluate the performance of the assay on a larger number of cerebrospinal fluid (CSF) specimens from patients suspected of having viral meningitis. STUDY DESIGN: Real-time enterovirus RT-PCR and viral culture were performed on CSF specimens received from March 2000 to November 2001. Patient records were retrospectively reviewed for final clinical diagnosis. RESULTS: From the 186 CSF specimens tested, culture was positive for enterovirus in 31 cases, whereas real-time RT-PCR detected enterovirus RNA in 45 CSF specimens. The sensitivity of real-time RT-PCR in relation to the clinical diagnosis of viral meningitis was 72.6%, whereas the sensitivity of viral culture reached only 57.4%. Enterovirus RNA was also found in a number of specimens with low leukocyte counts. CONCLUSIONS: We confirm that the real-time enterovirus RT-PCR assay for CSF specimens is significantly more sensitive than viral culture.

Rapid detection of enterovirus RNA in cerebrospinal fluid specimens with a novel single-tube real-time reverse transcription-PCR assay.

A single-tube real-time reverse transcription-PCR (RT-PCR) assay for enterovirus detection in cerebrospinal fluid (CSF) was developed based on a fluorogenic probe and primers directed to highly conserved sequences in the 5' untranslated region of the enterovirus genome. Quantitative detection of enterovirus genome was demonstrated in a linear range spanning at least 5 logs. Endpoint titration experiments revealed that the in-tube detection limit of the assay was 11.8 enterovirus genome equivalents (95% detection rate) corresponding in our current extraction protocol to 592 enterovirus genome equivalents per ml of CSF. Twenty CSF specimens not suspected of viral meningitis were all found to be negative, and no cross-reactivity with herpes simplex virus type 1 and type 2, varicella-zoster virus, rhinovirus type 53, and influenza viruses A and B was observed. Nineteen CSF specimens from 70 patients suspected of viral meningitis were determined to be positive by PCR (27.1%), whereas only 17 were found to be positive by viral culture (24.3%). The sensitivity of the assay was 100% and the specificity was 96.2% compared to viral culture. Data from the real-time RT-PCR assay were available within 4 h. Our data suggest that the novel real-time RT-PCR assay may offer a reliable but significantly faster alternative to viral culture. Owing to the elimination of postamplification detection steps, its conduct required considerably less hands-on time and was associated with a substantially reduced carryover risk compared to previously described PCR-based enterovirus detection assays.

Renal osteopontin protein and mRNA upregulation during acute nephrotoxicity in the rat.

BACKGROUND: The effect of segment-specific proximal tubular injury on spatio-temporal osteopontin (OPN) distribution was determined in two different nephrotoxic rat models to evaluate its conceivability with a possible role for OPN in acute renal failure (ARF). OPN gene expression was further determined in proximal and distal tubular cells to investigate the origin of increased renal OPN. METHODS: Renal OPN protein and mRNA expression were compared in the rat during mercuric-chloride- vs gentamicin-induced ARF using immunohistochemistry and in situ hybridization. RESULTS: Mercuric chloride primarily induced tubular injury and subsequent cell proliferation in proximal straight tubules (PST), whereas gentamicin predominantly injured proximal convoluted tubules (PCT). In both models, the distribution of OPN protein was associated with increased OPN mRNA levels in proximal as well as distal tubular cells. However, upregulation was delayed in the proximal tubular segment suffering most from injury, i.e. PCT in gentamicin ARF vs PST in mercuric-chloride ARF. OPN immunostaining at the apical cell membrane from distal tubules was in contrast to perinuclear vesicular staining in proximal tubular cells. CONCLUSIONS: OPN gene and protein expression is induced in both proximal and distal tubular cells during rat toxic ARF. The distinct subcellular localization in proximal vs distal tubular cells indicates differences in OPN processing and/or handling. The spatio-temporal distribution is consistent with a possible role in renal injury and regeneration.

Antibodies to both ICAM-1 and LFA-1 do not protect the kidney against toxic (HgCl2) injury.

