Frozen Kidney Sections Research Articles

Lack of Cannabinoid Type 2 Promoter Activity in Normal or Injured Kidneys Using a Cnr2-GFP Reporter Mouse.

Introduction: Although cannabinoid type 2 (CB2) receptor activity is known to promote diverse biological functions in the kidney, published data regarding CB2 receptor protein levels and cellular distribution within the kidney is inconsistent. The goal of the present study was to investigate the changes of CB2 in the kidney obtained from mice exposed to various forms of kidney injury using a genetic mouse model expressing green fluorescent protein (GFP) driven by the endogenous cannabinoid receptor 2 (Cnr2) promoter. Materials and Methods: Kidney injury was established in a genetic mouse model expressing green fluorescent protein (GFP) driven by the endogenous Cnr2 promoter. Kidney injury was initiated by either treatment with different chemicals [cisplatin or lipopolysaccharide (LPS)] or by unilateral ureteral obstruction (UUO). Changes in the detection of GFP were used as a proxy for CB2 levels and localization. Histological changes due to the injury stimuli were observed by time-related, morphological changes in kidney cytoarchitecture and blood parameters, such as serum creatinine levels. Cnr2 mRNA levels were detected by reverse transcription coupled to polymerase chain reaction (RT-PCR) while protein changes in the tissue lysates were measured by Western blot analysis. Cellular localization of GFP was detected by fluorescent microscopy. Results: Our data demonstrated that there was no band or a minimally detectable band for GFP using kidney lysates from vehicle- or cisplatin-treated mice. A similar lack of GFP was detected in the UUO kidney versus the contralateral control kidney. This is consistent with the low, albeit detectable levels of Cnr2 mRNA in the kidney samples from control or cisplatin treatment. In frozen kidney sections from vehicle and cisplatin-treated mice, GFP fluorescence was not detectable in tubular epithelia, glomeruli or blood vessels in the cortex. Instead, GFP was detected in rare cells within the interstitial space. A second chemical injury model using LPS found a similar lack of GFP protein levels and an absence of legitimate GFP fluorescence in the main cell types within the kidney. Conclusion: These findings suggest that Cnr2 promoter activity is minimally active in normal or injured kidneys, and that pharmacological manipulation of CB2 receptors may be associated with receptors being expressed in cells recruited to the kidney.

N-glycosylated IgG in patients with kidney transplants increases calcium/calmodulin kinase IV in podocytes and causes injury.

Transplant glomerulopathy (TG) is a major cause of late allograft loss. Increased urine podocin/creatinine ratio in TG signifies accelerated podocyte loss. The mechanisms that lead to podocyte injury in TG remain unclear. We report that IgG from kidney transplant recipients with TG, but not from those without TG, cause a reduction in the expression of nephrin, significant podocyte actin cytoskeleton, and motility changes. These changes are preceded by increased expression of calcium/calmodulin kinase IV (CAMK4). Mechanistically, we found that CAMK4 phosphorylates GSK3β (glycogen synthase kinase 3 beta), activates the Wnt pathway and stabilizes the nephrin transcriptional repressor SNAIL. Silencing neonatal Fc Receptor (FcRn) or CAMK4 prevented the podocyte-damaging effects of IgG from patients with TG. Furthermore, we show that removal of N-linked glycosyl residues from these IgG did not interfere with its entry into the podocytes but eliminated its ability to upregulate CAMK4 and cause podocyte injury. The translational value of these findings is signified by the fact that CAMK4 is increased in podocytes of patients with TG but not in those without TG despite other forms of renal dysfunction. Our results offer novel considerations to limit podocyte injury in patients with kidney transplants, which may lead to eventual glomerular destabilization and transplant glomerulopathy.

Molecular Imaging of the Glomerulus via Mesangial Cell Uptake of Radiolabeled Tilmanocept.

