Dimethyl fumarate decreases short-term but not long-term inflammation in a focal EAE model of neuroinflammation.

BACKGROUND: Dimethyl fumarate (DMF) is an oral immunomodulatory drug used in the treatment of autoimmune diseases. Here, we sought to study whether the effect of DMF can be detected using positron emission tomography (PET) targeting the 18-kDa translocator protein (TSPO) in the focal delayed-type hypersensitivity rat model of multiple sclerosis (fDTH-EAE). The rats were treated orally twice daily from lesion activation (day 0) with either vehicle (tap water with 0.08% Methocel, 200 µL; control group n = 4 (3 after week four)) or 15 mg/kg DMF (n = 4) in 0.08% aqueous Methocel (200 µL) for 8 weeks. The animals were imaged by PET using the TSPO tracer [18F]GE-180 in weeks 0, 1, 2, 4, 8, and 18 following lesion activation, and the non-displaceable binding potential (BPND) was calculated. Immunohistochemical staining for Iba1, CD4, and CD8 was performed in week 18, and in separate cohorts of animals, following 2 or 4 weeks of treatment. RESULTS: Using the fDTH-EAE model, DMF reduced the [18F]GE-180 BPND in the DMF-treated animals compared to control animals after 1 week of treatment (two-tailed unpaired t test, p = 0.031), but not in weeks 2, 4, 8, or 18 when imaged in vivo by PET. Immunostaining for Iba1 showed that DMF had no effect on the perilesional volume or the core lesion volume after 2 or 4 weeks of treatment, or at 18 weeks. However, the optical density (OD) measurements of CD4+ staining showed reduced OD in the lesions of the treated rats. CONCLUSIONS: DMF reduced the microglial activation in the fDTH-EAE model after 1 week of treatment, as detected by PET imaging of the TSPO ligand [18F]GE-180. However, over an extended time course, reduced microglial activation was not observed using [18F]GE-180 or by immunohistochemistry for Iba1+ microglia/macrophages. Additionally, DMF did affect the infiltration of CD4+ and CD8+ T-lymphocytes at the fDTH-EAE lesion.

Basal and cold-induced fatty acid uptake of human brown adipose tissue is impaired in obesity.

Fatty acids (FA) are important substrates for brown adipose tissue (BAT) metabolism, however, it remains unclear whether there exists a difference in FA metabolism of BAT between lean and obese healthy humans. In this study we evaluated supraclavicular BAT fatty acid uptake (FAU) along with blood perfusion in lean and obese subjects during cold exposure and at room temperature using positron emission tomography (PET)/computed tomography (CT). Additionally, tissue samples were taken from supraclavicular region (typical BAT region) from a subset of subjects to evaluate histological presence of BAT. Non-shivering cold stress elevated FAU and perfusion of BAT in lean, but not in obese subjects. Lean subjects had greater FAU in BAT compared to obese subjects during cold exposure and interestingly also at room temperature. The higher BAT FAU was related to younger age and several indicators of superior systemic metabolic health. The subjects who manifested BAT histologically had several folds higher BAT FAU compared to subjects with no such histological manifestation. Together, obese subjects have less active tissue in supraclavicular region both in basal and cold-activated state and the FA metabolism of BAT is blunted in obesity.

Cessation of anti-VLA-4 therapy in a focal rat model of multiple sclerosis causes an increase in neuroinflammation.

BACKGROUND: Positron emission tomography (PET) can be used for in vivo evaluation of the pathology associated with multiple sclerosis. We investigated the use of longitudinal PET imaging and the 18-kDa translocator protein (TSPO) binding radioligand [18F]GE-180 to detect changes in a chronic multiple sclerosis-like focal delayed-type hypersensitivity experimental autoimmune encephalomyelitis (fDTH-EAE) rat model during and after anti-VLA-4 monoclonal antibody (mAb) treatment. Thirty days after lesion activation, fDTH-EAE rats were treated with the anti-VLA-4 mAb (n = 4) or a control mAb (n = 4; 5 mg/kg, every third day, subcutaneously) for 31 days. Animals were imaged with [18F]GE-180 on days 30, 44, 65, 86 and 142. Another group of animals (n = 4) was used for visualisation the microglia with Iba-1 at day 44 after a 2-week treatment period. RESULTS: After a 2-week treatment period on day 44, there was a declining trend (p = 0.067) in [18F]GE-180-binding in the anti-VLA-4 mAb-treated animals versus controls. However, cessation of treatment for 4 days after a 31-day treatment period increased [18F]GE-180 binding in animals treated with anti-VLA-4 mAb compared to the control group (p = 0.0003). There was no difference between the groups in TSPO binding by day 142. CONCLUSIONS: These results demonstrated that cessation of anti-VLA-4 mAb treatment for 4 days caused a transient rebound increase in neuroinflammation. This highlights the usefulness of serial TSPO imaging in the fDTH-EAE model to better understand the rebound phenomenon.

