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Complement activation at a particular location is determined by the balance of activating and inhibitory proteins. Factor H is a key regulator of the alternative pathway of complement, and genetic or acquired impairments in Factor H are associated with glomerular injury. The human Factor H-related proteins (FHRs) comprise a family of five proteins that are structurally related to Factor H. Variations in the genes or expression levels of the FHRs are also associated with glomerular disease, although the mechanisms of glomerular protection/injury are incompletely understood. To explore the role of the FHRs on complement regulation/dysregulation in the kidney, we expressed and purified recombinant murine FHRs (FHRs A, B, C and E). These four distinct FHRs contain binding regions with high amino acid sequence homology to binding regions within Factor H, but we observed different interactions of the FHRs with Factor H binding ligands, including heparin and C3d. There was differential binding of the FHRs to the resident kidney cell types (mesangial, glomerular endothelial, podocytes, and tubular epithelial). All four FHRs caused complement dysregulation on kidney cell surfaces in vitro, although the magnitude of the effect differed among the FHRs and also varied among the different kidney cells. However, only FHR E caused glomerular complement dysregulation when injected in vivo but did not exacerbate injury when injected into mice with ischemic acute kidney injury, an alternative pathway-mediated model. Thus, our experiments demonstrate that the FHRs have unique, and likely context-dependent, effects on the different cell types within the kidney., (Copyright © 2022 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Properdin is the only known positive regulator of complement activation by stabilizing the alternative pathway convertase through C3 binding, thus prolonging its half-life. Recent in vitro studies suggest that properdin may act as a specific pattern recognition molecule. To better understand the role of properdin in vivo, we used an experimental model of acute anti-glomerular basement membrane disease with wild-type, C3- and properdin knockout mice. The model exhibited severe proteinuria, acute neutrophil infiltration and activation, classical and alternative pathway activation, and progressive glomerular deposition of properdin, C3 and C9. Although the acute renal injury was likely due to acute neutrophil activation, we found properdin deposition in C3-knockout mice that was not associated with IgG. Thus, properdin may deposit in injured tissues in vivo independent of its main ligand C3., (Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Severe ischemia reperfusion injury (IRI) results in rapid complement activation, acute kidney injury and progressive renal fibrosis. Little is known about the roles of the C5aR1 and C5aR2 complement receptors in IRI. In this study C5aR1-/- and C5aR2-/- mice were compared to the wild type in a renal IRI model leading to renal fibrosis. C5a receptor expression, kidney morphology, inflammation, and fibrosis were measured in different mouse strains one, seven and 21 days after IRI. Renal perfusion was evaluated by functional magnetic resonance imaging. Protein abundance and phosphorylation were assessed with high content antibody microarrays and Western blotting. C5aR1 and C5aR2 were increased in damaged tubuli and even more in infiltrating leukocytes after IRI in kidneys of wild-type mice. C5aR1-/- and C5aR2-/- animals developed less IRI-induced inflammation and showed better renal perfusion than wild-type mice following IRI. C5aR2-/- mice, in particular, had enhanced tubular and capillary regeneration with less renal fibrosis. Anti-inflammatory IL-10 and the survival/growth kinase AKT levels were especially high in kidneys of C5aR2-/- mice following IRI. LPS caused bone marrow-derived macrophages from C5aR2-/- mice to release IL-10 and to express the stress response enzyme heme oxygenase-1. Thus, C5aR1 and C5aR2 have overlapping actions in which the kidneys of C5aR2-/- mice regenerate better than those in C5aR1-/- mice following IRI. This is mediated, at least in part, by differential production of IL-10, heme oxygenase-1 and AKT., (Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Neutrophil extracellular traps (NETs) are auto-antigenic strands of extracellular DNA covered with myeloperoxidase (MPO) and proteinase3 (PR3) that can be a source for the formation of anti-neutrophil cytoplasmic autoantibodies (ANCAs). The presence of NETs was recently demonstrated in renal tissue of patients with ANCA-associated vasculitis (AAV). NET formation was enhanced in AAV, suggesting that MPO-ANCA could trigger NET formation, supporting a vicious circle placing NETs in the center of AAV pathogenesis. Here we investigated NET formation in 99 patients with AAV by a novel highly sensitive and automated assay. There was a significant excess of ex vivo NET formation in both MPO-ANCA- and PR3-ANCA-positive patients with AAV compared to healthy individuals. Excessive NET formation did not correlate with serum ANCA levels. Likewise, immunoglobulin G depletion had no effect on excessive NET formation in patients with AAV, indicating an ANCA-independent process. Next, we explored the relation of excessive NET formation to clinical disease in ten patients with AAV and showed that excessive NET formation was predominantly found during active disease, more so than during remission. Excessive NET formation was found in patients with AAV hospitalized for disease relapse but not during severe infection. Thus, excessive NET formation in AAV is independent of ANCA, and an excess of ex vivo NET formation was related to active clinical disease in patients with AAV and a marker of autoimmunity rather than infection., (Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Plasmacytoid dendritic cells (pDCs) are antigen presenting cells specialized in viral recognition through Toll-like receptor (TLR)7 and TLR9, and produce vast amounts of interferon alpha upon ligation of these TLRs. We had previously demonstrated a strong influx of pDCs in the tubulointerstitium of renal biopsies at the time of acute rejection. However, the role of human pDCs in mediating acute or chronic allograft rejection remains elusive. pDCs are thought to have a limited capacity to ingest apoptotic cells, critical for inducing CD4 + T cell activation via indirect antigen presentation and subsequent activation of antibody producing B cells. Here we tested whether the function of pDCs is affected by their presence within the graft. Maturation and interferon alpha production by pDCs was enhanced when cells were activated in the presence of viable HK2 renal epithelial cells. Importantly, soluble factors produced by cytomegalovirus-infected (primary) epithelial or endothelial cells enhanced pDC activation and induced their capacity to phagocytose apoptotic cells. Phagocytosis was not induced by free virus or soluble factors from non-infected cells. Activated pDCs showed an enhanced CD4 + and CD8 + T cell allostimulatory capacity as well as a potent indirect alloantigen presentation. Granulocyte Macrophage-Colony Stimulating Factor is one of the soluble factors produced by renal epithelial cells that, combined with TLR9 ligation, induced this functional capacity. Thus, pDCs present in the rejecting allograft can contribute to alloimmunity and potentially act as important orchestrators in the manifestation of acute and chronic rejection., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Dendritic cells are key players in renal allograft rejection and have been identified as an intrinsic part of the kidney. Here we quantified and phenotyped the dendritic cell populations in well-defined biopsies of 102 patients with acute renal allograft rejection in comparison with 78 available pretransplant biopsies. There was a strong increase in BDCA-1(+) and DC-SIGN(+) myeloid, BDCA-2(+) plasmacytoid, and DC-LAMP(+) mature dendritic cells in rejection biopsies compared with the corresponding pretransplant tissue. Mature dendritic cells were mostly found in clusters of lymphoid infiltrate and showed a strong correlation with the Banff infiltrate score. The presence of both myeloid and plasmacytoid dendritic cell subsets in the kidney during acute rejection correlated with interstitial fibrosis and tubular atrophy. Importantly, the myeloid dendritic cell density at the time of acute rejection was an independent risk factor for loss of renal function after the first year. Thus, acute renal allograft rejection is characterized by an influx of myeloid and plasmacytoid dendritic cells, strongly associated with local damage in the graft. Hence, the density of myeloid dendritic cells during acute rejection could be an important risk factor for the long-term development of chronic changes and loss of graft function.