An unmet need for the clinical management of chronic kidney disease is a predictive tool of kidney function during the first decade of the disease, when there is silent loss of glomerular function. The objective of this study was to demonstrate receptor-mediated binding of tilmanocept to CD206 within the kidney and provide evidence of kinetic sensitivity of this binding to renal function. Methods: Rats were positioned in a PET scanner with the liver and kidneys within the field of view. After an intravenous injection of 68Ga-IRDye800-tilmanocept, using 1 of 2 scaled molar doses (0.02 nmol/g, n = 5; or 0.10 nmol/g, n = 5), or coinjection (n = 3) of 68Ga-IRDye800-tilmanocept (0.10 nmol/g) and unlabeled tilmanocept (5.0 nmol/g), or a negative control, 68Ga-IRDye800-DTPA-galactosyl-dextran (0.02 nmol/g, n = 5), each animal was imaged for 20 min followed by a whole-body scan. Frozen kidney sections were stained for podocytes and CD206 using immunofluorescence. Molecular imaging of diabetic db/db mice (4.9 wk, n = 6; 7.3 wk, n = 4; 13.3 wk, n = 6) and nondiabetic db/m mice (n = 6) was performed with fluorescence-labeled 99mTc-tilmanocept (18.5 MBq, 2.6 nmol). Thirty minutes after injection, blood, liver, kidneys, and urine were assayed for radioactivity. Renal time-activity curves were generated. Results: Rat PET whole-body images and time-activity curves of 68Ga-IRDye800-tilmanocept demonstrated receptor-mediated renal accumulation with evidence of glomerular uptake. Activity within the renal cortex persisted during the 40-min study. Histologic examination demonstrated colocalization of CD206 and IRDye800-tilmanocept within the glomerulus. The glomerular accumulation of the coinjection and the negative control studies were significantly less than the CD206-targeted agent. The db/db mice displayed a multiphasic renal time-activity curve with high urinary bladder accumulation; the nondiabetic mice exhibited renal uptake curves dominated by a single phase with low bladder accumulation. Conclusion: This study demonstrated receptor-mediated binding to the glomerular mesangial cells and kinetic sensitivity of tilmanocept to chronic renal disease. Given the role of mesangial cells during the progression of diabetic nephropathy, PET or SPECT renal imaging with radiolabeled tilmanocept may provide a noninvasive quantitative assessment of glomerular function.

Deep Learning Global Glomerulosclerosis in Transplant Kidney Frozen Sections.

Transplantable kidneys are in very limited supply. Accurate viability assessment prior to transplantation could minimize organ discard. Rapid and accurate evaluation of intra-operative donor kidney biopsies is essential for determining which kidneys are eligible for transplantation. The criterion for accepting or rejecting donor kidneys relies heavily on pathologist determination of the percent of glomeruli (determined from a frozen section) that are normal and sclerotic. This percentage is a critical measurement that correlates with transplant outcome. Inter- and intra-observer variability in donor biopsy evaluation is, however, significant. An automated method for determination of percent global glomerulosclerosis could prove useful in decreasing evaluation variability, increasing throughput, and easing the burden on pathologists. Here, we describe the development of a deep learning model that identifies and classifies non-sclerosed and sclerosed glomeruli in whole-slide images of donor kidney frozen section biopsies. This model extends a convolutional neural network (CNN) pre-trained on a large database of digital images. The extended model, when trained on just 48 whole slide images, exhibits slide-level evaluation performance on par with expert renal pathologists. Encouragingly, the model's performance is robust to slide preparation artifacts associated with frozen section preparation. The model substantially outperforms a model trained on image patches of isolated glomeruli, in terms of both accuracy and speed. The methodology overcomes the technical challenge of applying a pretrained CNN bottleneck model to whole-slide image classification. The traditional patch-based approach, while exhibiting deceptively good performance classifying isolated patches, does not translate successfully to whole-slide image segmentation in this setting. As the first model reported that identifies and classifies normal and sclerotic glomeruli in frozen kidney sections, and thus the first model reported in the literature relevant to kidney transplantation, it may become an essential part of donor kidney biopsy evaluation in the clinical setting.

Can examination of tissue stained with Oil red O be postponed up to three months?

Abstract Introduction: As far as we know, there are no known studies on the durability of frozen tissue stained with Oil Red O. The purpose of this study was to examine if the lipid drops in Oil Red O stained tissue keep the original position and color over time (3 months). Further we examined if storage position of the stained tissue makes a difference. Method: We used ten frozen kidney sections stained with Oil Red O. Half of the samples were stored vertically and the other half horizontally, and photos of the same areas were taken within the first 24 hours after staining, and then after 48 hours, 72 hours, 7 days, 14 days, 1 month, 2 months and 3 months respectively. Results and conclusion: No changes in position of the lipids were observed. The color of the staining faded somewhat over time, but it was still possible to distinguish the positive sites from the negative.

Immunohistochemical staining of heme oxygenase-1 in frozen kidney sections

Objective To study the immunohistochemical staining of heme oxygenase-1 (HO-1)in frozen kidney sections and the interference factors.Methods High expression of HO-1 in the kidney of rats (n =24) was achieved through CoPP administration.The Avidin-Biotin Complex system (SP system)with (n =8) /without (n =8) egg white blocking was compared with the Dako EnVision system (n =8) in the staining of endogenous peroxydase,endogenous biotin and HO-1 in the frozen kidney sections of rats.Results CoPP induced HO-1 high expression in all 24 rat kidneys.There was no endogenous peroxydase in the normal rat kidneys,except for few area of large blood vessels,which could be blocked by 0.3％ H202 ·methanol or 3％ H202.Endogenous biotin was widely detected in frozen rat kidney sections,which could not be blocked by 20％ egg white and even be enlarged instead (n =8).The Dako EnVision system could effectively avoid the the non-specific staining of the endogenous biotin activity and showed a clean strong expression of HO-1 (n =8).Conclusion Endogenous biotin is the major interference factor of the immunohistochemical staining in frozen sections.The routine SP system with/without egg white blocking can not eliminate the interference.EnVision system gives an efficiency of the detection of HO-1 with a clean backgroud. Key words: Biotin ; Avidin ; Immunohistochemistry ; Rat