Recurrence of nephrotic syndrome after transplantation in CNF is due to autoantibodies to nephrin.

The novel gene NPHS1 is defective in the patients with congenital nephrotic syndrome of the Finnish type (CNF) leading to abnormal expression of the respective protein product nephrin in glomerular cells. CNF patients are treated with early nephrectomy and renal transplantation, but about 20% show recurrence of nephrotic syndrome (NS). We used indirect immunofluorescence microscopy and immunoblotting and an ELISA assay to search for circulating autoantibodies to nephrin, the protein defect in CNF patient kidneys. In serial serum samples gathered before and after recurrence of NS, we show an increased antibody titer to nephrin prior to the NS episode and a subsequent drop in antibody level after its successful treatment and reactivity of the high titer sera with glomeruli in indirect immunofluorescence microscopy as well. The results show that the transplantation treatment introduces a neoantigen inducing production of autoantibodies, which may be pathogenic for perturbation of the function of the glomerular filtration barrier.

Lipid peroxidation in human proteinuric disease.

BACKGROUND: While metabolically generated oxidants are produced locally in experimental glomerular diseases, little is still known of their significance and the respective scavenger systems in human glomerular diseases. METHODS: Here we studied kidneys from patients with congenital nephrotic syndrome of the Finnish type (CNF), a human model disease of isolated proteinuria. Expression of specific mRNAs for a major antioxidant system against lipoperoxidation [phospholipid hydroperoxide glutathione peroxidase (PHGPx)] and for mitochondrial proteins were studied in Northern blotting together with analysis of PHGPx in semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The respective proteins and lipoperoxide (LPO) adducts malonyldialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were analyzed in immunohistochemistry. RESULTS: PHGPx and the mitochondrially encoded subunits of cytochrome-c-oxidase were distinctly down-regulated within the glomeruli of CNF kidneys. These changes were confirmed in semiquantitative RT-PCR. Increases of lipoperoxidation products MDA and 4-HNE were constantly found in the glomeruli of CNF. In agreement with findings in CNF, similar results were obtained in biopsies from other human glomerular diseases. CONCLUSIONS: These findings suggest that local mitochondrial damage initiates LPO, which then causes deposition of the cytotoxic LPO products in glomeruli, as seen especially in CNF kidneys. Together with down-regulation of the local antioxidant protection, these may be important pathophysiologic mechanisms in human glomerular disease.

Interactions between mitochondrial proteins and lipid peroxidation products in the maintenance of the glomerular filtration barrier in the in vitro perfused kidney.

BACKGROUND: The fourth complex of the mitochondrial respiratory chain, cytochrome-c oxidase (CytC) consists of thirteen both mitochondrially and nuclearly encoded subunits, which are differently regulated in proteinuric kidneys. The effect of mitochondrial involvement on proteinuria is not known. METHODS: We set up an in vitro kidney perfusion model to study the direct effect of inhibitors of the mitochondrial respiratory chain, rotenone and antimycin A, on the glomerular filtration barrier by using immunohistochemistry and Northern blotting and quantitating the resulting proteinuria. RESULTS: Rapid onset of proteinuria and characteristic changes in CytC subunits were seen in the perfused kidneys. Urinary protein excretion increased significantly in the rotenone- and antimycin-A-treated groups during perfusion. Downregulation of CytC subunits I and IV was similarly found in the groups treated with rotenone and antimycin A, while increases in the lipid peroxidation (LPO) products malondialdehyde and 4-hydroxynonenal which reflect mitochondrial damage, were observed. CONCLUSIONS: These data show rapid changes in mitochondrial proteins and induction of proteinuria in response to exposure to mitochondrial inhibitors. Together with the concomitant increase in local LPO products, these results suggest that continuous maintenance of a proper energy balance is important to maintain the glomerular filtration barrier.

Nephrin in experimental glomerular disease.