BACKGROUND: The role of inflammatory leukocytes in acute renal failure (ARF) remains controversial and appears largely uninvestigated in toxic (in contrast to ischemic) ARF. METHODS: Female Wistar rats were injected with monoclonal antibodies (mAbs) directed to both the leukocyte function-associated antigen 1 (LFA-1) and the intercellular adhesion molecule 1 (ICAM-1). Doses (6 mg/kg of each mAb) were given 24 hours prior to the induction of acute tubular necrosis (ATN) by mercuric chloride administration (2 mg/kg, subcutaneously, day 0) and subsequently every 48 hours. Control rats similarly received either control antibody (12 mg/kg) or vehicle prior to and following the induction of ATN. Renal function was also measured from male Lewis rats that were similarly treated with anti-adhesion antibodies during exposure to 30 minutes of unilateral renal ischemia. RESULTS: Injected antibodies were demonstrated on peripheral blood leukocytes (flow cytometrical detection of mouse anti-LFA-1) and on endothelium (immunohistochemical staining of mouse anti-ICAM-1) and were measured in serum (enzyme-linked immunosorbent assay). Macrophages and T cells were prominent in the kidney of control treatment rats after HgCl2 injection, but anti-adhesion treatment clearly had prevented their infiltration. Notwithstanding, renal tubular injury was equally pronounced in all mercuric chloride treatment groups and so was the decline in renal function (serum creatinine, proteinuria). Tubular epithelial cell proliferation seemed slightly less pronounced and delayed in anti-adhesion treated rats. Kidneys from ischemia exposed rats were, however, functionally protected by identical anti-ICAM-1/anti-LFA-1 treatment. CONCLUSION: Prevention of cellular infiltration by mAbs to LFA-1 and ICAM-1 has no effect on renal morphology, function, or regeneration following mercuric chloride-induced ARF in the rat. This result contrasts with the functional protection of the rat kidney to ischemia/reperfusion injury by virtue of an identical antibody treatment protocol. Resolving that controversy should bring better insight in fundamental processes underlying different types of ARF, and will be the subject of further study.

Differences in osteopontin up-regulation between proximal and distal tubules after renal ischemia/reperfusion.

BACKGROUND: Osteopontin (OPN) is a highly acidic phosphoprotein containing an arginine-glycine-aspartic acid (RGD) cell adhesion motif. High OPN expression has been found in tissues with high cell turnover, and OPN up-regulation has been demonstrated in several models of renal injury, suggesting a possible role in tissue remodeling and repair. However, its exact function in the kidney remains unknown. In this study, the possible contribution of OPN to regeneration and repair in the kidney was explored by studying the time course and subcellular localization of OPN up-regulation after renal ischemia/reperfusion injury in different nephron segments and by investigating its relationship with tubular morphology. METHODS: Rats that underwent 60 minutes of left renal ischemia and a right nephrectomy sacrificed at 10 different time points (from 1 hr to 10 days after reperfusion) were compared with uninephrectomized rats at each time point. In renal tissue sections immunostained for OPN, proximal (PTs) and distal tubules (DTs) in both the renal cortex and outer stripe of the outer medulla (OSOM) were scored for the degree of OPN expression and tubular morphology. RESULTS: Kidneys of uninephrectomized rats showed no injury, and the localization and intensity of their OPN expression remained unaltered compared with normal rats. After ischemia/reperfusion, morphological damage was most severe in PTs of the OSOM, but all examined nephron segments showed a significant increase in OPN expression. The time course of OPN up-regulation was different in PTs and DTs. DTs in both cortex and OSOM rapidly increased their OPN expression, with a maximum at 24 hours after reperfusion followed by a slow decrease. In contrast, PTs showed a delayed increase in OPN staining, with a maximum after five to seven days, higher in the OSOM than in the cortex. In OSOM PTs, OPN expression was predominantly associated with morphological regeneration, whereas DTs showed a substantial OPN up-regulation without major morphological damage. PTs and DTs displayed a different subcellular OPN staining pattern: OPN staining in DTs was located to the apical side of the cell; PTs, however, presented a vesicular, perinuclear staining pattern. CONCLUSIONS: Our study found a different pattern of OPN up-regulation after renal ischemia/reperfusion in PTs versus DTs, both with regard to time course and subcellular localization. DTs show an early and persistent increase in OPN staining in the absence of major morphological injury, whereas OPN staining in PTs is delayed and is mostly associated with morphological regeneration. PTs show a vesicular, perinuclear OPN staining pattern, whereas DTs show OPN staining at the apical cell side.

Selective depletion of CD8-positive leukocytes does not alter mercuric chloride induced acute renal failure in the rat.

