Establishment of a novel triple-transgenic mouse: conditionally and liver-specifically expressing hepatitis C virus NS3/4A protease.

It is well known that NS3/4A protein plays crucial roles in the hepatitis C virus (HCV) replication. NS3/4A protein also results to virus-mediated immune evasion and persistence of infection through the interaction with host proteins. However, the lack of a suitable animal model hampers studies of HCV NS3/4A protein interaction with host proteins, which impacts immunopathology due to infection. Here, transgenic vector containing transcriptional regulation and Fluc reporter gene was constructed to conditionally express NS3/4A protein under the dual control of Tet-On regulatory system and Cre/LoxP gene-knockout system. NS3/4A transgenic founder mice were continuously crossed with Lap transgenic mice expressing reverse tetracycline-controlled transcriptional activator (rtTA), the NS3/4A/Lap double transgenic mouse lines with liver-specifically and conditionally expressing reporter (luciferase Fluc) under control of Tet-On system were established. The NS3/4A/Lap double transgenic mouse are mated with Lap/LC-1 double transgenic mouse with liver-specifically and conditionally expressing Cre recombinase under control of Tet-On system, NS3/4A/Lap/LC-1 triple transgenic mouse were generated. In vivo bioluminescent imaging, western blotting and immunohistochemical staining (IHS) was used to confirm that NS3/4A protein was strictly expressed in the liver of Doxycycline-induced triple transgenic mice. The results show that we established a triple-transgenic mouse model conditionally expressing the HCV NS3/4A protein under strict control of the Tet-On regulatory system and Cre/loxP system. This novel transgenic mouse model expressing NS3/4A in a temporally and spatially-specific manner will be useful for studying interactions between HCV NS3/4A protein and the host, also for evaluating NS3/4A protease inhibitors.

miR-21 is a key therapeutic target for renal injury in a mouse model of type 2 diabetes.

AIMS/HYPOTHESIS: As microRNA-21 (miR-21) plays a pathological role in fibrosis, we hypothesised that it may be a therapeutic target for diabetic nephropathy. METHODS: Abundance of miR-21 was examined in diabetic kidneys from db/db mice. The therapeutic potential of miR-21 in diabetic kidney injury was examined in db/db mice by an ultrasound-microbubble-mediated miR-21 small hairpin RNA transfer. In addition, the role and mechanisms of miR-21 in diabetic renal injury were examined in vitro under diabetic conditions in rat mesangial and tubular epithelial cell lines by overexpressing or downregulating miR-21. RESULTS: In db/db mice, a mouse model of type 2 diabetes, renal miR-21 at age 20 weeks was increased twofold compared with db/m (+) mice at the same age, and this increase was associated with the development of microalbuminuria and renal fibrosis and inflammation. More importantly, gene transfer of miR-21 knockdown plasmids into the diabetic kidneys of db/db mice at age 10 weeks significantly ameliorated microalbuminuria and renal fibrosis and inflammation at age 20 weeks, revealing a therapeutic potential for diabetic nephropathy by targeting miR-21. Overexpression of miR-21 in kidney cells enhanced, but knockdown of miR-21 suppressed, high-glucose-induced production of fibrotic and inflammatory markers. Targeting Smad7 may be a mechanism by which miR-21 regulates renal injury because knockdown of renal miR-21 restored Smad7 levels and suppressed activation of the TGF-ß and NF-κB signalling pathways. CONCLUSIONS/INTERPRETATION: Inhibition of miR-21 might be an effective therapy for diabetic nephropathy.

Expression profile of macrophage migration-inhibitory factor in human gingiva and reconstituted human gingival epithelia stimulated by Porphyromonas gingivalis lipopolysaccharide.