The Fc-α receptor (FcαR/CD89) is involved in IgA complex formation and may affect the development of IgA nephropathy (IgAN). In this study, we tested the genetic variations of the CD89 gene in relation to disease susceptibility in IgAN and the expression of soluble CD89 (sCD89) in sera of patients with IgAN and in controls. There was a significant difference between the levels of sCD89-IgA complexes, measured by sandwich enzyme-linked immunosorbent assay (ELISA), in 177 patients with IgAN with and without disease progression at the time of first diagnosis. No such difference was found in 42 patients with other renal diseases. The patients with IgAN without disease progression had stable but high levels of sCD89 over 5-15 years of follow-up in contrast to stable but low levels of sCD89 in the disease progression group. Moreover, levels of sCD89 complexes were correlated with one of the five CD89 genetic variants in 212 patients with IgAN and 477 healthy Caucasians; the single-nucleotide polymorphism (SNP) rs11084377 was significantly associated with a lower expression of sCD89. However, no association between CD89 gene polymorphisms and susceptibility to IgAN was detected. Thus, we found an association between the levels of sCD89-IgA complexes in serum and the severity of IgAN, and a possible genetic component in regulating the production or expression of sCD89.

Background: Renal tubular epithelial cells (TECs) play an active role in renal inflammation. Previous studies have demonstrated the capacity of TECs to modulate T-cell responses both positively and negatively. Recently, new costimulatory molecules [inducible T cell costimulator-L (ICOS-L) and B7-H1] have been described, which appear to be involved in peripheral T-cell activation., Methods: We characterized expression and regulation of costimulatory molecules on primary human TECs and the TEC line human kidney-2 (HK-2) with reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. Immunohistochemistry was performed on human kidney biopsies. The capacity of TECs to modulate T-cell activation was studied in TEC/T-cell cultures., Results: We demonstrate that TECs express ICOS-L and B7-H1 in vitro and in vivo. Stimulation with interferon-gamma (IFN-gamma) resulted in increased expression of B7-H1, whereas ICOS-L expression was marginally increased upon stimulation with CD40L, with no effect of interleukin (IL-1), IL-17, or tumor necrosis factor-alpha (TNF-alpha). Furthermore, we show that TECs are able to costimulate T cells that have received signal-1 using alphaCD3 antibodies, inducing strong IL-10 production, which was partially mediated by ICOS-L. In contrast, B7-H1 appeared to be involved in inhibition of proliferation and cytokine synthesis. In addition, TECs were able to alter the cytokine profile of fully activated T cells, which were incubated with alphaCD3 and alphaCD28 antibodies, resulting in low IFN-gamma and high IL-10 production. This activity appeared to be independent of ICOS-L and B7-H1., Conclusion: Interaction of tubular epithelial cells and kidney infiltrating T cells via ICOS-L and B7-H1 may change the balance of positive and negative signals to the T cells, leading to IL-10 production and limitation of local immune responses.

Background: IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide, characterized by mesangial IgA1 deposits. We have previously demonstrated that IgAN patients have a hampered IgA immune response after mucosal challenge with a neoantigen. Dendritic cells are critically involved in the initiation of humoral immune responses, not only via activation of T-helper cells, but also via direct effect on naïve B cells. The aim of this study was to investigate the capacity of dendritic cells from IgAN patients to regulate IgA production., Methods: Dendritic cells were generated by culturing monocytes for 7 days in the presence of interleukin (IL)-4 and granulocyte macrophage-colony-stimulating factor (GM-CSF). Dendritic cells from either IgAN patients (N= 12) or controls (N= 12) were cultured for 14 days with naïve B cells in the presence of CD40L-transfected mouse fibroblasts (L-CD40L cells) and medium with or without IL-2 or IL-10. Supernatants were tested for the presence of immunoglobulins by specific enzyme-linked immunosorbent assay (ELISA)., Results: In the presence of CD40L and IL-10, dendritic cells were able to increase immunoglobulin production by naïve B cells. Dendritic cells of IgAN patients induced significantly (P= 0.026) less IgA production than dendritic cells of control persons (2.30 microg/mL vs. 5.24 microg/mL), whereas no differences were found in the IgG and IgM production. When dendritic cells were replaced by supernatant of CD40L-stimulated dendritic cells of patients and controls, IgA production was increased, but no difference was seen between the two groups., Conclusion: In the present study we show that dendritic cells of IgAN patients have an impaired capacity to induce IgA production in naïve B cells, which might explain the observed IgA hyporesponse upon mucosal challenge with a neoantigen.