The process of injury and regeneration in different models of acute renal failure is accompanied by the transient interstitial accumulation of mononuclear leukocytes. The relationship between these accumulated cells and the onset and progression of the regeneration process resulting in the complete functional and morphological recovery is still a matter of debate. In this process cell subsets may either be selectively important or combine to a communicative network, signalling the cells at the site of injury. In a first trial to investigate this hypothesis, the CD8-positive subset of leukocytes, consisting mainly of cytotoxic and suppressor T lymphocytes and to a lesser extent of natural killer cells, was depleted in vivo in rats by means of a monoclonal antibody directed against CD8. Although the depletion obtained evidently prevented the infiltration of these cells into the renal interstitium, it could not influence neither the development nor the resolution of renal insufficiency in response to mercuric chloride administration as compared with control animals who had received an irrelevant isotype-matched monoclonal antibody. The extent of renal damage was unaffected as were onset and duration of renal epithelial cell proliferation. Consequently, these data do not support a major role for the CD8-positive cell subset per se in the development of acute nephrotoxic injury and subsequent regeneration.

Inflammatory cells in renal regeneration.

Much research has been performed to gain better insight into the regeneration process, responsible for the functional and morphological recovery after acute renal failure (ARF). Many investigators focused on endogenously produced polypeptide growth factors as the major mediators of tubular epithelial cell proliferation. However, arguments contradicting this hypothesis have recently gained more support. Indeed, the early decrease of renal epidermal growth factor (EGF) and insulinlike growth factor-1 (IGF-1) in different experimental models of ARF has been frequently shown at both the mRNA and protein level, while other growth factors could not be shown to increase. Moreover, the inaccessibility of the upregulated receptors for endogenously produced growth factors has encouraged research to seek alternative origins for the signals inducing renal regeneration. The accumulation of mononuclear leukocytes in the renal interstitium is a striking observation in renal failure. Where the interstitial disease, recognized by the persistent interstitial accumulation of leukocytes, is a better predictor of chronic renal failure and developing fibrosis, ARF distinguishes itself by the disappearance of the infiltrate when regeneration is complete. The existence of a regenerative potential provided by the network of inflammatory mononuclear leukocytes is supported by studies on tissue repair in different fields. This review discusses the infiltrating network of mononuclear leukocytes as a major participant in the regeneration process after acute renal failure, including the approach which can be followed to investigate this hypothesis.

Time course of growth factor expression in mercuric chloride acute renal failure.

BACKGROUND: Renal EGF expression decreases in varying models of acute renal failure (ARF). We found previously that the loss of distal tubular EGF during gentamicin ARF is strongest in the cortex, where proximal tubular injury was most severe. To gain more insight into the mechanism underlying this apparent anatomical association, renal growth factor expression was investigated during mercuric chloride ARF, in which proximal tubular injury is most severe in the outer stripe of the outer medulla (OSOM). METHODS: Endogenous renal growth factor expression was investigated by RNA hybridization and by immunohistochemistry in a rat model of mercuric chloride ARF. In addition we determined temporal and spatial profiles of tubular injury, cell proliferation, and mononuclear cell infiltration during the 3-week observation period. RESULTS: Serum creatinine values were maximal 2 days after treatment and were again normalized at day 6. Tubular injury was most severe in the PST and maximal at day 2. Cell proliferation was also higher in the PST and maximal at day 4. Three weeks after treatment, normal renal morphology was restored. Increased numbers of mononuclear cells appeared transiently in the renal interstitium from day 1 on. Most of these cells were macrophages and T lymphocytes; macrophages surrounded preferentially the severely injured PST in the OSOM. In analogy to gentamicin ARF, renal EGF and IGF-I gene expression were decreased early in the setting of mercuric chloride ARF. The decrease in distal tubular EGF staining was most pronounced in the OSOM, i.e. the anatomical area where mercuric-chloride-induced proximal tubular injury was most severe. CONCLUSIONS: Renal EGF and IGF-I gene expression decreases strongly during mercuric chloride ARF. The spatial association between the initial decrease of distal tubular EGF expression and the zone of major proximal tubular injury could originate from metabolic alterations secondary to oxygen starvation. A possible role of mononuclear cells remains to be determined.

Hyperplasia, hypertrophy, and phenotypic alterations in the distal nephron after acute proximal tubular injury in the rat.