BACKGROUND AND OBJECTIVE: Macrophage migration-inhibitory factor (MIF) plays crucial roles in the recruitment and activation of macrophages as well as in helping to kill bacteria. This study investigated the expression profile of MIF in human gingiva under different periodontal conditions and its expression patterns induced by Porphyromonas gingivalis lipopolysaccharide (LPS) in gingival epithelia. MATERIAL AND METHODS: Gingival tissue samples were collected from deep pockets and clinically healthy sites of 22 nonsmoking subjects with chronic periodontitis. The expression of MIF mRNA and protein was evaluated using real-time PCR and immunohistochemistry, respectively. The in vitro study analyzed the effects of P. gingivalis LPS on the expression of MIF in a reconstituted human gingival epithelia (RHGE) model. RESULTS: In gingival epithelia, MIF protein was diffusely expressed from the basal layer to the granular and spinous layers; whereas, in the underlying connective tissues, MIF was observed around the dilated blood vessels in the deep-pocket tissues. A significantly lower level of expression of MIF mRNA and an increased level of expression of MIF protein were found in deep-pocket tissues compared with clinically healthy tissues. Expression of MIF mRNA in the RHGE model was significantly down-regulated by P. gingivalis LPS. CONCLUSION: The present study suggests that MIF expression may be related to periodontal conditions and that its expression profile could be modulated by P. gingivalis LPS. MIF may play a role in periodontal pathogenesis.

Kidney-targeting Smad7 gene transfer inhibits renal TGF-ß/MAD homologue (SMAD) and nuclear factor κB (NF-κB) signalling pathways, and improves diabetic nephropathy in mice.

AIMS/HYPOTHESIS: The TGF-ß/MAD homologue (SMAD) and nuclear factor κB (NF-κB) signalling pathways have been shown to play a critical role in the development of renal fibrosis and inflammation in diabetic nephropathy. We therefore examined whether targeting these pathways by a kidney-targeting Smad7 gene transfer has therapeutic effects on renal lesions in the db/db mouse model of type 2 diabetes. METHODS: We delivered Smad7 plasmids into the kidney of db/db mice using kidney-targeting, ultrasound-mediated, microbubble-inducible gene transfer. The histopathology, ultrastructural pathology and pathways of TGF-ß/SMAD2/3-mediated fibrosis and NF-κB-dependent inflammation were evaluated. RESULTS: In this mouse model of type 2 diabetes, Smad7 gene therapy significantly inhibited diabetic kidney injury, compared with mice treated with empty vectors. Symptoms inhibited included: (1) proteinuria and renal function impairment; (2) renal fibrosis such as glomerular sclerosis, tubulo-interstitial collagen matrix abundance and renal inflammation, including Inos (also known as Nos2), Il1b and Mcp1 (also known as Ccl2) upregulation, as well as macrophage infiltration; and (3) podocyte and endothelial cell injury as demonstrated by immunohistochemistry and/or electron microscopy. Further study demonstrated that the improvement of type 2 diabetic kidney injury by overexpression of Smad7 was associated with significantly inhibited local activation of the TGF-ß/SMAD and NF-κB signalling pathways in the kidney. CONCLUSIONS/INTERPRETATION: Our results clearly demonstrate that kidney-targeting Smad7 gene transfer may be an effective therapy for type 2 diabetic nephropathy, acting via simultaneous modulation of the TGF-ß/SMAD and NF-κB signalling pathways.

Transmutation of long-lived fission products in an advanced nuclear energy system.

Disposal of long-lived fission products (LLFPs) produced in reactors has been paid a lot attention for sustainable and clean nuclear energy. Although a few transmutation means have been proposed to address this issue, there are still scientific and/or engineering challenges to achieve efficient transmutation of LLFPs. In this study, we propose a novel concept of advanced nuclear energy system (ANES) for transmuting LLFPs efficiently without isotopic separation. The ANES comprises intense photoneutron source (PNS) and subcritical reactor, which consist of lead-bismuth (Pb-Bi) layer, beryllium (Be) layer, and fuel, LLFPs and shield assemblies. The PNS is produced by bombarding radioactive cesium and iodine target with a laser-Compton scattering (LCS) γ-ray beam. We investigate the effect of the ANES system layout on transmutation efficiency by Monte Carlo simulations. It is found that a proper combination of the Pb-Bi layer and the Be layer can increase the utilization efficiency of the PNS by a factor of ~ 10, which helps to decrease by almost the same factor the LCS γ-beam intensity required for driving the ANES. Supposing that the ANES operates over 20 years at a normal thermal power of 500 MWt, five LLFPs including 99Tc, 129I, 107Pd, 137Cs and 79Se could be transmuted by more than 30%. Their effective half-lives thus decrease drastically from ~ 106 to less than 102 years. It is suggested that this successful implementation of the ANES paves the avenue towards practical transmutation of LLFPs without isotopic separation.