Abstract 551: Inhibition of Sphingosine-1-phosphate Receptor 2 Activation Protects against Renal Ischemia-Reperfusion Injury via Reduction of Reactive Oxygen Species

Sphingosine-1-phosphate (S1P) is a potent vasoconstrictor of the preglomerular microvasculature. Our recent studies indicate that S1P-mediated afferent arteriolar vasoconstriction is enhanced in ischemia-reperfusion (IR) induced renal injury and S1P2 receptor protein expression is increased in renal microvessels. Reactive oxygen species (ROS) play a critical role in the development of IR renal injury. We hypothesized that inhibition of S1P2 receptor activation protects against renal IR injury via ROS reduction. Renal IR injury was induced by bilateral renal artery occlusion for 60 min followed by 24 hrs reperfusion. In some rats, IR was induced during treatment with the selective S1P2 receptor antagonist, JTE-013 (0.1mg/kg BW, IP) prior to ischemia and repeated at the time of reperfusion. After reperfusion, plasma was collected and kidneys were harvested for histological analysis and ROS detection. Plasma creatinine (Cr) concentration was markedly increased in IR rats (4.2±0.1 mg/dl, n=6) compared to sham controls (1.1±0.1 mg/dl, n=5, p<0.05). JTE-013 treatment (n=4) significantly reduced plasma Cr in IR rats (2.0±0.3 mg/dl, p<0.05). Histological analysis revealed no significant cell damage in control kidney sections stained with hematoxylin and eosin whereas severe cell damage was detected in IR kidneys, including cell necrosis, disruption of the plasma membrane, nuclear pyknosis, cellular vacuolization, and the presence of luminal casts and sloughed cells in proximal tubules, particularly in the outer stripe of the outer medulla. Additionally, there was significant trapping of erythrocytes in the medulla. In contrast, JTE-013 treated IR rats showed less necrosis and cell damage in the proximal tubules. Renal ROS levels were assessed in frozen kidney sections using the fluorogenic probe, H 2 DCFDA. Fluorescence intensity was markedly increased in IR rats (18.7±2.4 in cortex and 19.0±2.5 in medulla) compared to control (10.9±0.7 and 9.6±0.7, p<0.05, respectively). JTE-013 treatment reduced ROS levels to values not different from control (13.5±1.1 and 15.4±1.5, p>0.05 vs. control, respectively). These data suggest that blockade of S1P2 receptors protects against renal IR injury by reducing tissue ROS accumulation.

In Vivo Applied Positively Charged FITC-Labeled Peptide Conjugates Show Artificial Relocation in Frozen Sections

Our original aim was to evaluate in vivo nuclear uptake of a conjugate containing the SV40 T Antigen nuclear localization sequence (NLS), 6 aminohexanoic acids alternating with 7 arginines, the fluorescence dye fluorescein isothiocyanate (FITC) and the gadolinium chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). During the confocal laser scanning microscopy examination of frozen sections of the kidneys of mice injected intravenously with the conjugate we found unexpected conjugate relocation. In the dry frozen sections no nuclear staining was found. After covering the sections with physiological saline and a coverslip, the conjugate was relocated to the cell nuclei. Causation of this effect by FITC and DOTA could be excluded with appropriate controls. In the assumption that other positively charged conjugates behave in the same manner we tested two further conjugates in vivo. An octaarginine coupled to a lysine carrying FITC on the -amino-group was found in the cell nuclei of the frozen kidney sections before and after covering the slices with saline. By contrast, a conjugate containing myristoylated HIV-Tat was found not to stain the cell nuclei but only the cytoplasm before and after covering with saline. This demonstrates that not all positively charged conjugates are relocated to the cell nucleus after covering the frozen sections with saline. This was confirmed by direct incubation of frozen sections from untreated kidneys with the same conjugates and additional positively charged FITC conjugates. By coincubating the FITC conjugates with a cytoplasm directed rhodamine conjugate on untreated kidney sections we found that such conjugates could be used as nuclear FITC counterstain. In summary, one should bear in mind that positively charged conjugates applied in vivo can relocate artificially after covering with saline. Keywords: Morphological artifacts, Frozen Sections, FITC-Labeled Peptide Conjugates, Artificial Relocation, SV40 T Antigen nuclear localization sequence, nuclear uptake, FITC conjugates, DOTA, glioma cells, N-terminally myristoylated HIV 1 Tat peptide sequence

