Expression of nestin in the podocytes of normal and diseased human kidneys

The complex cyto-architecture of the podocyte is critical for glomerular permselectivity. The present study characterizes the expression of nestin, an intermediate filament protein, in human kidneys. In normal kidneys, nestin was detected at the periphery of glomerular capillary loops. Colabeling showed nestin was expressed in WT1-positive cells. Within the podocyte, nestin immunoreactivity was present in the cell body and primary process. This was supported by immunoelectron microscopy. Nestin also colocalized with vimentin in the periphery of capillary loops but not in the mesangium. Nestin was not detected in other structures of the adult human kidney. To determine the potential role of nestin in proteinuria, nestin was examined in kidney biopsies from patients with or without proteinuria. These patients were diagnosed with IgA nephropathy with mild mesangial expansion but without proteinuria, IgA nephropathy with proteinuria, membranous nephropathy (MN), and focal segmental glomerular sclerosis (FSGS). The distribution of nestin in these biopsies was similar to that in the normal kidney. Semiquantitative analysis of immunostaining showed that glomerular nestin expression in IgA nephropathy without proteinuria was not different from normal kidney; however, nestin expression in kidneys of patients with IgA nephropathy and proteinuria, or MN and FSGS with proteinuria was significantly reduced compared with normal kidney (P < 0.01). Reduced nestin mRNA expression in the patients with IgA nephropathy with proteinuria and FSGN was also observed by quantitative real-time PCR. These studies suggest that nestin may play an important role in maintaining normal podocyte function in the human kidney.     

IL-6 and its role in IgA nephropathy development

IL-6 is considered one of the well characterized cytokines exhibiting homeostatic, pro- and anti-inflammatory activities, depending on the receptor variant and the induced intracellular cis- or trans-signaling responses. IL-6-activated pathways are involved in the regulation of cell proliferation, survival, differentiation, and cell metabolism changes.Deviations in IL-6 levels or abnormal response to IL-6 signaling are associated with several autoimmune diseases including IgA nephropathy (IgAN), one of most frequent primary glomerulonephritis worldwide. IgAN is associated with increased plasma concentration of IL-6 and increased plasma concentration of aberrantly galactosylated IgA1 immunoglobulin (Gd-IgA1). Gd-IgA1 is specifically recognized by autoantibodies, leading to the formation of circulating immune complexes (CIC) with nephritogenic potential, since CIC deposited in the glomerular mesangium induce mesangial cells proliferation and glomerular injury. Infection of the upper respiratory or digestive tract enhances IL-6 production and in IgAN patients is often followed by the macroscopic hematuria.This review recapitulates general aspects of IL-6 signaling and summarizes experimental evidences about IL-6 involvement in the etiopathogenesis of IgA nephropathy through the production of Gd-IgA1 and regulation of mesangial cell proliferation.     

Defective oral tolerance promotes nephritogenesis in experimental IgA nephropathy induced by oral immunization.

Oral tolerance, an important feature of the mucosal immune system, appears to protect against immune-mediated disease by blunting production of systemic IgG and IgM antibody directed toward immunogens chronically present at mucosal surfaces. In this study, we explored the role of oral tolerance and mucosal immunoregulation in an experimental model of IgA nephropathy (IgAN), an important form of nephritis in humans. Cyclophosphamide and estradiol were used to inhibit the expression of oral tolerance, which otherwise develops after chronic oral presentation of Ag. BALB/c mice given drinking water containing 0.1% bovine gamma globulin (BGG) continuously for 14 wk were randomly assigned to groups given either 2 mg of cyclophosphamide i.p., 2 mg of estradiol s.c. or both drugs. Groups of control mice received neither BGG nor drugs. In three separate experiments, a low percentage of saline-treated orally immunized mice had microscopic hematuria (0 to 20%), as did nonimmunized controls (0 to 20%). However, 58 to 83% of mice given estradiol and/or cyclophosphamide at appropriate times developed significant hematuria. If drugs were given at suboptimal times, only 25 to 56% of mice developed hematuria. Drug-treated immunized mice also had more serum IgG and IgM anti-BGG antibodies than control and saline groups. Immunofluorescence showed significantly more glomerular deposits of IgG, IgM, and C3 in drug-treated immunized mice compared to saline-treated immunized and normal untreated control mice. Hematuria and glomerular deposits of IgG, IgM, and C3 paralleled serum IgG and IgM antibody. All immunized mice showed significant mesangial IgA and BGG deposits and there were no differences in such deposits between saline- and drug-treated immunized mice. We suggest that blunting of oral tolerance with promotion of systemic IgG and IgM antibody production leads to nephritis in chronically orally immunized mice and that glomerular immune complexes containing IgG and/or IgM promote complement deposition and hematuria in IgAN. Analogous defects in oral (or more generally mucosal) tolerance could play a role in the genesis of symptomatic human IgAN.     