C-reactive protein promotes diabetic kidney disease in a mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: Although C-reactive protein (CRP) has been implicated as a risk factor in diabetes, its pathogenic importance in diabetic kidney disease (DKD) remains unclear. The present study investigated the potential role of CRP in DKD. METHODS: Diabetes was induced by streptozotocin in human CRP transgenic and wild-type mice for assessment of kidney injury at 24 weeks by real-time PCR, immunohistochemistry and western blot analysis. In vitro, the pathogenic effect of CRP was investigated using human kidney tubular epithelial cells cultured with high glucose and/or CRP. RESULTS: We found that CRP transgenic mice developed much more severe diabetic kidney injury than wild-type mice, as indicated by a significant increase in urinary albumin excretion and kidney injury molecule-1 abundance, enhanced infiltration of macrophages and T cells, and upregulation of pro-inflammatory cytokines (IL-1ß, TNFα) and extracellular matrix (collagen I, III and IV). Enhanced renal inflammation and fibrosis in CRP transgenic mice was associated with upregulation of CRP receptor, CD32a, and over-activation of the TGF-ß/SMAD and nuclear factor κB signalling pathways. In vitro, CRP significantly upregulated pro-inflammatory cytokines (IL-1ß, TNFα, monocyte chemoattractant protein-1 [MCP-1]) and pro-fibrotic growth factors (TGF-ß1, connective tissue growth factor [CTGF]) via CD32a/64. CRP was induced by high glucose, which synergistically promoted high glucose-mediated renal inflammation and fibrosis. CONCLUSIONS/INTERPRETATION: CRP is not only a biomarker, but also a mediator in DKD. Enhanced activation of TGF-ß/SMAD and nuclear factor κB signalling pathways may be the mechanisms by which CRP promotes renal inflammation and fibrosis under diabetic conditions.

The pathogenic role of macrophage migration inhibitory factor in immunologically induced kidney disease in the rat.

Macrophage migration inhibitory factor (MIF) plays a pivotal role in the inflammatory response in endotoxemia and in the delayed-type hypersensitivity response, but its potential as a regulator of immunologically induced disease is unknown. We have addressed this issue by administering a neutralizing anti-MIF antibody in a rat model of immunologically induced crescentic anti-glomerular basement membrane (GBM) glomerulonephritis. Six individual experiments using paired inbred littermates were performed. Rats were primed with rabbit immunoglobulin on day -5 and then injection with rabbit anti-rat GBM serum on day 0. Pairs of animals were treated with anti-MIF or a control monoclonal antibody from the time of anti-GBM serum administration until being killed 14 d later. Control antibody-treated animals developed severe proteinuria and renal function impairment with severe histological damage due to marked leukocytic infiltration and activation within the kidney. In contrast, anti-MIF treatment substantially reduced proteinuria, prevented the loss of renal function, significantly reduced histological damage including glomerular crescent formation, and substantially inhibited renal leukocytic infiltration and activation (all P <0.001 compared with control treatment). Inhibition of renal disease by anti-MIF treatment was attributed to preventing the marked upregulation of interleukin-1beta, leukocyte adhesion molecules including intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, and inducible nitric oxide synthase expression seen in the control antibody-treated animals. This inhibition of progressive renal injury was mirrored by the complete suppression of the skin delayed-type hypersensitivity response to the challenge antigen (rabbit IgG). Interestingly, anti-MIF treatment did not effect the secondary antibody response or immune deposition within the kidney, indicating that MIF participates in cellular-based immunity in this primed macrophage-dependent anti-GBM glomerulonephritis. In conclusion, this study has demonstrated a key regulatory role for MIF in the pathogenesis of immunologically induced kidney disease. These results argue that blocking MIF activity may be of benefit in the treatment of human rapidly progressive glomerulonephritis, and suggest that MIF may be important in immune-mediated disease generally.