BACKGROUND: The recently identified gene NPHS1 with its mutations causing congenital nephrotic syndrome of the Finnish type (CNF) is highly promising in providing new understanding of pathophysiology of proteinuria. Earlier we cloned a rat NPHS1 homologue, as well as characterized and raised antibodies to the respective protein product nephrin. METHODS: Changes in the expression levels of nephrin-specific mRNA in commonly used experimental models of proteinuria were examined using semiquantitative reverse transcription-polymerase chain reaction, immunofluorescence, and immunoelectron microscopy (IEM) of nephrin. RESULTS: Notably, a 40% down-regulation of the nephrin-specific mRNA of cortical kidney was seen already at day 3 after induction of the puromycin aminonucleoside nephrosis (PAN), while no major elevation of urinary protein secretion was seen at this stage. A further decrease of 80% of nephrin message was seen at the peak of proteinuria at day 10. A similar decrease of up to 70% from the basal levels was seen in mercuric chloride-treated rats. Changes in the protein expression paralleled those of the mRNA in indirect immunofluorescence. Interestingly, a remarkable plasmalemmal dislocation from the normal expression site at the interpodocyte filtration slits could be observed in IEM. CONCLUSIONS: Nephrin appears to be an important causative molecule of proteinuria and shows a remarkable redistribution from the filtration slits to the podocyte plasma membrane, especially in PAN.

Mitochondrial dysfunction in congenital nephrotic syndrome.

The molecular mechanisms maintaining the kidney glomerular filtration barrier remain poorly understood. Recent evidence suggests that mitochondrial dysfunction is a characteristic feature of kidney glomeruli in congenital nephrotic syndrome of the Finnish type (CNF). Here we searched for detailed functional evidence of mitochondrial lesion in CNF kidneys. We used histochemical and immunohistochemical methods, quantitative measurement of mitochondrial DNA, and superoxide production to characterize the mitochondrial function. The results unequivocally show down-regulation of mitochondria-encoded respiratory chain components, whereas the respective nuclearly encoded subunits were close to normal. These results give detailed evidence of distinct mitochondrial dysfunction and of the resulting abnormal production of reactive oxygen species in CNF and suggest a critical role for mitochondria in maintaining the glomerular permeability barrier.

Nephrin localizes at the podocyte filtration slit area and is characteristically spliced in the human kidney.

Defects in the newly reported gene NPHS1 in chromosome 19 cause the massive proteinuria of Finnish type congenital nephrotic syndrome (CNF). Together with its gene product, nephrin, NPHS1 is providing new understanding of the pathophysiological mechanisms of glomerular filtration. Here we show the characteristic splicing of NPHS1 mRNA in the normal and CNF kidneys and localize nephrin exclusively in the glomerulus and to the filtration slit area by light and immunoelectron microscopy. These results indicate that nephrin is a new protein of the interpodocyte filtration slit area with a profound role in the pathophysiology of the filtration barrier.

Cloning and expression of the rat nephrin homolog.

Despite of the increased availability of genetically modified mouse strains, the experimental models in the rat have provided the most widely employed and versatile models for the study of renal pathophysiology and functional genetics. The identification of the human gene mutated in the congenital nephrotic syndrome of the Finnish type (NPHS1) has recently been reported, and its protein product has been termed nephrin. Here we report the molecular cloning and characterization of rat nephrin cDNA. Rat nephrin cDNA has an open reading frame of 3705 bp, shows 82% sequence identity with human nephrin cDNA, and shows characteristic rat-specific splicing variants. The translated nucleotide sequence has 89% sequence identity at the amino acid level. The signal sequence, glycosylation, and cysteine localization patterns are nearly identical to those of human nephrin. As in the human, the rat nephrin transcript is expressed in a tissue-restricted pattern. Antipeptide antibodies raised to the intracellular nephrin-specific domain identified immunoreactivity exclusively within the rat kidney glomerulus by indirect immunofluorescence. Initial results with semiquantitative reverse transcriptase-polymerase chain reaction analysis showed a remarkable down-regulation of nephrin-specific mRNA in the puromycin nephrosis of the rat.

Altered gene expression and functions of mitochondria in human nephrotic syndrome.