Uncoupling of Glomerular IgA Deposition and Disease Progression in Alymphoplasia Mice with IgA Nephropathy

Previous clinical and experimental studies have indicated that cells responsible for IgA nephropathy (IgAN), at least in part, are localized in bone marrow (BM). Indeed, we have demonstrated that murine IgAN can be experimentally reconstituted by bone marrow transplantation (BMT) from IgAN prone mice in not only normal mice, but also in alymphoplasia mice (aly/aly) independent of IgA⁺ cells homing to mucosa or secondary lymphoid tissues. The objective of the present study was to further assess whether secondary lymph nodes (LN) contribute to the progression of this disease. BM cells from the several lines of IgAN prone mice were transplanted into aly/aly and wild-type mice (B6). Although the transplanted aly/aly showed the same degree of mesangial IgA and IgG deposition and the same serum elevation levels of IgA and IgA-IgG immune-complexes (IC) as B6, even in extent, the progression of glomerular injury was observed only in B6. This uncoupling in aly/aly was associated with a lack of CD4⁺ T cells and macrophage infiltration, although phlogogenic capacity to nephritogenic IC of renal resident cells was identical between both recipients. It is suggested that secondary LN may be required for the full progression of IgAN after nephritogenic IgA and IgA/IgG IC deposition.     

Glomerular Deposition of Properdin in Henoch-Sch?nlein Syndrome and Idiopathic Focal Nephritis

Biopsy of renal tissue from four patients with idiopathic focal nephritis and three patients with Henoch-Schönlein syndrome showed that C3 and properdin were deposited with IgA in the glomerular mesangium, C1q could not be detected. These observations suggest that glomerular injury in disorders characterized by mesangial deposits of IgA and C3 is mediated via the properdin system.     

Glomerular pathology in surviving pigs experimentally infected with African swine fever virus.

Twelve miniature pigs were inoculated with an attenuated African swine fever virus to study glomerular involvement in surviving pigs. In acute phase, kidneys were severely affected and displayed a glomerular capillary thrombosis with fibrin deposition in vascular lumen, detected by immunofluorescence. Fibrin-positive deposits were progressively cleared between one to three months after infection in surviving pigs. The histological picture in kidneys of surviving pigs, up to one post-infection year, showed a focal and segmental glomerulonephritis with hyalinosis, and IgM and C3 deposition was detected by immunofluorescence. Its pathogeny as an evolutive stage of acute glomerular injury is pointed out.     

Thyroid antigen-antibody nephritis: possible involvement of fucosyl-GM1 as the antigen

Hyperthyroidism, microscopic hematuria, and proteinuria developed in an 11-year-old girl. Proteinuria decreased during treatment of hyperthyroidism with an antithyroid drug. On admission, serum anti-thyroglobulin antibody, antimicrosomal antibody, and immune complex were present. The thyrotropin binding inhibitory immunoglobulin (TBII) level was low. On the other hand, an antibody to the ganglioside component (fucosyl-GM1) was detected by an enzyme linked immunosolvent assay (ELISA). A thyroid biopsy specimen showed massive lymphocytic infiltration and interstitial fibrosis. A renal biopsy specimen showed marked proliferation of mesangial cells and increased mesangial matrix with focal segmental capillary wall abnormality. Electron microscopec studies demonstrated mild paramesangial dense deposits. By indirect immunofluorescence, granular glomerular basement membrane and mesangial staining were not detected with rabbit antibody to thyroglobulin, but were detected with rabbit antibody to fucosyl GM1. Fucosyl GM1 was also seen along the basilar aspect of the thyroid follicular epithelial cells. These observation suggests the development of glomerulonephritis mediated by thyroid antigen, particularly ganglioside component.     