Immunogenicity and protective efficacy in rhesus monkeys of a recombinant ORF2 protein from hepatitis E virus genotype 4.

Several antigens derived from hepatitis E virus (HEV) genotype 1 strains have shown immunogenicity and efficacy against hepatitis E in primates and humans. However, the protective effect of a vaccine derived from HEV genotype 4 has not been studied. This study aimed to evaluate the immunogenicity and protective efficacy of the T1-ORF2 (56 kDa) capsid protein derived from HEV strain T1 (genotype 4) in rhesus monkeys. Two doses (40 microg) of alum-absorbed T1-ORF2 capsid protein were administered 4 weeks apart. Seroconversion occurred in all immunized monkeys 1-2 weeks after the first dose. The peak levels of anti-HEV IgG appeared at 2-3 weeks after the second dose and ranged from 5.7 to 196.0 U/ml. All monkeys showed an anamnestic antibody response to the second dose. Control monkeys immunized with saline remained negative for HEV antibodies throughout the pre-challenge period. The immunized monkeys were challenged intravenously with HEV genotypes 1 and 4. Monkeys immunized with T1-ORF2 were protected from infection and hepatitis after challenge with 5 x 10(4) genome equivalents of HEV, regardless of the genotype. After challenge with 5 x 10(5) genome equivalents of HEV genotype 4, the monkeys immunized with T1-ORF2 had a shorter period of raised alanine aminotransferase levels and a shorter duration of fecal shedding compared to control monkeys immunized with saline. In conclusion, these results suggest that, in rhesus monkeys, the T1-ORF2 capsid protein of HEV genotype 4 has similar cross-protective effects to other candidate vaccines derived from HEV genotype 1.

Oncogene functions of FHL2 are independent from NF-kappaBIalpha in gastrointestinal cancer.

Four and a half of LIM-only protein 2 (FHL2) is an adaptor protein that can interact with many transcription factors and thus plays a variety of biological functions. Previous studies by our group have demonstrated that suppression of FHL2 was capable of inducing tumor cell differentiation, and inhibiting the growth of experimental gastric and colon cancers. Therefore, FHL2 appears to function as an oncogene. In order to further explore the mechanisms of how FHL2 is involved in tumorigenesis, we attempted to test whether FHL2 has any direct association with nuclear factor (NF-kappaB), the most important transcription factor involved in apoptosis, inflammation, and carcinogenesis. Using an Yeast Two Hybrid (Y2H) screening system, we have shown that FHL2 may have an interaction with NF-kappaBIalpha, the coding gene for IkappaBalpha which is the most potent endogenous inhibitor for NF-kappaB activation. However, subsequent studies using co-immunoprecipitation and co-localization failed to confirm the Y2H finding. Down-regulation of FHL2 by FHL2-siRNA down-regulated the expression of NF-kappaB p65. We therefore concluded that under the physiological condition, FHL2 may activate NF-kappaB pathway, even though such an activation may not be mediated by a direct binding of FHL2 to NF-kappaB inhibitor protein IkappaB.

Monocyte chemoattractant protein-1 promotes macrophage-mediated tubular injury, but not glomerular injury, in nephrotoxic serum nephritis.