Chronic rejection is currently the most prevalent cause of renal transplant failure. Clinically, chronic rejection presents by chronic transplant dysfunction, characterized by a slow loss of function, often in combination with proteinuria and hypertension. The histopathology is not specific in most cases but transplant glomerulopathy and multilayering of the peritubular capillaries are highly characteristic. Several risk factors have been identified such as young recipient age, black race, presensitization, histoincompatability, and acute rejection episodes, especially vascular rejection episodes and rejections that occur late after transplantation. Chronic rejection develops in grafts that undergo intermittent or persistent damage from cellular and humoral responses resulting from indirect recognition of alloantigens. Progression factors such as advanced donor age, renal dysfunction, hypertension, proteinuria, hyperlipidemia, and smoking accelerate deterioration of renal function. At the tissue level, senescence conditioned by ischemia/reperfusion (I/R) may contribute to the development of chronic allograft nephropathy (CAN). The most effective option to prevent renal failure from chronic rejection is to avoid graft injury from both immune and nonimmune mechanism together with nonnephrotoxic maintenance immunosuppression.

Primary IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis, leading to progressive renal failure in almost one third of the patients. The disease is characterized by mesangial deposits of IgA. The pathogenesis of IgAN remains incompletely understood. The basic abnormality of this disorder lies within the IgA immune system rather than in the kidney. Elevated levels of IgA and IgA-containing complexes are found in sera of most patients with IgAN, but increased levels alone are not sufficient to develop IgAN. Therefore abnormal physicochemical properties of circulating IgA, such as size, charge, and glycosylation may play a role. This is supported by the presence of altered glycosylation of serum and mesangial IgA in patients with IgAN. Although the precise origin and nature of the mesangial IgA deposits are still uncertain, they contain at least in part macromolecular IgA, which may be derived from circulating IgA-containing complexes. Recently, novel insights have been obtained in the molecular composition of circulating high-molecular-weight IgA, which might include complexes with underglycosylated IgA1 and IgA-CD89 complexes. In this review various aspects of macromolecular IgA in relation to IgAN will be discussed.

Background: Pentraxin 3 (PTX3) is a prototypic long pentraxin with structural similarities in the C-terminal domain to the classical short pentraxins C-reactive protein (CRP) and serum amyloid P component. PTX3 is suggested to play an important role in the innate resistance against pathogens, regulation of inflammatory reactions, and clearance of apoptotic cells. Unlike the classic pentraxins, PTX3 is mainly expressed extrahepatically. The present study was designed to investigate the expression of PTX3 by human proximal renal tubular epithelial cells (PTECs)., Methods: PTECs were cultured in the presence or absence of inflammatory cytokines. PTX3 mRNA expression was measured by reverse transcription-polymerase chain reaction (RT-PCR) in human kidney and PTECs. PTX3 protein levels in PTEC cultures were quantified by enzyme-linked immunosorbent assay (ELISA)., Results: PTX3 mRNA was shown to be constitutively expressed in human kidney. Constitutive expression and production of PTX3 was shown in primary mesangial cells, in primary PTECs, and in renal fibroblasts. Further analysis showed that interleukin (IL)-1 and tumor necrosis factor-alpha (TNF-alpha) stimulation strongly enhanced the expression and production of PTX3 in PTECs in a dose- and time-dependent manner. In addition, activation of PTECs with IL-17 and CD40L, respectively, but not with IL-6 or IL-4, resulted in strongly increased production of PTX3, whereas granulocyte macrophage-colony-stimulating factor (GM-CSF) inhibited IL-1-induced PTX3 production. PTX3 produced by PTEC is functionally active in binding C1q., Conclusion: These results indicate that PTX3 is expressed and released by PTECs and that in proinflammatory conditions PTX3 production is up-regulated. Local expression of PTX3 may play a role in the innate immune response and inflammatory reactions in the kidney.