IgA nephropathy in adults: immunohistologic findings and clinical course

Laboratory examination of specimens from 123 consecutive renal biopsies performed at Victoria General Hospital, Halifax revealed six cases of mesangial deposition, predominantly of IgA, unassociated with systemic disorders. Immunohistologic examination showed deposits of only IgA in one specimen, IgA and IgG in two and IgA, IgG and IgM in three. Glomerular deposits of C3 were seen in five of the specimens, and properdin was seen in three. Glomeruli in all the specimens showed increased matrix and increased numbers of cells in the mesangium. Electron microscopy revealed deposits in the mesangium or capillary wall in all five of the specimens so studied. All six patients had proteinuria, four had microscopic hematuria, and three had hypertension; in one patient the disease progressed to renal failure.     

Uteroglobin is essential in preventing immunoglobulin A nephropathy in mice.

The molecular mechanism(s) of immunoglobulin A (IgA) nephropathy, the most common primary renal glomerular disease worldwide, is unknown. Its pathologic features include hematuria, high levels of circulating IgA-fibronectin (Fn) complexes, and glomerular deposition of IgA, complement C3, Fn and collagen. We report here that two independent mouse models (gene knockout and antisense transgenic), both manifesting deficiency of an anti-inflammatory protein, uteroglobin (UG), develop almost all of the pathologic features of human IgA nephropathy. We further demonstrate that Fn-UG heteromerization, reported to prevent abnormal glomerular deposition of Fn and collagen, also abrogates both the formation of IgA-Fn complexes and their binding to glomerular cells. Moreover, UG prevents glomerular accumulation of exogenous IgA in UG-null mice. These results define an essential role for UG in preventing mouse IgA nephropathy and warrant further studies to determine if a similar mechanism(s) underlies the human disease.     

Complement activation contributes to both glomerular and tubulointerstitial damage in adriamycin nephropathy in mice

Adriamycin nephropathy is a model of focal segmental glomerulosclerosis, characterized by proteinuria and progressive glomerulosclerosis and tubulointerstitial damage. In this study, we examined the role of complement in the etiology of adriamycin nephropathy in mice. We used mice deficient in C1q, factor D, C3, and CD59, and compared them with strain-matched controls. C3 deposition occurred in the glomeruli of wild-type mice as early as 48 h following a single i.v. injection of adriamycin. C3-deficient mice developed significantly less proteinuria and less podocyte injury at day 3 postadriamycin than controls, suggesting that complement is important in mediating the early podocyte injury. At later time points, C3-deficient mice were protected from glomerulosclerosis, tubulointerstitial injury, and renal dysfunction. Factor D-deficient mice were also protected from renal disease, confirming the importance of alternative pathway activation in this model. In contrast, C1q-deficient mice developed similar disease to controls, indicating that the complement cascade was not activated via the classical pathway. CD59-deficient mice, which lack adequate control of C5b-9 formation, developed significantly worse histological and functional markers of renal disease than controls. Interestingly, although more C9 deposited in glomeruli of CD59-deficient mice than controls, in neither group was tubulointerstitial C9 staining apparent. We have demonstrated for the first time that alternative pathway activation of complement plays an important role in mediating the initial glomerular damage in this in vivo model of focal segmental glomerulosclerosis. Lack of CD59, which regulates the membrane attack complex, led to greater glomerular and tubulointerstitial injury.     