Monocyte chemoattractant protein-1 (MCP-1) is upregulated in renal parenchymal cells during kidney disease. To investigate whether MCP-1 promotes tubular and/or glomerular injury, we induced nephrotoxic serum nephritis (NSN) in MCP-1 genetically deficient mice. Mice were analyzed when tubules and glomeruli were severely damaged in the MCP-1-intact strain (day 7). MCP-1 transcripts increased fivefold in MCP-1-intact mice. MCP-1 was predominantly localized within cortical tubules (90%), and most cortical tubules were damaged, whereas few glomerular cells expressed MCP-1 (10%). By comparison, there was a marked reduction (>40%) in tubular injury in MCP-1-deficient mice (histopathology, apoptosis). MCP-1-deficient mice were not protected from glomerular injury (histopathology, proteinuria, macrophage influx). Macrophage accumulation increased adjacent to tubules in MCP-1-intact mice compared with MCP-1-deficient mice (70%, P < 0.005), indicating that macrophages recruited by MCP-1 induce tubular epithelial cell (TEC) damage. Lipopolysaccharide-activated bone marrow macrophages released molecules that induced TEC death that was not dependent on MCP-1 expression by macrophages or TEC. In conclusion, MCP-1 is predominantly expressed by TEC and not glomeruli, promotes TEC and not glomerular damage, and increases activated macrophages adjacent to TEC that damage TEC during NSN. Therefore, we suggest that blockage of TEC MCP-1 expression is a therapeutic strategy for some forms of kidney disease.

De novo expression of macrophage migration inhibitory factor in atherogenesis in rabbits.

Macrophage migration inhibitory factor (MIF) has been shown to play an important role in macrophage-mediated diseases. We investigate the potential role of MIF in atherogenesis using a hypercholesterolemic rabbit model. New Zealand White rabbits fed with a 2% cholesterol diet developed hypercholesterolemia and early fatty streaks at 1 month. The lesions became advanced at 3 months and were associated with de novo MIF expression by vascular endothelial cells (VECs) and smooth muscle cells (SMCs), as demonstrated by immunohistochemistry, reverse transcriptase-polymerase chain reaction, and in situ hybridization. By contrast, there was no increase in MIF levels in rabbits fed a normal diet. In early atherogenesis, marked upregulation of MIF mRNA and protein by VECs and some intimal cells were closely associated with CD68(+) monocyte adhesion onto and subsequent migration into subendothelial space. Of significance, the accumulation of macrophages was exclusively localized to areas of strong MIF expression, which may be associated with the macrophage-rich fatty streak lesion formation. Upregulation of MIF by SMCs is transient during atherogenesis. Importantly, strong MIF expression by activated macrophages may be responsible for the development of foam cell-rich lesions. Finally, the ability of MIF to induce intercellular adhesion molecule-1 expression by VECs implicates its pathogenic role in atherogenesis. In conclusion, the present study provides the first demonstration that MIF is markedly upregulated during atherogenesis. Upregulation of MIF by VECs and SMCs may play a role in macrophage adhesion, transendothelial migration, accumulation, and, importantly, transformation into foam cells. Furthermore, strong MIF expression by macrophages may both initiate and amplify the atherogenesis process.

Macrophage migration inhibitory factor expression in male and female ethanol-fed rats.

Macrophage migration inhibitory factory (MIF) regulates macrophage accumulation at sites of injury and can promote the inflammatory response. We studied MIF expression in the intragastric feeding rat model for alcoholic liver injury. Male and age-matched female rats were fed ethanol or dextrose with fish oil. Two groups of male rats were fed medium-chain triglycerides with ethanol or dextrose. Analysis of liver histopathology, lipid peroxidation, endotoxin, mRNA, and immunohistochemistry for MIF, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) were carried out. Male and female rats fed fish oil and ethanol showed necroinflammatory liver injury and had the highest expression of MIF, TNF-alpha, and IFN-gamma in the liver. Decreased levels of MIF protein were seen in rats with higher endotoxin levels, suggesting that preformed MIF is released into the circulation. MIF is an important mediator of the inflammatory response in alcoholic liver disease and a potential therapeutic target.

A novel method of microwave treatment for detection of cytoplasmic and nuclear antigens by flow cytometry.