Chronic allograft nephropathy includes chronic calcineurin nephrotoxicity, recurrent and de novo glomerulonephritis and a group of disorders with graft dysfunction of unknown etiology designated chronic rejection. Review of risk factors of the latter category show that the chronic rejection lesions emerge in organs that have undergone injury. Despite the relevance of nonalloantigen-dependent progression factors in the tissue injury, alloantigen-dependent factors predominate in the pathogenesis. Lately, B cell responses have received increasing interest in transplant rejection and include responses against both major histocompatibility complex (MHC) and tissue-specific antigens, mainly on the endothelium and in the glomeruli. These humoral responses are thought to be involved in the development of vascular and glomerular lesions. Furthermore, at the tissue level, markers of senescence are found in the tubular epithelium contributing to the lesions of tubular atrophy and interstitial fibrosis.

Renal tubular epithelial cells (TEC) are thought to play an active role in tubulointerstitial inflammation. Various immune and non-immune factors activate TEC to produce a variety of cytokines and chemokines, contributing to attraction of inflammatory cells to the kidney. The proinflammatory transcription factor nuclear factor-kappaB (NF-kappaB) appears to be a key player in these responses and tubular expression of NF-kappaB has been demonstrated in vitro and in vivo. Although glucocorticoids are known to inhibit NF-kappaB activation at different levels, the proinflammatory capacity of TEC was not inhibited. In contrast, glucocorticoids seemed to enhance the profibrotic response of TEC, emphasizing the cell-type specific characteristics of glucocorticoid action. We propose that specific inhibition of NF-kappaB activation in TEC might be an attractive strategy for therapeutic intervention in renal inflammation.

Background: Various renal cell types have been shown to contribute to the excessive matrix deposition observed in diabetic nephropathy. The present study examined the effect of high ambient glucose and transforming growth factor-beta1 (TGF-beta1) on matrix production by human renal fibroblasts., Methods: Human renal fibroblasts (TK173) were used to examine the effects of high glucose and TGF-beta1 on fibronectin and collagen type III expression. Stable transfectants were generated of TK173 cells expressing a dominant negative TGF-beta type II receptor. Matrix components were measured in enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR)., Results: Fibronectin secretion by renal fibroblasts was increased upon exposure to high glucose, but with delayed kinetics compared to TGF-beta1-induced fibronectin. Exposure to high glucose resulted in an increased secretion of latent TGF-beta1. However, treatment with neutralizing pan-specific anti-TGF-beta antibodies could not attenuate the effects of glucose. Furthermore, collagen type III was up-regulated by high glucose, but not by TGF-beta1. Importantly, fibroblasts expressing a dominant negative TGF-beta type II receptor were defective in TGF-beta1-induced fibronectin production, whereas glucose-induced fibronectin and collagen type III were unaffected., Conclusions: These data show that in renal fibroblasts exposure to high glucose can increase matrix production independent of endogenous TGF-beta1. Although glucose activation is accompanied by an increased production of latent TGF-beta1, which can have an important role in vivo, the data suggest involvement of alternative growth factors in the mechanism by which hyperglycemic conditions can modulate matrix accumulation in diabetic nephropathy.

Background: The presence of IgA together with the myeloid IgA-receptor FcalphaRI/CD89 in the circulation of patients with IgA nephropathy (IgAN) has been suggested as a specific pathogenic factor for mesangial deposition. However, in a recent study we found these complexes also in serum samples from healthy subjects. To investigate whether these circulating complexes are specific for IgAN, the levels and characteristics of IgA-CD89 complexes were analyzed in patients with IgAN and healthy controls., Methods: Specific ELISAs with different poly- and monoclonal antibodies and a sensitive dot-blot method were used to measure IgA-CD89 levels in serum and purified IgA samples obtained from healthy volunteers (N = 30) and patients with IgAN (N = 35). Fractionated samples of purified IgA were used to compare the size characteristics of the IgA-CD89 complexes., Results: Almost all CD89 in serum of patients with IgAN and controls was associated with high molecular weight IgA. Quantitative analysis of IgA-CD89 complexes in purified IgA revealed no significant difference between patients with IgAN and controls. No correlation was found between levels of IgA-CD89 complexes and clinical parameters associated with progressive IgAN., Conclusions: CD89 in the circulation is found mainly linked to high molecular weight IgA. The presence of these complexes is not specific for IgAN. Therefore, if IgA-CD89 complexes are involved in the pathogenesis of primary IgA nephropathy, additional factors are required to explain the IgA-CD89 complex-mediated renal inflammation.

