Abdalla Rifai

Phenotypic Characterization of Cytokine Expression in Patients With IgA Nephropathy

To identify the cytokines that play a relevant role in the pathogenesis of IgA nephropathy, we analyzed and compared the gene expression of proinflammatory cytokines, immuno-regulatory cytokines, and growth factors in peripheral blood mononuclear cells (PBMC). Expression of IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IFN-γ, TGF-β, TNF-α, and PDGF was examined in 28 patients with IgA nephropathy (IgAN), 20 patients with non-IgA mesangial proliferative glomerulo-nephritis (mesPGN), and 19 healthy controls. Compared with healthy controls, a significant number of IgAN and mesPGN patients showed increased expression of IL-1β, IL-4, IL-10, IL-12, and IFN-γ. The cytokine profile of renal tissue of 10 IgAN and 5 mesPGN biopsies was simultaneously analyzed and compared with that of PBMC. The proinflammatory IL-1α and growth factor PDGF-B were expressed more in renal tissues than in PBMC. Furthermore, the renal profile of IL-α, IFN-γ, and TNF-α expression was associated with the expression of IFN-γ PBMC. The serum level of IFN-γ of IgAN correlated significantly (P = 0.0003) with that of IL-12, suggesting a potential role for cross-stimulation. More importantly, expression of IFN-γ in PBMC and the elevated serum level correlated with the decline in glomerular filtration rate (P = 0.0012) and severity of renal histopathologic grade. To elucidate the role of leukocytes in renal cytokine expression, surface markers of T cells (CD3), monocytes (CD14), natural killer cells (CD16), and B cells (CD19) were also examined in renal tissues. The prominent renal expression of CD3, CD14, and CD16 implicates the leukocytes as the major source of proinflammatory cytokines in IgAN. Collectively, these findings indicate that IFN-γ plays a prominent role in an interactive network of cytokines that contribute to the pathogenesis and progression of IgA nephropathy.

Characterization and evaluation of detoxification functions of a nontumorigenic immortalized porcine hepatocyte cell line (HepLiu).

Primary porcine hepatocytes (PPH) are currently used in research and therapeutic applications as the biological component of extracorporeal liver assist devices to overcome the shortage of human hepatocytes. However, their finite life span and typically rapid loss of functions limit their utility. An immortalized, nontumorigenic, highly differentiated porcine hepatocyte cell line was developed in our laboratory to resolve these disadvantages. PPH were transfected with simian virus 40 (SV40) T antigen under the control of the SV40 early promoter. From the established 69 clones, 23 clones displaying hepatocyte-like morphology were screened for diazepam metabolism. One clone, HepLiu D63, has been maintained in culture for > 2 years, through more than 60 passages and 240 divisions. Albumin protein, present in early passages, was lost at later passages, but albumin transcript still was detectable in later passages. Carbamoyl phosphate synthetase, a gateway enzyme of the urea cycle, was consistently detectable in HepLiu cells. Cytokeratin 18, a characteristic marker of primary hepatocytes, was detected by both immunofluorescent staining and Western blot in HepLiu cells. Furthermore, maintenance of P450 functions in HepLiu cells was evidenced by diazepam and 7-ethoxycoumarin metabolites measured by HPLC. Phase II conjugative function was measured as acetaminophen glucuronidation. P450 dealkylase was demonstrated microscopically by the conversion of a nonfluorescent substrate to a fluorescent product. Both Northern blot analysis and immunofluorescent staining showed SV40 T antigen expression in the nuclei of HepLiu cells. No tumor formation occurred when HepLiu cells were injected into severe combined immunodeficient (SCID) mice nor was the TA1 (a tumor marker) mRNA expressed, even in later passages. This immortalized, nontumorigenic, highly functional cell line may provide a valuable tool for drug/toxicological studies, liver biologic regulation studies, and artificial liver support systems.