Flow cytometry has recently become a useful technique for the quantitative analysis of cytoplasmic and nuclear antigens. We report here a rapid, simple, reproducible, and sensitive method for the simultaneous detection of cytoplasmic and nuclear antigens by flow cytometry. This technique involves the treatment of cell suspensions with 60 s of microwave oven heating after fixation with 2% paraformaldehyde. Following this treatment a number of cytoplasmic and nuclear antigens were detected on the human myelomonocytic cell line U937 (CD68, PCNA and Ki-67), peripheral blood leukocytes from both normal donors and leukemia patients (CD68, lipocortin-1 and PCNA) and a rat mesangial cell line 1097 (desmin, alpha-smooth muscle actin) using a standard indirect immunofluorescent staining with mouse monoclonal antibodies (mAbs). There are several advantages of this technique over the routinely used methods currently available. Firstly, microwave treatment is a rapid, simple, and reproducible method, which largely reduces both time and cost expenditure, and makes this technique widely available for flow cytometric analysis in many areas of diagnostic and research purposes. Secondly, microwave treatment produces optimal results for simultaneous detection of both cytoplasmic (CD68, lipocortin-1, desmin, alpha-smooth actin) and nuclear (PCNA, Ki67) antigens. Thirdly, microwave treatment also produces a discrete profile for DNA content analysis. Finally, microwaving retains a clear discrimination between cells and debris as measured by light scatter. This study demonstrates that microwave treatment is a powerful technique which will be particularly applicable to flow cytometric analysis in the detection of many cytoplasmic and nuclear antigens.

Deoxyspergualin suppresses local macrophage proliferation in rat renal allograft rejection.

Deoxyspergualin (DSP) is a potent immunosuppressive drug that is able to both prevent and reverse acute allograft rejection. Although there is good evidence that DSP can inhibit T and B lymphocyte responses, the effect of this drug upon monocyte function is controversial. In the current study, substantial local proliferation of inflammatory macrophages (41.6 +/- 5.5% of ED1+ cells) within acutely rejecting rat renal allografts was identified by expression of the proliferating cell nuclear antigen. Treatment of animals with DSP not only reduced macrophage accumulation within the tissue, but it also significantly inhibited local proliferation of macrophages within the graft (26.4 +/- 5.6% of ED1+ cells, P < 0.05 vs. untreated). This appeared to be, at least in part, a direct effect of DSP upon macrophages since the drug also inhibited growth of 2 monocytic cell lines (RC-2A and U937) in vitro. However, DSP treatment had no effect upon LPS-induced monocyte IL-1 beta, TNF alpha, and IL-6 mRNA and protein production, indicating that this drug is not a general inhibitor of monocyte function. In conclusion, this study has demonstrated that local proliferation of macrophages within the kidney is a prominent feature of acute allograft rejection and that inhibition of this response is one mechanism whereby DSP exerts its potent immunosuppressive actions.

Macrophage migration inhibitory factor expression in human renal allograft rejection.

BACKGROUND: Macrophage migration inhibitory factor (MIF) plays a pivotal role in immune-mediated diseases. Despite the long-standing association of MIF with the delayed-type hypersensitivity response, the potential role of MIF in allograft rejection is unknown. METHODS: MIF expression was assessed by in situ hybridization and immunohistochemistry staining in 62 biopsies of human renal allograft rejection and in normal human kidney. RESULTS: MIF mRNA and protein is constitutively expressed in normal kidney, being largely restricted to tubular epithelial cells, some glomerular epithelial cells, and vascular smooth muscle cells. In both acute and chronic renal allograft rejection, there was marked up-regulation of MIF mRNA and protein expression by intrinsic kidney cells such as tubular epithelial cells and vascular endothelial and smooth muscle cells. There was also MIF expression by infiltrating macrophages and T cells. Of note, macrophage and T cell infiltrates were largely restricted to areas with marked up-regulation of MIF expression, potentially contributing to the development of severe tubulitis and intimal or transmural arteritis. Quantitative analysis found that increased MIF expression in allograft rejection gave a highly significant correlation with macrophage and T cell accumulation in both the glomerulus and interstitium (P<0.001). In addition, the number of MIF+ tubules and interstitial MIF+ cells correlated significantly with the severity of allograft rejection (P<0.01), and the loss of renal function (P<0.01). In contrast, no up-regulation of renal MIF expression and no leukocyte accumulation was seen in allograft biopsies without evidence of rejection. CONCLUSIONS: This is the first study to demonstrate that local MIF expression is up-regulated during allograft rejection. The association between up-regulation of MIF expression, macrophage and T cell infiltration and the severity of renal allograft rejection suggests that MIF may be an important mediator in the process of allograft rejection.