Background: The prognosis of primary renal disease is often dependent on the degree of tubulointerstitial scarring. Scarring is caused by proliferation and excessive matrix production of renal fibroblasts and possibly other cellular elements. Transforming growth factor-beta (TGF-beta) is the most important cytokine for the induction of matrix synthesis in the kidney. However, its effects on renal fibroblast proliferation have not been determined. We have recently demonstrated that the expression of basic fibroblast growth factor (FGF-2) is robustly up-regulated in human kidneys with tubulointerstitial fibrosis and that FGF-2 is a potent inducer of fibroblast proliferation. The present study examined the interaction between TGF-beta 1 and FGF-2 in human renal fibroblasts., Methods: Experiments were performed on a transformed medullary fibroblast line and on primary cortical kidney and skin fibroblasts isolated from human biopsies. mRNA levels of FGF-2 and TGF-beta 1 were analyzed by Northern blot analyses. Changes in protein expression were examined by immunoblots and enzyme-linked immunosorbent assay (ELISA). Bromodeoxyuridine incorporation assays and cell counts were used to analyze cell proliferation. The expression of cell cycle-regulatory proteins cyclin-dependent kinase (cdk) 2 and the cdk inhibitor p27(kip1) were determined by immunoblots., Results: Stimulation of renal fibroblasts with FGF-2 resulted in no change of TGF-beta 1 mRNA expression, whereas incubation of the cells with TGF-beta 1 induced FGF-2 mRNA up to 3.51 +/- 0.21-fold after six hours. This increase could be blocked almost completely by the addition of cyclohexamide, indicating that the process is in large part dependent on protein synthesis. The up-regulation in FGF-2 mRNA expression was paralleled by de novo detection of FGF-2 protein in the supernatant, peaking after 12 to 24 hours, as determined by Western blot and ELISA, whereas cellular protein was only increased up to 2.1-fold. Interestingly, both methods detected release of FGF-2 protein to the supernatant already at three hours, indicating a role for TGF-beta1 in directly releasing preformed FGF-2. Since TGF-beta 1 induced FGF-2, which results in fibroblast proliferation, we hypothesized that TGF-beta1 may cause fibroblast proliferation mediated by FGF-2. This hypothesis was verified by cell proliferation assays demonstrating that stimulation of renal fibroblasts with TGF-beta1 resulted in an up to 3.21 +/- 0.28-fold increase in bromodeoxyuridine incorporation and a 1.95 +/- 0.16-fold increase in cell number after 72 hours. This mitogenic effect of TGF-beta1 could be blocked completely by the addition of a neutralizing antibody to FGF-2 or the tyrosine kinase inhibitor tyrphostin AG1296, which blocks FGF receptor (FGFR) tyrosine kinase activity. Conversely, a neutralizing antibody to epidermal growth factor (EGF) or the tyrphostin B42, which inhibits EGF receptor signal transduction, had no effect. Interestingly, a neutralizing antibody to PDGF had only minor effects in primary kidney fibroblasts but reduced TGF-beta 1-induced proliferation considerably in primary skin fibroblasts. Finally, TGF-beta1-induced proliferation in kidney fibroblasts was paralleled by a robust increase in cdk 2 protein expression up to 72 hours, whereas p27(kip1), whose activity is maintained by TGF-beta in epithelial cells, was down-regulated up to 48 hours., Conclusions: Our studies demonstrate, to our knowledge for the first time, that TGF-beta1 induces proliferation in human renal fibroblasts and that this process is mediated largely by FGF-2. The induction of proliferation by TGF-beta 1 via induction of FGF-2 may play an important role in the autonomy of renal fibroblast growth and thus in the pathogenesis of human fibrogenesis.