A Protein Toxin from the Sea Anemone Phyllodiscus semoni Targets the Kidney and Causes a Severe Renal Injury with Predominant Glomerular Endothelial Damage

Envenomation by the sea anemone Phyllodiscus semoni causes fulminant dermatitis and, rarely, acute renal failure in humans. Here, we investigated whether the venom extracted from the nematocysts (PsTX-T) was nephrotoxic when administered intravenously in rats and whether PsTX-T induced activation of the complement system. Although small dose of PsTX-T induced acute tubular necrosis in rats resembling pathology seen in patients, kidneys displayed glomerular injury with glomerular endothelial damage, thrombus formation, mesangiolysis, and partial rupture of glomerular basement membrane, accompanied by severe tubular necrosis at 24 hours after administration of 0.03 mg of PsTX-T per animal, similar to the glomerular findings typical of severe hemolytic uremic syndrome. The early stage injury was accompanied by specific PsTX-T binding, massive complement C3b, and membrane attack complex deposition in glomeruli in the regions of injury and decreased glomerular expression of complement regulators. A pathogenic role for complement was confirmed by demonstrating that systemic complement inhibition reduced renal injury. The isolated nephrotoxic component, a 115-kd protein toxin (PsTX-115), was shown to cause identical renal pathology. The demonstration that PsTX-T and PsTX-115 were highly nephrotoxic acting via induction of complement activation suggests that inhibition of complement might be used to prevent acute renal damage following envenomation by P. semoni.

Development of Immunoglobulin A Nephropathy- Like Disease in β-1,4-Galactosyltransferase-I-Deficient Mice

Beta4 galactosylation of glycoproteins plays important roles in protein conformation, stability, transport, and clearance from the circulation. Recent studies have revealed that aberrant glycosylation causes various human diseases. Here we report that mice lacking beta-1,4-galactosyltransferase (beta4GalT)-I, which transfers galactose to the terminal N-acetylglucosamine of N- and O-linked glycans in a beta-1,4 linkage, spontaneously developed human immunoglobulin A nephropathy (IgAN)-like glomerular lesions with IgA deposition and expanded mesangial matrix. beta4GalT-I-deficient mice also showed high serum IgA levels with increased polymeric forms as in human IgAN. IgAN is the most common form of glomerulonephritis, and a significant proportion of patients progress to renal failure. However, pathological molecular mechanisms of IgAN are poorly understood. In humans, abnormal character of serum IgA, especially serum IgA1 with aberrant galactosylation and sialylation of O-glycans in its hinge region is thought to contribute to the pathogenesis of IgAN. Mouse IgA has N-glycans but not O-glycans, and beta4-galactosylation and sialylation of the N-glycans on the serum IgA from beta4GalT-I-deficient mice was completely absent. This is the first report demonstrating that genetic remodeling of protein glycosylation causes IgAN. We propose that carbohydrates of serum IgA are involved in the development of IgAN, whether the carbohydrates are O-glycans or N-glycans.

The sialoadhesin (CD169) expressing a macrophage subset in human proliferative glomerulonephritis

Sialoadhesin (Sn; CD169) is a lectin-like receptor whose expression is restricted to subsets of tissue and inflammatory macrophages. We have previously identified accumulation of Sn+ macrophages as an important marker of disease progression versus remission in rat mesangial proliferative nephritis. The current study examined the significance of Sn+ macrophages in human proliferative glomerulonephritis. Frozen kidney sections from normal adult human kidney (n = 4) and pediatric nephropathy (n = 40) were stained for total macrophages (CD68+ cells), Sn+ macrophages, CD3+ T-cells and collagen type I by immunofluorescence. Leukocyte infiltration and the severity of glomerular lesions and interstitial damage were scored. A second protocol biopsy was performed in 27 cases and clinical and biopsy-based data obtained. Sn+ macrophages were absent from glomeruli in normal adult human kidney and in thin basement membrane disease (n = 4), but were detected in 4 of 9 cases of purpura nephritis; 7 of 17 IgA nephropathy; 5 of 5 membranoproliferative glomerulonephritis, and 5 of 5 lupus nephritis. Sn+ macrophages were localized in areas of focal glomerular and interstitial damage. Two-colour immunostaining confirmed that Sn+ cells are a subset of total CD68+ macrophages. The number of glomerular Sn+ macrophages correlated with the degree of proteinuria and glomerular lesions (r = 0.44, P = 0.0045 and r = 0.82, P<0.0001; respectively), while interstitial Sn+ macrophages correlated with the degree of proteinuria and interstitial damage (r = 0.59, P<0.0001 and r = 0.75, P<0.0001; respectively). Combined immunostaining revealed that interstitial Sn+ macrophages and CD3+ T-cells co-localized in areas of tubulointerstitial damage with increased type I collagen deposition. There was significant correlation between the number of interstitial Sn+ macrophages and CD3+ T-cells (r = 0.74, P<0.0001). Most patients responded to a 2 year period of glucocorticoid therapy with a reduction in proteinuria and glomerular lesions and this correlated with the reduction in the number of glomerular Sn+ macrophages. This study has identified Sn+ cells as a macrophage subset whose accumulation in the kidney correlates with proteinuria and histologic damage. These results, together with recent findings from animal studies, suggest that Sn+ macrophages may play an important role in progressive renal disease.