Increase of HLA-DR-positive natural killer cells in peripheral blood from patients with IgA nephropathy☆

Previous in vivo and in vitro studies have presented various abnormalities of cellular immunity in patients with IgA nephropathy (IgAN). In the present study, we described increased expression of HLA-DR antigens on peripheral natural killer cells (NK cells) in relation to altered cytokine interactions. The numbers of HLA-DR expressing NK cells were enumerated by two-color flow cytometry and found to be significantly increased in patients with IgAN. Peripheral blood mononuclear cells were then fractionated into pure NK cells by a magnetic cell-sorting system and analyzed concerning expression of messages of interleukin (IL)-2, IL-4, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). Among the four cytokines, only the IFN-gamma message was significantly increased in patients NK cells. Furthermore, intensity of the IFN-gamma message in NK cells showed positive correlation with the percentage of HLA-DR-positive NK cells from the same patient. Then we assayed serum levels of IL-2, IL-12, and IFN-gamma by enzyme-linked immunosorbent assay (ELISA) and the levels of IL-12 and IFN-gamma showed positive correlations with HLA-DR expression on NK cells. Creatinine clearance of the patients was reevaluated 36 months later, and patients with high HLA-DR on NK cells tended to show faster deterioration of renal function than patients with lower HLA-Dr expression. On the basis of these findings, we suggested that HLA-DR-positive NK cells in patients with IgAN form an activated population that produces IFN-gamma, and this unique cell population may be maintained by multiple factors and be involved in the development and progression of IgAN.

Characteristics of nephritogenic IgA immune complexes.

Studies were undertaken to elucidate the characteristics of IgA immune complexes responsible for glomerular deposition and complement activation. Monomeric IgA antidinitrophenyl (DNP) and the antigen DNP-Ficoll tended to form small soluble complexes that circulated in the blood without localizing in glomeruli of experimental animals. In contrast, polymeric IgA formed intermediate and large-sized complexes that caused glomerular immune deposits. Although covalently cross-linked large-sized IgA oligomers deposited in the glomeruli, they failed to induce concomitant deposition of complement components. Only the presence of a complement-activating antigen endowed the glomerular IgA immune deposits with the ability to activate complement. Thus, the size of the IgA complexes plays a primary role in initiating glomerular localization, and the nature of the antigen causes a secondary phase of inflammation.

IgA nephropathy and mesangial cell proliferation: shared global gene expression profiles

SUMMARY: It is well established that mesangial cell proliferation plays a major role in glomerular injury and progressive renal injury. The expression of a number of different genes has been reported in proliferative mesangial cells in culture. However, the relevance of these genes to renal injury in general and IgA nephropathy (IgAN) remains to be established. Assessment of gene activity on a global genome‐wide scale is a fundamental and newly developed molecular strategy to expand the scope of clinical investigation from a single gene to studying all genes at once in a systematic pattern. Capitalizing on the recently developed methodology of high cDNA array hybridization, the simultaneous expression of thousands of genes in primary human proliferating mesangial cells was monitored and compared with renal tissue of IgAN. Complex [α‐33P]‐labelled cDNA targets were prepared from cultured mesangial cells, remnant tissue from five IgAN renal biopsies and four nephrectomies (controls). Each target was hybridized to a high‐density array of 18u2003326 paired target genes. The radioactive hybridization signals were analysed by phosphorimager. Approximately 8212u2003±u2003530 different gene transcripts were detected per target. Close to 5% (386u2003±u200390 genes) were full‐length mRNA human transcripts (HT) and the remainder were expressed sequence tags (EST). Using a relational database, electronic subtraction was performed and matching was carried out to allow identification of 203 HT with shared expression in proliferative mesangial cells and IgAN renal biopsies. In addition hierarchical clustering analysis was performed on the HT of IgAN and controls to establish differential expression profiles of mesangial HT in IgAN and controls. Collectively the presented data constitutes a preliminary renal bioinformatics database of the transcriptional profiles in IgAN. More importantly, the information may help to speed up the discovery of genes underlying human IgAN.