Hyperuricemia exacerbates chronic cyclosporine nephropathy.

BACKGROUND: Hyperuricemia frequently complicates cyclosporine (CSA) therapy. The observation that longstanding hyperuricemia is associated with chronic tubulointerstitial disease and intrarenal vasoconstriction raised the hypothesis that hyperuricemia might contribute to chronic CSA nephropathy. METHODS: CSA nephropathy was induced by the administration of CSA (15 mg/kg/day) for 5 and 7 weeks to rats on a low salt diet (CSA group). The effect of hyperuricemia on CSA nephropathy was determined by blocking the hepatic enzyme uricase with oxonic acid (CSA-OA). Control groups included rats treated with vehicle (VEH) and oxonic acid alone (OA). Histological and functional studies were determined at sacrifice. RESULTS: CSA treated rats developed mild hyperuricemia with arteriolar hyalinosis, tubular injury and striped interstitial fibrosis. CSA-OA treated rats had higher uric acid levels in association with more severe arteriolar hyalinosis and tubulointerstitial damage. Intrarenal urate crystal deposition was absent in all groups. Both CSA and CSA-OA treated rats had increased renin and decreased NOS1 and NOS3 in their kidneys, and these changes are more evident in CSA-OA treated rats. CONCLUSION: An increase in uric acid exacerbates CSA nephropathy in the rat. The mechanism does not involve intrarenal uric acid crystal deposition and appears to involve activation of the renin angiotensin system and inhibition of intrarenal nitric oxide production.

A novel, simple, reliable, and sensitive method for multiple immunoenzyme staining: use of microwave oven heating to block antibody crossreactivity and retrieve antigens.

We report a simple and reliable method for detection of two or more antigens within tissue sections by indirect immunoenzyme staining using mouse monoclonal antibodies (MAbs). This technique involves treating sections with two 5-min microwave (MW) oven heatings between sequential rounds of three-layer immunoenzyme staining (mouse MAb, goat anti-mouse IgG, and mouse PAP or mouse APAAP) and color development. Discrete staining of cell surface, cytoplasmic, and nuclear antigens was evident within individual cells. This technique has a number of advantages over those currently available. First, MW treatment denatures bound antibody molecules, thereby completely blocking crossreactivity between sequential rounds of staining. This allows the use of primary (and other) antibodies raised in the same species and the use of a sensitive three-layer staining method. Second, antigen retrieval after MW treatment markedly increases the sensitivity of cytoplasmic and nuclear antigen detection. Third, inactivation of peroxidase and alkaline phosphatase enzymes present in PAP and APAAP complexes prevents inappropriate color development. Finally, this method can be used in both paraformaldehyde-fixed cryostat sections and formalin-fixed paraffin tissue sections. In conclusion, this is a simple, reliable, and sensitive technique that will be useful in many areas of diagnosis and research.

A simple, reliable, and sensitive method for nonradioactive in situ hybridization: use of microwave heating to improve hybridization efficiency and preserve tissue morphology.