The molecular basis of glomerular permselectivity remains largely unknown. The congenital nephrotic syndrome of the Finnish type (CNF) characterized by massive proteinuria already present but without extrarenal symptoms is a unique human disease model of pure proteinuria. In search of genes and pathophysiologic mechanisms associated with proteinuria, we used differential display-PCR to identify differences in gene expression between glomeruli from CNF and control kidneys. A distinctly underexpressed PCR product of the CNF kidneys showed over 98% identity with a mitochondrially encoded cytochrome c oxidase (COX I). Using a full-length COX I cDNA probe, we verified down-regulation of COX I mRNA to 1/4 of normal kidney values on Northern blots. In addition, transcripts of other mitochondrially encoded respiratory chain complexes showed a similar down-regulation whereas the respective nuclearly encoded complexes were expressed at comparable levels. Additional studies using histochemical, immunohistochemical, in situ hybridization, RT-PCR, and biochemical and electron microscopic methods all showed a mitochondrial involvement in the diseased kidneys but not in extrarenal blood vessels. As a secondary sign of mitochondrial dysfunction, excess lipid peroxidation products were found in glomerular structures in CNF samples. Our data suggest that mitochondrial dysfunction occurs in the kidneys of patients with CNF, with subsequent lipid peroxidation at the glomerular basement membrane. Our additional studies have revealed similar down-regulation of mitochondrial functions in experimental models of proteinuria. Thus, mitochondrial dysfunction may be a crucial pathophysiologic factor in this symptom.

Podocalyxin in rat platelets and megakaryocytes.

Podocalyxin is a membrane protein of rat podocytes and endothelial cells. It has not been described in other cell types, and no amino acid or DNA sequence data are available about it. Here we show that podocalyxin antigens are present in rat platelets and megakaryocytes. In resting platelets, the antigens are mainly intracellular but become surface exposed after thrombin stimulation, as shown by immunofluorescence and flow cytometry. By Western blotting, platelet podocalyxin has an apparent Mr of 140,000. Cytocentrifuge slides of rat bone marrow show that anti-podocalyxin antibodies recognize large polyploid cells also expressing CD62P, indicating that the cells are megakaryocytes. From a rat glomerular cDNA library we isolated a clone covering the carboxyl-terminal nucleotides of rat podocalyxin. Its putative transmembrane or intracellular domains are 100% or >93% identical, respectively, with the human and rabbit podocalyxin-like proteins. The truncated extracellular domain extends to include two of the four conserved cysteines shared by podocalyxin-like proteins. By Northern blotting, a 5.5-kb renal cortical transcript is seen. By in situ hybridization, cRNA probes recognize podocytes, endothelial cells, and megakaryocytes, and by reverse transcription polymerase chain reaction, platelets are shown to contain podocalyxin mRNA. Our results show that rat podocalyxin is a homologue of the previously cloned podocalyxin-like proteins and suggest that also in mammals podocalyxin has a role in hematopoiesis, as previously shown in the chicken.

mRNA differential display analysis of nephrotic kidney glomeruli.

BACKGROUND: Differential display RT-PCR (DDRT-PCR) is a new powerful technique for identification and characterization of altered gene expression in eukaryotic cells and tissues. We studied here changes in kidney glomerular gene expression in patients with congenital nephrotic syndrome of the Finnish type (CNF), an inherited kidney disease with heavy proteinuria already in utero. METHODS: Using the DDRT-PCR approach and isolated glomeruli from removed human kidneys, we compared the gene expression patterns of normal human and CNF glomeruli. Differential expression of candidate genes was verified by Northern blotting, and the corresponding PCR fragments were sequenced and compared to known sequences in databanks. RESULTS: We found several genes and sequence tags with altered expression in nephrotic glomeruli including fragments with close homologies to cytochrome c oxidase subunit I, integrin-linked kinase, insulin-like growth factor II receptor and eotaxin, and also clones resembling anchyrin and cadherin-like consensus sequences. CONCLUSION: All the sequences identified are of interest in respect to pathogenesis of proteinuria. Furthermore, this study reveals potentially new members to known gene families with tissue and cell type-specific expression.

Striatal presynaptic dopamine function in type 1 alcoholics measured with positron emission tomography.