Background: Renal tubular epithelial cells are a central cell type in tubulointerstitial inflammation because they can produce inflammatory mediators such as cytokines and chemokines. Several signals derived from either monocytes or activated T cells have been reported to regulate the activation of tubular epithelial cells. We studied this regulation in more detail by combined treatment with CD40 ligand and the proinflammatory cytokine interleukin-1 (IL-1) in vitro., Methods: The regulation of cytokine and chemokine production was studied in primary cultures of human proximal tubular epithelial cells (PTECs). PTECs were activated by coculture with CD40L-transfected murine fibroblasts in combination with recombinant human cytokines. The production of IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1), and RANTES were measured by specific enzyme-linked immunosorbent assay., Results: The combined activation of PTECs with CD40L and IL-1 resulted in strong synergistic effects on the production of IL-6, IL-8, and RANTES, whereas only an additive stimulation of MCP-1 production was observed. The effects were specific for IL-1 and could be neutralized by the addition of the IL-1R antagonist. Both IL-1alpha and IL-1beta showed similar effects on cytokine production by PTECs. The effects of IL-1 were dose dependent, and kinetic experiments showed that synergistic effects were observed after 24 hours of activation and remained present for at least five days. Reverse transcription-polymerase chain reaction analysis showed that human PTECs could express both IL-1alpha and IL-1beta. The activation of PTECs with IL-1 resulted in an up-regulation of CD40 expression on these cells., Conclusions: A complex network of regulation exists for the production of cytokines and chemokines by PTECs. The combined treatment results in strong synergistic effects on IL-6, IL-8, and RANTES production. This strengthens the potential role of tubular epithelial cells in inflammatory responses within the kidney.

Previously it has been demonstrated that human proximal tubular epithelial cells (PTEC) are able to produce chemokines (such as IL-8 and MCP-1) and complement components (such as C2, C3, C4 and factor H), and that production of these proteins is regulated by pro-inflammatory cytokines such as interleukin-1 alpha (IL-1alpha), tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). Since TGF-beta is also expressed in the renal interstitium during inflammation, we investigated the effect of TGF-beta on the production of chemokines and complement components by PTEC in culture. Transforming growth factor-beta 1 up-regulated IL-8 production by an average of 4.17 +/- 1.0 fold. macrophage chemoattractant phagocyte (MCP-1) production, on the other hand, was down-regulated by TGF-beta 1 by an average of 2.2 +/- 0.7 fold. The production of C3 and C4 was also down-regulated after incubation with TGF-beta 1 (1.9 +/- 0.3- and 3.0 +/- 1.2-fold, respectively). All effects were dose- and time-dependent and were found to be specific for TGF-beta 1, as assessed by inhibition of the effect with a neutralizing antibody against TGF-beta 1. These data, together with the knowledge that TGF-beta, chemokines and complement components play a role in several types of renal disease, suggest that TGF-beta is involved in the regulation of local expression of chemokines and complement components by tubular cells.

Interstitial infiltration by mononuclear cells is a hallmark of most inflammatory kidney diseases, and the degree of infiltration is associated with disease progression. It has been demonstrated that proximal tubular epithelial cells (PTEC) are an important source of different cytokines/chemokines and thereby play a central role in the regulation of the local inflammatory response. CD40 is a cell surface receptor involved in immune regulation for which the ligand is expressed on activated T cells. By different staining methods, CD40 was found expressed in cryosections on the basolateral side of tubuli, as well as on the surface of an SV40-transformed PTEC line (PTEC-TRL) and on primary PTEC cultures. Cross linking CD40 receptor on these cultured cells, using a CD40L-transfected mouse fibroblast, resulted in strong up-regulation of the production of the chemokines IL-8, MCP-1 and RANTES. For IL-8 and MCP-1 production, the stimulation index after CD40 activation ranged from two- to sevenfold. Much stronger effects were observed for RANTES production, where levels remained undetectable (< 0.1 ng/ml) in non-stimulated cultures, whereas CD40 activation resulted in a strong production reaching 5 ng/ml in a 72-hour culture period. These data suggest that CD40L-CD40 interactions between infiltrating activated T cells and PTEC might be an important factor in the regulation of interstitial infiltration within the kidney.