Podocan, a Novel Small Leucine-rich Repeat Protein Expressed in the Sclerotic Glomerular Lesion of Experimental HIV-associated Nephropathy

Growing evidence suggests that human immunodeficiency virus (HIV)-1 infection of podocytes plays a central role in the glomerular disease of HIV-associated nephropathy (HIVAN). As an approach to identify host genes involved in the pathogenesis of the sclerotic glomerular lesion in HIVAN, representational difference analysis of cDNA was used to identify differentially expressed genes in HIV-1 transgenic and nontransgenic podocytes. We isolated a novel member of the small leucine-rich repeat (SLR) protein family, podocan, that is expressed at high levels in the HIV-1 transgenic podocytes. In normal embryonic kidney, a 3.2-kb podocan transcript was detected at low levels, and expression increased dramatically within 24 h following birth. Expression of a 2.3-kb transcript became evident after birth and gradually increased to 50% of the total podocan RNA in the mature kidney. Phylogenetically, podocan represents a new class in the SLR protein gene family, an expanding protein family sharing homology with the small leucine-rich repeat proteoglycans. The 3.2-kb transcript encodes a predicted 611-amino acid secretory protein with 20 leucine-rich repeats, a unique N-terminal cysteine-rich cluster pattern and a highly acidic C-terminal domain. In situ hybridization of normal kidney revealed podocan mRNA expression in podocytes and likely vascular endothelial cells within the glomerulus. The immunohistochemical staining pattern of podocan protein in normal kidney glomeruli was consistent with that of the glomerular basement membrane, and staining was markedly increased in sclerotic glomerular lesions in the transgenic HIVAN model. Thus, podocan defines a new class within the SLR protein family and is a previously unrecognized component of the sclerotic glomerular lesion that develops in the course of experimental HIVAN.

Pattern of prepro-endothelin-1 expression revealed by reporter-gene activity in kidneys of erythropoietin-overexpressing mice.

Transgenic overexpression of erythropoietin (Epo) in mice increases haematocrit to a mean of 80% in adult mice, leading to an increase in blood viscosity and volume. As a consequence, renal tissue endothelin-1 (ET-1) concentrations are significantly elevated in erythropoietin-overexpressing (Epo+) mice (mean+/-S.E.M; Epo+, 798+/-71; Epo-, 400+/-25 pg/g tissue; P<0.01). To investigate the pattern of expression of the primary translation product of the ET-1 gene, prepro-ET-1, in kidneys of (Epo+) mice, we generated crossbred mice overexpressing the human EPO gene with mice carrying a reporter gene construct expressing the LacZ gene under the control of the human prepro-ET-1 promotor sequence (LacZ+/Epo+). For comparison, we generated (LacZ+/Epo-) mice from the same strains. After Bluo-Gal staining of frozen kidney sections (n=10 in each group), intracellular blue precipitates as indicators of prepro-ET-1 promotor activity were detectable in tubular and vascular endothelium and glomerular cells in (LacZ+/Epo-) as well as (LacZ+/Epo+) mice. Comparison of the amount of blue precipitates by semiquantitative scoring showed a significant increase in reporter gene activity in tubular epithelium of (LacZ+/Epo+) mice (mean+/-S.E.M.; LacZ+/Epo+, 1.64+/-0.18; LacZ+/Epo-, 1.00+/-0.19; P<0.05). Reporter gene activity was not significantly elevated in epithelium of small intrarenal arteries of (LacZ+/Epo+) mice (mean+/-S.E.M.; LacZ+/Epo+, 0.86+/-0.14; LacZ+/Epo-, 0.38+/-0.21; P=0.08) and was similar in glomerular cells (mean+/-S.E.M.; LacZ+/Epo+, 1.28+/-0.16; LacZ+/Epo-, 1.14+/-0.21; P=0.6). The main source of elevated ET-1 tissue concentration in kidneys of (Epo+) mice therefore seems more likely to be tubular than vascular endothelium or glomerular cells.