Molecular basis of IgA nephropathy

IgA nephropathy (IgAN) is the most common glomerulonephritis worldwide and remains an important cause of end-stage renal failure. However, the basic molecular mechanism(s) underlying abnormal IgA synthesis, selective mesangial deposition with ensuing mesangial cell proliferation and extracellular matrix expansion remains poorly understood. Notably, the severity of tubulointerstitial lesions better predicts the renal progression than the degree of glomerular lesions. The task of elucidating the molecular basis of IgAN is made especially challenging by the fact that both environmental and genetic components likely contribute to the development and progression of IgAN. This review will summarize the earlier works on the structure of the IgA molecule, mechanisms of mesangial IgA deposition and pathophysiologic effects of IgA on mesangial cells following mesangial deposition. Recently, a series of important advances in the area of communication between the glomerular mesangium and renal tubular cells have emerged. These novel findings regarding the molecular pathogenesis of IgAN will be helpful in designing future directions for therapy.     

Dimeric and polymeric IgA, but not monomeric IgA, enhance the production of IL-6 by human renal mesangial cells

Depositions of IgA in the renal glomerular mesangial area are a hallmark of IgA nephropathy, and are thought to be crucial for the onset of inflammation processes in IgA nephropathy. In this report we show that human mesangial cells (MC) in vitro bind IgA and that binding of IgA enhances the production of IL-6 by MC. Furthermore we show that the size of IgA is crucial in its capability to enhance IL-6 production. Monomeric IgA does not affect basic IL-6 production, whereas dimeric and polymeric IgA enhance IL-6 production up to 3- to 9-fold respectively. Additional studies demonstrate that enhanced IL-6 production by MC is not accompanied by increased proliferation of human mesangial cells, a finding which is distinct from that found with rat mesangial cells. Taken together, these fmdings suggest that deposition of dimeric and polymeric IgA in the mesangial area of human kidneys in IgA nephropathy may amplify local inflammation.     

The long pentraxin PTX3 is synthesized in IgA glomerulonephritis and activates mesangial cells

The long pentraxin PTX3 has been recently involved in amplification of the inflammatory reactions and regulation of innate immunity. In the present study we evaluated the expression and role of PTX3 in glomerular inflammation. PTX3 expression was investigated in the IgA, type I membranoproliferative, and diffuse proliferative lupus glomerulonephritis, which are characterized by inflammatory and proliferative lesions mainly driven by resident mesangial cells, and in the membranous glomerulonephritis and the focal segmental glomerular sclerosis, where signs of glomerular inflammation are usually absent. We found an intense staining for PTX3 in the expanded mesangial areas of renal biopsies obtained from patients with IgA glomerulonephritis. The pattern of staining was on glomerular mesangial and endothelial cells. Scattered PTX3-positive cells were also detected in glomeruli of type I membranoproliferative glomerulonephritis. The concomitant expression of CD14 suggests an inflammatory origin of these cells. Normal renal tissue and biopsies from patients with the other glomerular nephropathies studied were mainly negative for PTX3 expression in glomeruli. However, PTX3-positive cells were detected in the interstitium of nephropathies showing inflammatory interstitial injury. In vitro, cultured human mesangial cells synthesized PTX3 when stimulated with TNF-alpha and IgA and exhibited specific binding for recombinant PTX3. Moreover, stimulation with exogenous PTX3 promoted mesangial cell contraction and synthesis of the proinflammatory lipid mediator platelet-activating factor. In conclusion, we provide the first evidence that mesangial cells may both produce and be a target for PTX3. The detection of this long pentraxin in the renal tissue of patients with glomerulonephritis suggests its potential role in the modulation of glomerular and tubular injury.     

Expression of Fc alpha/mu receptor by human mesangial cells: a candidate receptor for immune complex deposition in IgA nephropathy.