Profiling the IgA nephropathy renal transcriptome: analysis by complementary DNA array hybridization

SUMMARY: A comprehensive profile of genes expressed at the mRNA level (transcriptome) in human renal tissue is important for elucidating the pathogenesis and treatment of IgA nephropathy (IgAN). The recent development of complementary DNA (cDNA) array hybridization allows the parallel monitoring of thousands of genes expressed in renal tissue. High‐density cDNA containing 18u2003326 paired unique human cDNA gene probes were used to quantitatively analyse the expression of genes in renal biopsies of IgAN and controls. Computational analysis was used to cluster genes according to similarity in pattern of gene expression. Through relational database analysis, we determined 1901 genes that were commonly expressed in four selected IgAN renal biopsies and four controls. Further analysis of the expressed genes by self‐organizing maps and hierarchical clustering revealed a genomic profile unique to severely affected IgAN patients. These findings represent a comprehensive preliminary molecular index of genes transcribed in IgAN. More importantly, this molecular approach may accelerate the discovery of pathogenic mechanisms underlying the most common form of glomerulonephritis worldwide.

Immunopathogenesis of experimental IgA nephropathy

ConclusionsThe ultimate purpose of this review of experimental IgA nephropathy has been to identify the important immunopathogenetic factors that need to be elucidated en route to clear understanding of clinical IgA nephropathy. Summarizing from the discussion above, key experimental findings include the following.All the experimental models propound glomerular IgA-IC deposits as an end product of IC formation. The molecular form of IgA plays an important role in causing IC formation. Monomer IgA can only form small-sized complexes without any propensity for glomerular deposition. In contrast, polymeric IgA can form large-sized complexes that get entrapped in the mesangium. Free antigenic determinants in the deposited polymeric IgA complexes may serve as a binding site for circulating monomer IgA. Hence IgA-IC deposits may represent a continuous process of entrapped circulating macromolecules and in situ formed complexes.The liver has a major responsibility of maintaining the circulation free of IgA-IC. Under normal conditions, IgA-specific receptor on sinusoidal cells removes rapidly large-sized complexes. Hepatobiliary dysfunction or saturation of the elimination process may induce or contribute to glomerular IgA-IC deposition. Exacerbating factors under these conditions may also include heightened IgA immune response to alimentary and microbial antigens due to celiac disease or alcohol consumption.Macromolecules of purified human or murine IgA, free in solution or insolublized as renal deposits, lack the ability to activate the complement system. Glomerular C3 deposits that parallel the IgA pattern are due to complement activation by the antigen component of the IgA-IC deposits. Experimental and clinical evidence does not support the assumption that the complement system is essential for renal injury.The search for the inductor of tissue injury in the glomerular IgA-IC deposits revealed the antigen as the main proponent of damage. Comparative and correlative studies demonstrated the antigen as an elicitor of mesangial cell proliferation, inflammatory cell infiltration, fibrin deposition, and proteinuria production. This inflammatory process may also culminate from a cascade of engendered cytokines and eicosanoids.Although the extrapolation of experimental findings to IgA nephropathy is still in a developmental stage, astonishing progress has been made. In some instances, experimental investigations blazed the way for human studies that mutually reciprocated with seminal clinical observations providing essential clues and guides for the research to follow. Comparative analysis of the clinical and experimental literature on IgA nephropathy reveals parallel and complementary tracks that are converging on the preeminent objective of understanding and treatment of human renal disease.

Pathogenesis of IgA Immune Complex-Mediated Glomerulonephritis

In a period of less than ten years, complementary experimental and clinical observations have contributed greatly to our understanding of various aspects of IgA immune complexes and their pathogenetic potential in IgA nephropathy. The primary mechanism of glomerular IgA deposition has been discussed in the light of experimental data. These findings indicate that the molecular form of IgA is critical for the formation of large- and intermediate-sized complexes that result in glomerular deposition. Glomerular IgA deposits, however, appear to develop in a continuum of preformed polymeric IgA complexes and in situ formed monomeric IgA complexes. Removal of IgA immune complexes from the circulation is mediated by the liver, with no evidence of any removal dysfunction in IgA nephropathy patients. IgA macromolecules are unable to activate the complement pathway in vitro or as a glomerular deposit. Complement activation, however, is shown to be an antigen-mediated process. Participation of the complement system in induction of glomerular injury associated with IgA nephropathy needs further investigation. Experimental evidence supports the hypothesis that in IgA immune complex-mediated nephropathy, the repertoire of IgA immune complexes that induce mesangial proliferation and glomerular sclerosis consists of IgA-bound nephritogenic antigens which determine the extent and outcome of glomerular injury.