Differential tissue targeting and pathogenesis of verotoxins 1 and 2 in the mouse animal model

Both verotoxin (VT)1 and VT2 share the same receptor, globotriaosyl ceramide (Gb(3)). Although VT1 is slightly more cytotoxic in vitro and binds Gb(3) with higher affinity, VT2 is more toxic in mice and may be associated with greater pathology in human infections. In this study we have compared the biodistribution of iodine 125 ((125)I)-VT1 and (125)I-VT2 versus pathology in the mouse. (125)I-VT1 whole-body autoradiography defined the tissues targeted. VT1 and VT2 tissue distribution, clearance, and tissue binding sites were compared. The effect of a soluble receptor analogue, adamantylGb(3), on VT2/Gb3 binding and in vivo pathology was assessed. (125)I-VT1 autoradiography identified the lungs and nasal turbinates as major, previously unrecognized, targets, while kidney cortex and the bone marrow of the spine, long bones, and ribs were also significant targets. VT2 did not target the lung, but accumulated in the kidney to a greater extent than VT1. The serum half-life of VT1 was 2.7 minutes with 90% clearance at 5 minutes, while that of VT2 was 3.9 minutes with only 40% clearance at 5 minutes. The extensive binding of VT1, but not VT2, within the lung correlated with induced lung disease. Extensive hemorrhage into alveoli, edema, alveolitis and neutrophil margination was seen only after VT1 treatment. VT1 targeted lung capillary endothelial cells. Identical tissue binding sites (subsets of proximal/distal tubules and collecting ducts) for VT1 and VT2 were detected by toxin overlay of serial frozen kidney sections. Glucosuria was found to be a new marker of VT1- and VT2-induced renal pathology and positive predictor of outcome in the mouse, consistent with VT-staining of proximal tubules. Lung Gb3 migrated on thin-layer chromatography (TLC) faster than kidney Gb(3), suggesting a different lipid composition. AdamantylGb(3), a soluble Gb(3) analogue, competed effectively for Gb3 binding by VT1 and VT2 in vitro. However, the effect in the mouse model (only measured against VT2, due to the lower LD(50), a concentration required for 50% lethality) was to increase, rather than reduce, pathology and further reduce the VT2 serum clearance rate. Additional renal pathology was seen in VT2 + adamantylGb(3)-treated mice. The lung is a preferential (Gb(3)) "sink" for VT1, which explains the relatively slower clearance of VT2 and subsequent increased VT2 renal targeting and VT2 mortality in this animal model.

Endothelin and Endothelin A/B Receptors Are Increased after Ischaemic Acute Renal Failure

Background/Aims: Endothelin (ET) has been implicated as an indirect mediator of injury following acute renal ischaemia (ARI). The purpose of this study was to localize and quantitate ET and ET_A and ET_B receptors following ARI. Methods: A model of ARI, well characterized previously, was produced by 45 min occlusion of the renal pedicle of unilaterally nephrectomized female Sprague-Dawley rats. Animals were sacrificed 1, 2, 4, 8, 16, 32 and 64 days after ischaemia (n = 6). Corresponding control groups with unilateral nephrectomy but no ischaemia were sacrificed after 0, 8 and 64 days. Immunohistochemistry for ET-1, -2 and -3 was performed. Tissue ET levels were calculated by RIA (femtomoles per kidney). Receptor ligand binding studies for ET_A and ET_B receptors were performed by autoradiography on frozen kidney sections and quantitated by densitometry (relative optical density per square millimetre). Results: The concentration of tissue ET increased from 24 h after ischaemia and remained significantly increased for the duration of the study, reaching a maximum at 8 days. There was a small increase in the non-ischaemic 8-day control group, but this returned to basal levels by day 64. The increase in tissue ET 8 days after ischaemia was localized by immunohistochemistry to renal medullary interstitial cells, damaged tubules at the corticomedullary junction and peritubular capillaries surrounding these damaged tubules. Increases in cortical ET_A and ET_B receptors were evident 24 h after ischaemia and were maximal 8 days after ischaemia, before returning to basal levels at 16 days. After a small increase 24 h after ischaemia, medullary ET_A receptors decreased on day 4 before returning to basal levels on day 8 after ischaemia. Medullary ET_B receptors, however, decreased on day 4 after ischaemia and remained low throughout the duration of the study. Conclusion: The previously reported amelioration of pathological changes resulting from the use of ET receptor antagonists after ARI may be related to the quantitative and qualitative changes in tissue ET and ET receptors observed in this study.