Recent in vivo studies have shown low dopamine D2 receptor and dopamine transporter densities among late onset (type 1) alcoholics. We have now studied 6-[18F]-FDOPA (FDOPA) uptake in 10 type 1 alcoholics and eight matched controls to test the hypothesis that striatal presynaptic dopamine function is lower among alcoholics. Markedly low FDOPA uptake (Ki) was observed in the left caudate of two alcoholic patients, but the mean striatal uptake values of the patient group were higher than those of the control group. The greatest difference was observed in the mean FDOPA intake in the left putamen, which was 28% higher in the patient group (t = 3.00, P = 0.008, d.f. = 16, independent samples t-test), and in the right caudate (difference 36%, t = 2.87, P = 0.01). The elevated FDOPA uptake in putamen and caudate correlated with poor Wisconsin Card Sorting Test (WCST) performance (error %) among alcoholics (correlation coefficients from 0.49 to 0.56), which suggests that the magnitude of presynaptic dopamine function alteration correlates with the degree of disability to modify one's behavior. The results do not give support to the hypothesis of generally decreased striatal dopamine turnover in type 1 alcoholism, but on the contrary indicate an increased presynaptic dopamine function. This may represent a compensatory mechanism to low postsynaptic DA function. The low presynaptic DA function observed in the left caudate of two patients suggests that type 1 alcoholism may be a heterogeneous disorder.

Morphologic changes suggesting abnormal renal differentiation in congenital nephrotic syndrome.

Retrograde differentiation (or dedifferentiation) has recently been proposed as a pathogenetic mechanism involved also in various renal diseases. Here we studied whether evidence of these mechanisms can be found in the kidneys of patients with congenital nephrotic syndrome of the Finnish type (CNF). These patients show isolated massive proteinuria but no primary symptoms from any other organ systems. For the analysis we used antibody markers of early (fibronectin, stem cell factor, Wilms' tumor gene product, cytokeratin) and later (laminin, midgestation and kidney, heparin binding growth-associated molecule) stages of nephron differentiation as well as for apoptosis (acridine orange staining), rescue from apoptosis (anti-Bcl-2 antibodies) and cell proliferation (antibodies to proliferating cell nuclear antigen). In the peritubular spaces atypically organized areas were found which appeared positive with markers of low stages of differentiation, but neither abnormal cell proliferation nor activation of the apoptotic pathway could be detected. As morphologic signs of abnormal tissue organization, we found clusters of tightly compacted and large glomeruli corresponding to the size of two to three normal glomeruli. However, all individual glomerular cell compartments (mesangial, endothelial, visceral epithelial cells) appeared balanced in relative cell numbers. Together these results may indicate abnormal early mesenchymoepithelial tissue interaction leading to excessive and poorly organized formation of glomeruli. This could be causally related also to the serious functional immaturity of CNF kidneys presented as isolated proteinuria.

Relationship of serum leptin to total and truncal body fat.

1. In this study we investigated the relationship between serum leptin levels and body fat distribution in a random sample of women of widely ranging age and body mass index. Anthropometry and dual energy X-ray absorptiometry were used to measure body fat and its distribution. 2. Leptin levels (log transformed) were not significantly correlated with age, but were significantly positively correlated (P < 0.001) with most anthropometric measures except waist-to-hip circumference ratio. The strongest correlations were with total grams of body fat and percentage body fat (r = 0.68 and 0.76 respectively, P < 0.001). When corrected for percentage body fat the partial correlation coefficients for all other measures became non-significant. The correlation with truncal body fat fell significantly from 0.66 to -0.05 after correction, but the partial correlation with total body fat remained significant (P < 0.005) when grams of truncal fat were controlled for (r = 0.21). 3. These results indicate that the relationship of serum leptin to percentage body fat is the strongest, and that truncal body fat, although the most metabolically active, does not appear to have an independent association with serum leptin.

Expression of nine developmental stage-specific genes in human kidney and cultured renal cells.

BACKGROUND: Several genes transiently expressed during the maturation of the metanephrogenic mesenchyme have been reported in recent years while there is accumulating evidence of a reverted developmental pathway during tissue damage and loss of function. METHODS: Here we studied the expression of nine genes associated with kidney maturation from samples of normal human fetal, juvenile and adult kidneys and cultured glomerular cells using Northern blotting analysis. Subsequently, kidneys from patients with congenital nephrotic syndrome of the Finnish type (CNF), presenting with heavy proteinuria, and Wilms' tumor tissue were studied for the corresponding expression pattern for evidence of dedifferentiation/persistence of a fetal expression pattern. RESULTS: All the genes studied had their distinct expression pattern within the tissues and cells tested. No conclusive evidence of dedifferentiation could be obtained in the samples from CNF kidneys, whereas, as expected, the gene expression pattern of Wilms' tumor tissue was highly similar to that of fetal kidney tissue. CONCLUSION: Some genes thought to be only transiently expressed during kidney maturation were, however, constantly found in the samples from fetal to newborn and adult kidneys.

Mechanisms of proteinuria: vascular permeability factor in congenital nephrotic syndrome of the Finnish type.