IgA nephropathy is characterized by the deposition of IgA immune complexes in the glomerular mesangium, but the mechanisms responsible for this are not well understood. Human mesangial cells (HMCs) can bind IgA but do not express known IgA receptors. We show here that primary HMCs express mRNA for a novel receptor, the Fc alpha/mu receptor (Fcalpha/muR), and that receptor expression is upregulated by IL-1. We also detected mRNA for a novel receptor variant in HMCs that may encode a soluble form of the receptor. Fcalpha/muR was expressed in a heterologous system which showed that the receptor was approximately 58 kDa in weight and was only minimally N-glycosylated. As predicted from the characteristics of the murine homologue, the expressed human Fcalpha/muR was able to bind IgA and IgM, but not IgG. These results suggest that Fcalpha/muR may be the receptor responsible for mesangial IgA deposition in IgA nephropathy.     

Schistosoma japonicum: Immunopathology of nephritis in Macaca fascicularis

Macaca monkeys experimentally infected with Schistosoma japonicum developed a chronic progressive kidney lesion characterized by an increase of mesangial matrix, local glomerular hypercellularity, and local thickening of glomerular basement membrane. Immunofluorescence studies revealed the localization of IgG, IgM, IgA, and IgE immunoglobulins mostly in the mesangial area of the glomeruli accompanied by the deposition of Schistosoma antigens. By electron microscopy, in addition to the local thickening of the glomerular basement membrane, dense homogeneous deposits and those with moth-eaten appearance were detected in the mesangial matrix. These findings suggest that worms in the bloodstream continuously release antigenic materials that stimulate host's antibody response belonging to various immunoglobulin classes including IgE. The produced antibodies and antigens would form immune complexes that deposited in the glomeruli. The increased vascular permeability caused by antigen-IgE antibody interaction may play an important role in the deposition of immune complexes and in the rapid development of kidney injury.     

Downregulated expression in high IgA (HIGA) mice and the renal protective role of meprinbeta

This study discusses the critical role of the metalloproteinase meprinbeta in the progression of glomerulonephritis. Using a microarray technique, the gene expression profiles in glomeruli isolated from high serum IgA (HIGA) mice with a purity of 97% or greater were examined. HIGA mice are a valid model of human IgA nephropathy (IgAN), with the typical pathological features of this condition, including a consistently high serum IgA level as well as dominant mesangial IgA deposition and mesangial enlargement. Among the many upregulated/downregulated genes after the development of IgAN, the downregulation of meprinbeta was intriguing. The expression level of the meprinbeta gene at 40 weeks of age was 52% of that observed at 8 weeks of age (prior to the development of IgAN), although in the control BALB/c mice, a 2.19-fold elevation was seen. These results were also confirmed by semi-quantitative RT-PCR and immunostaining analyses. As meprinbeta is a subunit of metalloproteinase meprins (meprin A, meprin B) and meprins are capable of proteolytically degrading extracellular matrix (ECM) components and proteolytically processing bioactive peptides, the downregulation of meprinbeta may contribute to the progression of glomerulonephritis and the eventual glomerular scarring. This working hypothesis was examined using an in vivo meprinbeta inhibition study. The inhibition of meprins by actinonin exacerbated some parameters of renal injury in mice afflicted with anti-glomerular basement membrane (anti-GBM) antibody-associated nephritis. These in vitro and in vivo results suggest that meprinbeta may play a protective role against the progression of renal injury through the degradation of ECM and bioactive peptides.     

Fatty acid binding protein 3 as a potential mediator for diabetic nephropathy in eNOS deficient mouse

In human diabetic nephropathy, glomerular injury was found to comprise lipid droplets, suggesting that abnormal lipid metabolism might take place in the development of diabetic glomerular injury. However, its precise mechanism remains unclear. Fatty acid binding protein (FABP) is currently considered as a key molecule for lipid metabolism. Since diabetic eNOS knockout (KO) mouse is considered to be a good model for human diabetic nephropathy, we here investigated whether FABP could mediate glomerular injury in this model. We found that glomerular injuries were associated with inflammatory processes, such as macrophage infiltration and MCP-1 induction. Microarray assay with isolated glomeruli revealed that among 10 isoforms in FABP family, FABP3 mRNA was most highly expressed in diabetic eNOSKO mice compared to non-diabetic eNOSKO mice. FABP3 protein was found to be located in the mesangial cells. Overexpression of FABP3 resulted in a greater response to palmitate, a satulated FA, to induce MCP-1 in the rat mesangial cells. In turn, the heart, a major organ for FABP3 protein in normal condition, failed to alter its expression level under diabetic condition in either wild type or eNOSKO mice. In conclusion, FABP3 is induced in the mesangial cells and likely a mediator to induce MCP-1 in the diabetic nephropathy.     