CD28 knockout mice as a useful clue to examine the pathogenesis of chronic graft-versus-host reaction

CD28 knockout mice as a useful clue to examine the pathogenesis of chronic graft-versus-host reaction.BackgroundInjection of BALB/c or DBA/2 spleen cells into F1 C57BL/6 (B6) hybrids induces a graft-versus-host reaction (GVHR) of a chronic stimulatory type that results in clinical and pathologic manifestations that resemble the human systemic lupus erythematosus (SLE). The aim of the present study was to examine the role of a major T-cell costimulatory signal receptor, CD28, in the production of autoantibody and the development of an immune complex glomerulonephritis, which are common in SLE pathology.MethodsFor this purpose, CD28-deficient (CD28KO) mice were used for the source of donor lymphocytes. Chronic GVHR was induced by an injection of BALB/c or BALB. CD28KO donor cells into normal BCF1 mice. Serum titers of anti-dsDNA antibodies were assessed by enzyme-linked immunosorbent assay (ELISA) and major histocompatibility complex (MHC) class II antigen expression on B cells were tested by flow cytometry. In addition, depositions of immunoglobulin (Ig) were examined by direct immunofluorescence staining on frozen kidney sections.ResultsWhen (BALB/c × B6)F1 mice were injected with parental BALB/c lymphocytes, serum anti-dsDNA titer was significantly increased in association with nonspecific B-cell activation and IgG deposition in the glomerular basement membrane. In sharp contrast, none of these signs were observed in F1 mice, which were injected with CD28KO spleen cells.ConclusionThe CD28-mediated T-cell costimulatory pathway plays a pivotal role in the development of polyclonal B-cell activation, autoantibody production, and an immune complex glomerulonephritis. We propose that CD28KO mice are useful clues in examining the pathogenesis of experimental lupus nephritis. CD28 knockout mice as a useful clue to examine the pathogenesis of chronic graft-versus-host reaction. Injection of BALB/c or DBA/2 spleen cells into F1 C57BL/6 (B6) hybrids induces a graft-versus-host reaction (GVHR) of a chronic stimulatory type that results in clinical and pathologic manifestations that resemble the human systemic lupus erythematosus (SLE). The aim of the present study was to examine the role of a major T-cell costimulatory signal receptor, CD28, in the production of autoantibody and the development of an immune complex glomerulonephritis, which are common in SLE pathology. For this purpose, CD28-deficient (CD28KO) mice were used for the source of donor lymphocytes. Chronic GVHR was induced by an injection of BALB/c or BALB. CD28KO donor cells into normal BCF1 mice. Serum titers of anti-dsDNA antibodies were assessed by enzyme-linked immunosorbent assay (ELISA) and major histocompatibility complex (MHC) class II antigen expression on B cells were tested by flow cytometry. In addition, depositions of immunoglobulin (Ig) were examined by direct immunofluorescence staining on frozen kidney sections. When (BALB/c × B6)F1 mice were injected with parental BALB/c lymphocytes, serum anti-dsDNA titer was significantly increased in association with nonspecific B-cell activation and IgG deposition in the glomerular basement membrane. In sharp contrast, none of these signs were observed in F1 mice, which were injected with CD28KO spleen cells. The CD28-mediated T-cell costimulatory pathway plays a pivotal role in the development of polyclonal B-cell activation, autoantibody production, and an immune complex glomerulonephritis. We propose that CD28KO mice are useful clues in examining the pathogenesis of experimental lupus nephritis.

CD28 knockout mice as a useful clue to examine the pathogenesis of chronic graft-versus-host reaction

Injection of BALB/c or DBA/2 spleen cells into F1 C57BL/6 (B6) hybrids induces a graft-versus-host reaction (GVHR) of a chronic stimulatory type that results in clinical and pathologic manifestations that resemble the human systemic lupus erythematosus (SLE). The aim of the present study was to examine the role of a major T-cell costimulatory signal receptor, CD28, in the production of autoantibody and the development of an immune complex glomerulonephritis, which are common in SLE pathology. For this purpose, CD28-deficient (CD28KO) mice were used for the source of donor lymphocytes. Chronic GVHR was induced by an injection of BALB/c or BALB. CD28KO donor cells into normal BCF1 mice. Serum titers of anti-dsDNA antibodies were assessed by enzyme-linked immunosorbent assay (ELISA) and major histocompatibility complex (MHC) class II antigen expression on B cells were tested by flow cytometry. In addition, depositions of immunoglobulin (Ig) were examined by direct immunofluorescence staining on frozen kidney sections. When (BALB/c x B6)F1 mice were injected with parental BALB/c lymphocytes, serum anti-dsDNA titer was significantly increased in association with nonspecific B-cell activation and IgG deposition in the glomerular basement membrane. In sharp contrast, none of these signs were observed in F1 mice, which were injected with CD28KO spleen cells. The CD28-mediated T-cell costimulatory pathway plays a pivotal role in the development of polyclonal B-cell activation, autoantibody production, and an immune complex glomerulonephritis. We propose that CD28KO mice are useful clues in examining the pathogenesis of experimental lupus nephritis.