Vascular permeability factor (VPF) is the most potent known mediator of vessel wall permeability. In the kidney, it is expressed preferentially in the glomerular visceral epithelial cells. The present study was designed to clarify the proposed role of VPF in diseases with increased glomerular permeability as here exemplified by the congenital nephrotic syndrome of the Finnish type (CNF). For this, we studied the expression levels and the sites of synthesis of VPF and its kinase-insert domain receptor (KDR) in kidneys of patients with CNF using Northern and in situ hybridization techniques and immunohistologic staining with anti-VPF antibody. In addition, we extended the study to include analysis of fetal kidney tissue and cultured glomerular cells of normal and CNF kidneys. In CNF and in normal kidneys VPF was localized in the visceral epithelial aspect of the glomeruli and in the collecting ducts, as also earlier described. A new finding was its localization also in the juxtaglomerular area. The VPF receptor KDR was found in glomeruli in the endothelial cells, but it was not detected in the peritubular capillaries. no consistent differences in the levels of VPF or KDR mRNAs or in their sites of production were seen in CNF and control samples. Also the distribution of VPF antigen in the CNF kidneys and normal kidneys was similar. Thus, we propose that VPF and KDR are not directly involved in the pathogenesis of the proteinuria in CNF.

Sphingolipid activator proteins in a human hereditary renal disease with deposition of disialogangliosides.

Congenital nephrotic syndrome of the Finnish type is a recessively inherited renal disease with glomerular deposits of the disialoganglioside O-acetyl-GD3. Sphingolipid activator proteins (saposins) stimulate the degradation of glycosphingolipids by lysosomal enzymes, and defects in saposins cause accumulation of substrate lipids in the affected tissues in lysosomal storage disease. Here we report a study of the role of saposins in the accumulation of O-acetyl-GD3 in kidneys of congenital nephrotic syndrome patients. At the mRNA level, the expression of saposin precursor in diseased kidneys appeared normal, and the nucleotide sequence analysis of cDNA clones did not reveal abnormalities in the prosaposin gene. Immunohistologically, saposins were localized mainly to the epithelial cells of the distal renal tubules or to the parietal epithelial cells of glomeruli. In the nephrotic syndrome kidneys, the staining pattern was highly granular and appeared mostly in the apical part of the epithelial lining, unlike the control kidneys. These results show that a major site of ganglioside metabolism is located in the distal nephron. Furthermore, these results suggest that saposins are not directly involved in the metabolism of the terminal sialic acids of disialogangliosides in the nephrotic syndrome kidneys.

Decrease of glomerular disialogangliosides in puromycin nephrosis of the rat.

Puromycin aminonucleoside nephrosis (PAN) is a model for human minimal change nephropathy induced in rats by injection of puromycin. In PAN, defective sialylation of a major sialoprotein of podocytes, podocalyxin, has been demonstrated and the consequent decrease of anionic charge suggested as a causative factor for increased glomerular permeability and proteinuria. Whether defective sialylation is a general feature of PAN affecting also glomerular glycosphingolipids is not known. We have shown that rat glomeruli are rich in disialogangliosides GD3 and O-acetyl GD3, the functions of which are not known. Here, we made a sequential analysis of the glomerular gangliosides, especially of GD3 and its O-acetyl derivative in acute PAN using immunohistochemical and biochemical techniques and compared the results with another rat model of glomerular disease, Heymann nephritis. The prominent immunohistochemical finding was the almost total disappearance of glomerular O-acetyl GD3 and a substantial decrease of its precursor GD3 peaking at 10 days after injection of puromycin. Segmental areas lacking these gangliosides remained in glomeruli still at 30 days after injection. The response was dose dependent. Semiquantitative analysis by thin layer chromatograms showed that O-acetyl GD3 was decreased by 41% already at 3 days and by 60% at 10 days after injection of puromycin. Also GD3, the immediate precursor of O-acetyl GD3, was decreased by 20 and 19%, respectively, at 3 and 10 days after injection. At 3 days after injection, overt proteinuria had not started. At these times, no other changes were observed in the glomerular gangliosides. The decrease of glomerular GD3 and O-acetyl GD3 indicates a decrease of GD3 synthase activity and perhaps of O-acetyltransferase activity in PAN nephrosis. As these changes preceded the overt proteinuria, they may have a causal relationship to it. In the glomeruli of Heymann nephritic rats, no similar changes were seen, suggesting that the sialylation defect is not due to proteinuria but is a consequence of targeted puromycin action on cells.