The digestion of fixed tissue sections is a critical step in the optimization of any in situ hybridization protocol. We describe a novel application of microwave oven heating to optimize mRNA detection in paraformaldehyde-fixed tissues by in situ hybridization using digoxigenin-labeled probes. This technique replaces protease digestion of fixed tissue sections with 10 min of microwave pretreatment, followed by either conventional hybridization or hybridization involving microwave incubation. This new technique has several advantages over the standard protease treatment-based methods presently in use. (a) Microwave oven heating is a simple, rapid, and highly reproducible technique. (b) Microwave pretreatment significantly increased the hybridization signal and reduced the background compared to conventional protease digestion. Consequently, the hybridization time required to obtain optimal mRNA detection was reduced to 30 min. (c) Ten minutes of microwave pretreatment produced an optimal hybridization signal in six different tissues using a variety of probes, demonstrating the general applicability of this technique. (d) Microwave heating of the probe during the hybridization step itself further reduced the hybridization time and substantially enhanced the hybridization signal obtained from proteinase K-digested tissue. (e) Microwave pretreatment caused no discernible loss of fine cell structure and tissue morphology compared to untreated tissue sections. In conclusion, microwave oven heating can replace the complicated strategies and poor reproducibility of protease treatment of tissue sections, resulting in a simple, rapid, more reliable and sensitive method that has general applicability for in situ hybridization.

Characteristics of albumin processing during renal passage in anti-Thy1 and anti-glomerular basement membrane glomerulonephritis.

Recent studies have shown that glomerular-filtered albumin appears to be processed by two distinct cellular pathways. The major pathway, a high-capacity retrieval pathway, returns most of the filtered albumin to the blood supply intact. The albumin not taken up by the retrieval pathway is degraded by lysosomes during renal passage and excreted as fragments in urine. We studied the interplay of the albumin retrieval pathway and the degradation pathway in the disease models of anti-Thy1 nephritis, a model of mild proteinuria, and anti-glomerular basement membrane (anti-GBM) disease, a model of severe proteinuria. This is achieved by investigating the integrity of urinary albumin and its excretion rate. Total albumin excretion (intact plus fragments) did not change significantly in the rats with anti-Thy1 nephritis. However, it was established that intact albumin excretion had a strong positive correlation with increasing total-protein excretion, which showed that the degradation pathway was being predominantly affected in this disease. For the rats with anti-GBM disease, total protein excretion increased 26-fold compared with the control group, and intact albumin excretion increased 250-fold. The profound changes in albumin excretion in anti-GBM disease are consistent with inhibition primarily of the retrieval pathway.

Transferrin but not albumin mediates stimulation of complement C3 biosynthesis in human proximal tubular epithelial cells.

Complement is increasingly implicated in the pathogenesis of progressive renal disease resulting from persistent proteinuria. We have previously shown that apical serum proteins stimulate C3 in cultured human proximal tubular epithelial cells (PTECs), and that the stimulant is a nonalbumin compound of 30 to 100 kd. We postulated in this study that transferrin and apotransferrin, also important components of proteinuric urine in this molecular-weight range, might be the culprit. Human PTECs were obtained by differential sieving of renal cortical tissue from the normal pole of tumor nephrectomy specimens and characterized to be predominantly of proximal tubular origin. Complement C3 messenger RNA (mRNA) expression was analyzed in confluent growth-arrested PTEC monolayers in media containing different concentrations (2.5 to 20 mg/mL) of transferrin by reverse transcription and polymerase chain reaction. Pure human albumin was used as a control protein. C3 protein secretion was detected and quantified by a sandwich enzyme-linked immunosorbent assay on cell culture supernatants after distinct time points. Transferrin enhanced the rate of C3 secretion in a dose-dependent manner, reaching maximal stimulation at doses of 10 mg/mL. Selected experiments using the Transwell technique showed that C3 release was predominantly apical in the resting state. The addition of 10 mg/mL of transferrin apically but not basolaterally stimulated both apical and basolateral C3 secretion and increased the basolateral-apical ratio of C3 secretion from 0.45 +/- 0.16 to 0.93 +/- 0.24 (P: < 0.02). Constitutive C3 mRNA expression was upregulated by transferrin in a time- and dose-dependent fashion, reaching a peak after 24 hours. A similar degree of C3 upregulation was reproduced when iron-poor transferrin, apotransferrin, was used instead. These results indicate that C3 synthesis in PTECs is upregulated by transferrin, for which protein rather than iron moiety may account for the observed effects. These findings provide evidence linking proteinuria with overexpression of tubular complement.