Increased expression of cytoplasmic tail‐containing form of gp600/megalin in active Heymann nephritis

Active Heymann nephritis of rat, an autoimmune glomerular disease, is a model of human membranous glomerulonephropathy. The major target autoantigen of this disease is a 600 kD renal glycoprotein, variously named gp600/gp330/megalin/LRP2. It has been shown previously that there is a marked increase in the gp600/megalin mRNA level in glomeruli of rats with this disease. A rabbit antibody was prepared against a peptide located at the very end of the cytoplasmic tail of rat gp600/megalin. Affinity purified anti-peptide antibody was monospecific and reacted only with gp600/megalin on the immunoblots of total kidney proteins. Immunofluorescence microscopy on frozen kidney sections probed with this antibody revealed the presence of the cytoplasmic tail-containing form of gp600/megalin in both proximal tubules and glomeruli. Expression in glomeruli of normal kidneys was less than in tubules but was clearly detectable. In active Heymann nephritis kidneys, expression of this form was increased most remarkably in glomeruli. These results, showing increased expression of the cytoplasmic tail-containing form in active Heymann nephritis glomeruli, contradict previous reports of its absence. This is the first study to show that there is increased expression of the cytoplasmic tail-containing form of gp600/megalin in the glomeruli of rats with this disease and its role in active Heymann nephritis is discussed. Copyright © 2000 John Wiley & Sons, Ltd.

Increased expression of cytoplasmic tail-containing form of gp600/megalin in active Heymann nephritis.

Active Heymann nephritis of rat, an autoimmune glomerular disease, is a model of human membranous glomerulonephropathy. The major target autoantigen of this disease is a 600 kD renal glycoprotein, variously named gp600/gp330/megalin/LRP2. It has been shown previously that there is a marked increase in the gp600/megalin mRNA level in glomeruli of rats with this disease. A rabbit antibody was prepared against a peptide located at the very end of the cytoplasmic tail of rat gp600/megalin. Affinity purified anti-peptide antibody was monospecific and reacted only with gp600/megalin on the immunoblots of total kidney proteins. Immunofluorescence microscopy on frozen kidney sections probed with this antibody revealed the presence of the cytoplasmic tail-containing form of gp600/megalin in both proximal tubules and glomeruli. Expression in glomeruli of normal kidneys was less than in tubules but was clearly detectable. In active Heymann nephritis kidneys, expression of this form was increased most remarkably in glomeruli. These results, showing increased expression of the cytoplasmic tail-containing form in active Heymann nephritis glomeruli, contradict previous reports of its absence. This is the first study to show that there is increased expression of the cytoplasmic tail-containing form of gp600/megalin in the glomeruli of rats with this disease and its role in active Heymann nephritis is discussed.

Pentoxifylline attenuates experimental mesangial proliferative glomerulonephritis

Accumulation of glomerular macrophages, proliferation of mesangial cells (MCs), and deposition of extracellular matrix proteins are pathobiological hallmarks of glomerulonephritis. We previously reported that a clinically available nonselective inhibitor of cyclic 3',5'-nucleotide phosphodiesterase, pentoxifylline (PTX), inhibits proliferation of cultured rat MCs, as well as collagen production by these cells. In this study, we investigated the in vivo effects of PTX on rat anti-Thy1 disease, a model of mesangial proliferative nephritis. Anti-Thy1 nephritis was induced in Sprague-Dawley rats by injecting mouse anti-rat Thy1 antibodies intravenously. Nephritic rats were randomly assigned to receive PTX (0.1 g/kg/day) or vehicle (phosphate-buffered saline) and were sacrificed at various time points. Paraffin kidney sections were stained with hematoxylin and periodic acid-Schiff reagents for glomerular histology. Frozen kidney sections were stained by monoclonal antibodies against proliferating cell nuclear antigen, ED-1, and alpha-smooth muscle actin and were visualized by color development from a horseradish peroxidase reaction. Monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), and various extracellular matrix mRNAs were analyzed by Northern blotting. Urine protein concentrations were determined by Lowry's method. Nephritic rats treated with PTX excreted less urinary protein on day 5 of nephritis than vehicle-treated nephritic rats. In periodic acid-Schiff-stained kidneys from PTX-treated nephritic rats, there was attenuation of both glomerular cellularity and glomerular sclerosis compared with vehicle-treated nephritic rats. PTX decreased the augmented glomerular mRNA levels of MCP-1 and ICAM-1 at two hours and on day 1 of nephritis. Immunoreactive staining showed that PTX reduced the number of proliferating glomerular macrophages on days 1, 2, and 3, but not at two hours of nephritis, compared with vehicle-treated nephritic rats. On day 5, PTX decreased the number of activated proliferating MCs and attenuated the glomerular mRNA levels of type I (alpha1), type III (alpha1), and type IV (alpha1) collagen and fibronectin compared with vehicle-treated nephritic rats. The administration of PTX to rats with anti-Thy1 disease reduces accumulation and proliferation of glomerular macrophages, attenuates proteinuria, suppresses activation and proliferation of MCs, and ameliorates glomerular sclerosis. These results suggest that PTX may have a suppressive effect in acute phases or relapses of mesangial proliferative glomerulonephritis.