BACKGROUND: Little is known about the impact of acute proximal tubular injury and dysfunction on the distal nephron. EXPERIMENTAL DESIGN: Selective necrosis of the kidney proximal convoluted tubule (PCT) was induced in rats by subcutaneous injection of the aminoglycoside gentamicin during 2 days. Damage and repair were measured until complete morphologic recovery after 10 days. Special attention was given to structural and biochemical alterations in the distal nephron. RESULTS: In control animals, cellular turnover, measured by immunohistochemical staining for proliferating cell nuclear antigen, was higher in distal than in proximal tubules. After injury, the strongly increased cell proliferation in regenerating necrotic PCT was preceded by an equally important proliferation in the distal tubules of the cortex and outer stripe of the outer medulla in the absence of necrosis but displaying enhanced apoptosis. Yet, epithelial vimentin expression was restricted to regenerating PCT. A temporary loss in the amount of immunostainable epidermal growth factor in the distal nephron was paralleled by a similar reduction in Tamm- Horsfall protein and transferrin receptor staining and in peanut and Helix pomatia lectin binding. Furthermore, the epithelial area/nucleus in the cortical distal tubules was increased by 71%, 6 days after the onset of acute renal failure; this hypertrophic condition was confirmed ultrastructurally. After full recovery of the PCT, a second burst in proliferative activity occurred in the hypertrophic distal segments in the absence of apoptosis. In the regenerated PCT, an excess cell number was accompanied by increased apoptotic activity. CONCLUSIONS: Development of distal tubular hypertrophy after PCT necrosis may be a compensatory response to a transient loss of proximal tubular function. The early reduction in staining for epidermal growth factor and other distal tubular markers in the presence of apoptosis and hyperplasia indicates transient phenotypic simplification and implies that renal epidermal growth factor is unlikely to control PCT regeneration.

Epidermal growth factor in acute renal failure.

Epidermal growth factor (EGF) is produced in large amounts in the kidney in the form of a membrane-bound high molecular weight precursor. This precursor is inserted in the apical plasma membrane of the EGF-producing cells, which are localized in the thick ascending limb and distal convoluted tubule in mouse and rat kidney, and probably also in human kidney. High levels of EGF are excreted in urine, although renal tissue contains little mature EGF. It modulates renal cell proliferation and differentiation in vitro, but the role of the distal tubular EGF and/or its precursor in vivo is unknown. The expression of EGF in the kidney and its liberation into the urine are quickly abolished during several types of drug- or ischemia-induced acute renal failure and also in ureteral obstruction. Moreover, its expression is restored only after morphological and functional recovery of the kidney. This absence of EGF in conditions in which its mitogenic properties would be most appropriate suggests that the EGF of renal origin is not acting as a mitogen during kidney regeneration. Nevertheless, since the number of EGF receptors, which are localized at the basolateral cell surface in most nephron segments, is increased in regenerating renal epithelia, EGF of systemic origin or other members of the EGF family of growth factors, released from infiltrated inflammatory cells at the sites of injury, could enhance cellular proliferation by interacting with the EGF receptor. Administration of EGF indeed has a mildly beneficial effect on recovery from acute renal injury.

Altered growth factor expression during toxic proximal tubular necrosis and regeneration.

Growth factor expression was investigated during the regenerative response after toxic proximal tubular necrosis. Therefore, gentamicin was administered to rats to achieve an experimental model, characterized by the appearance of segment-specific proximal tubular necrosis, that is followed by a regenerative response leading to functional and morphological recovery in a limited time. Four days after the administration of the highest dose, serum creatinine rose to a mean value of 5.8 mg/dl and returned to normal values ten days after the treatment. The S1-S2 segment of the proximal tubules in the cortex became clearly affected by severe toxic necrosis one day after the treatment, while maximal necrosis was observed at days 2 to 4. Only minor injuries were noticed in the other renal compartments. The proliferative response started in the interstitial cells first. The major proliferative wave was localized in the convoluted part of the proximal tubules at days 6 to 8, although proliferation was also prominent among non-proximal tubular cells. A profound interstitial infiltration of leukocytes, including macrophages and T lymphocytes, was observed. Ten days after the treatment the functional and morphological recovery were completed. Slot blot hybridization revealed a decreased EGF and IGF-I mRNA expression from the start of the observation period. While IGF-I mRNA had regained its normal expression at day 10, EGF mRNA was still below control levels. The PDGF-B transcript became more abundant towards the end of our observation. No major changes in the expression of TGF-alpha, TGF-beta 1 and c-fos were detected. Renal EGF-immunoreactivity disappeared from the luminal plasma membrane of the distal tubular cells analogous to the results obtained at the messenger level. However, EGF-staining was lost in the cortex first, hence a topographical association between the loss of EGF-immunoreactivity in the distal tubules and the observed necrotic lesions in the proximal tubules was found. Immunoreactive EGF was never observed in proximal tubular cells from normal, injured or regenerating rat kidneys. We conclude that in this experimental rat model, EGF and IGF-I mRNA expression is decreased during the regenerative response upon severe toxic tubular necrosis. No evidence for a participation of EGF or IGF-I of renal origin in the recovery of the kidney is found.