Mediators and mechanisms of radiation nephropathy

Normal tissue radiation injury occurs after sufficient irradiation, thus limiting the curative potential of x-ray therapy. In the kidney, radiation injury results in fibrosis and, ultimately, renal failure. The mediators of fibrosis in radiation nephropathy have received scant attention. Therefore, we evaluated the sequential presence of alpha smooth muscle actin (alphasma), fibrin, collagen, and TGFbeta1 in a porcine model of radiation nephropathy using 9.8 Gy single-dose local kidney irradiation. During the 24-week study, there was progressive and significant collagen accumulation in glomeruli and in interstitium. In glomeruli, this was associated with significant mesangial alphasma expression by 2 weeks after irradiation, a further rise at 4 weeks, and then a gradual fall to baseline. Glomerular fibrin deposition was significant by 4 weeks after irradiation, and remained elevated thereafter. There was little or no glomerular TGFbeta1 expression at any time point. Tubular fibrin deposition was significant at 4 weeks after irradiation but declined thereafter. There was little or no tubulo-interstitial alphasma expression at any time after irradiation. At 6 weeks after irradiation, there was a significant peak of tubular epithelial TGFbeta1 expression that declined thereafter. The early glomerular injury is evident as mesangial alphasma expression but is not proceeded by TGFbeta1 expression. There is sustained glomerular fibrin deposition with deposition of fibrin in tubular lumens, suggesting that tubular fibrin derives and flows out from injured glomerular tufts. We conclude that i) alphasma expression is an early marker of glomerular radiation injury, presaging scarring; ii) fibrin deposition is involved in glomerular and tubular radiation injury; and iii) TGFbeta1 is not an early event in radiation nephropathy, and not apparent in glomeruli in this model, but may correlate with later tubulo-interstitial fibrosis. Thus, the mediators of scarring in this model differ according to time after injury and also according to the affected tissue compartment.     

Glomerulonephritis with mesangial deposits of IgA unassociated with systemic disease.

Typical features of IgA-associated nephritis were found in renal biopsies from 16 of 355 consecutive patients. Generalized segmental mesangial proliferation was noted in biopsies from most patients, and dense deposits were detected by electron microscopy in mesangial regions of approximately 50% of biopsies. Immunofluorescent studies showed IgA to be the predominant immunoglobulin in glomueruli; IgG was present in less than 50% of biopsies and IgM in only 12%. The serum IgA value was significantly increased (P les than 0.001) in 50% of patients and the mean IgA/IgG ratio was significantly increase (P less than 0.001) for the patient group as a whole, which suggests a selective increase in IgA. Mesangial deposits of C3 were present in 15 of 16 biopsies and properdin was noted in all biopsies tested; C4 was not demonstrated in any biopsy. This suggests activation of the alternative complement pathway. The results of this study support the concept that IgA-associated nephritis is a unique condition that in some patients gives rise to idiopathic recurrent hematuria. Although the prognosis is good in the majority of patients, the renal disease may progress.     

Pathogenic significance of IgA receptor interactions in IgA nephropathy

IgA nephropathy (IgAN), the most common primary glomerulonephritis worldwide, frequently progresses to renal failure. The pathogenesis of this disease involves the deposition of undergalactosylated IgA1 complexes in the glomerular mesangium. How the IgA1 complexes are generated and why they are deposited in the mesangium remains unclear. We propose a model wherein two types of IgA receptors participate in sequential steps to promote the development of IgAN, with FcalphaRI (CD89) being initially involved in the formation of circulating IgA-containing complexes and, subsequently, transferrin receptor (CD71) in mediating mesangial deposition of IgA1 complexes.