Late onset of treatment with a chemokine receptor CCR1 antagonist prevents progression of lupus nephritis in MRL-Fas(lpr) mice

Slowly progressive renal injury is the major cause for ESRD. The model of progressive immune complex glomerulonephritis in autoimmune MRL(lpr/lpr) mice was used to evaluate whether chemokine receptor CCR1 blockade late in the disease course can affect progression to renal failure. Mice were treated with subcutaneous injections of either vehicle or BX471, a nonpeptide CCR1 antagonist, three times a day from week 20 to 24 of age [corrected]. BX471 improved blood urea nitrogen levels (BX471, 35.1 +/- 5.3; vehicle, 73.1 +/- 39.6 mg/dl; P < 0.05) and reduced the amount of ERHR-3 macrophages, CD3 lymphocytes, Ki-67 positive proliferating cells, and ssDNA positive apoptotic cells in the interstitium but not in glomeruli. Cell transfer studies with fluorescence-labeled T cells that were pretreated with either vehicle or BX471 showed that BX471 blocks macrophage and T cell recruitment to the renal interstitium of MRL(lpr/lpr) mice. This was associated with reduced renal expression of CC chemokines CCL2, CCL3, CCL4, and CCL5 and the chemokine receptors CCR1, CCR2, and CCR5. Furthermore, BX471 reduced the extent of interstitial fibrosis as evaluated by interstitial smooth muscle actin expression and collagen I deposits, as well as mRNA expression for collagen I and TGF-beta. BX471 did not affect serum DNA autoantibodies, proteinuria, or markers of glomerular injury in MRL(lpr/lpr) mice. This is the first evidence that, in advanced chronic renal injury, blockade of CCR1 can halt disease progression and improve renal function by selective inhibition of interstitial leukocyte recruitment and fibrosis.     

Retinoic acid treatment protects MRL/lpr lupus mice from the development of glomerular disease

BACKGROUND: Retinoic acid (tRA) is an active metabolite of vitamin A with potent anti-inflammatory properties. We analyzed the effects of tRA on the development of lupus nephritis in MRL/lpr mice. METHODS: MRL/lpr mice received chow supplemented with vehicle or tRA (daily 10 mg/kg) from 8 to 14 weeks until their sacrifice. MRL/wt mice served as an additional control. RESULTS: tRA-treated MRL/lpr mice showed reduced lymphoadenopathy and splenomegaly as compared to vehicle-treated controls. Treatment reduced proteinuria to almost basal levels. Plasma IgG and anti-DNA antibodies increased comparably in both vehicle and tRA-treated mice. Vehicle-treated mice showed characteristic renal lesions. In contrast tRA-treated mice showed almost normal glomerular histology with a pronounced reduction in endocapillary cell proliferation. T-cell and macrophage infiltrates were reduced after tRA treatment within glomeruli and interstitium as compared to vehicle-treated animals. In spite of this, immune complex and complement deposition were comparable in both groups. Adoptively transferred T cells from vehicle-treated to tRA-treated MRL/lpr mice did not induce renal lesions or proteinuria. These beneficial effects of tRA treatment were associated with reduced renal expression of chemokines and inflammatory cytokines. Surprisingly, renal transforming growth factor-beta (TGF-beta) mRNA levels of tRA-treated mice were elevated, possibly indicating that TGF-beta acts as an anti-inflammatory signal in this lupus model. CONCLUSION: tRA treatment reduces lymphoproliferation and glomerulonephritis in MRL/lpr mice. This occurs in spite of unaltered anti-DNA titers and glomerular immune complex deposition, and cannot be overcome by T-cell transfer from nephritic MRL/lpr mice.     

Differential expression of beta-chemokines MCP-1 and RANTES and their receptors CCR1, CCR2, CCR5 in acute rejection and chronic allograft nephropathy of human renal allografts

BACKGROUND: The beta-chemokines MCP-1 (CCL2) and RANTES (CCL5) have been shown to play important roles in acute renal transplant rejection (AR) and chronic allograft nephropathy (CAN). The potential relationship of expression of these chemokines, their chemokine receptors CCR1, CCR2, CCR5, and the cell populations of inflammatory infiltrate, histological and clinical diagnoses were investigated in biopsies at the time of AR and compared with biopsies of CAN. METHODS: In 24 renal transplant biopsies with AR (n = 15) and CAN (n = 9), the expression of MCP-1 and RANTES, their receptors CCR1, CCR2, and CCR5 and the infiltration with monocytes/macrophages and T cells were studied. RESULTS: As previously described, chemokine and chemokine receptor expression was found mainly in mononuclear cells infiltrating the interstitium and glomeruli. In the tubulointerstitial area and glomeruli the expression of MCP-1, RANTES, and their receptors correlated with an infiltration by monocytes/macrophages. Biopsies with CAN revealed a lower expression of MCP-1, RANTES, CCR1, CCR2 and CCR5 in tubulointerstitial cells, and a significantly lower infiltration with MRP14-positive monocytes/macrophages than biopsies with AR. In AR, MCP-1 and CCR1 showed a lower expression compared to RANTES, CCR2, and CCR5. CONCLUSIONS: The positive correlation between chemokines and chemokine receptors and infiltrating leukocytes during acute rejection, the lower but detectable expression of MCP-1, RANTES, CCR1, CCR2 and CCR5 in CAN, and the differences in the quantity of expression between the different chemokines and chemokine receptors point to a complex regulation of chemokine expression in renal allografts. Since chemokines are not only involved in inflammation but also in tissue regeneration, this could have impact on the development of CAN.     

Obstructive nephropathy in the mouse: progressive fibrosis correlates with tubulointerstitial chemokine expression and accumulation of CC chemokine receptor 2- and 5-positive leukocytes

The infiltration of leukocytes plays a major role in mediating tubulointerstitial inflammation and fibrosis in chronic renal disease. CC chemokines participate in leukocyte migration and infiltration into inflamed renal tissue. Because CC chemokine-directed leukocyte migration is mediated by target cell expression of a group of CC chemokine receptors, this study examined the expression of CC chemokines and their receptors during initiation of tubulointerstitial fibrosis after unilateral ureteral obstruction in C57BL/6 mice. Obstructed kidneys developed hydronephrosis, tubular cell damage, interstitial inflammation, and fibrosis. From days 2 to 10, a progressive interstitial influx of F4/80+ macrophages and CD3+ lymphocytes occurred (macrophages, 4-fold; lymphocytes, 20-fold at day 10, compared with contralateral control kidneys). In parallel, the number of activated fibroblast-specific protein 1+ fibroblasts and interstitial collagen IV accumulation increased from days 2 to 10. The mRNA expression of CC chemokines (predominantly monocyte chemoattractant protein-1 [MCP-1]/CCL2, RANTES/CCL5) and their receptors CCR1, CCR2, CCR5 increased progressively from days 2 to 10. By in situ hybridization, a prominent interstitial mRNA expression of MCP-1 and RANTES and their receptors CCR2 and CCR5 localized to interstitial mononuclear cell infiltrates. MCP-1 and RANTES expression was also seen in tubular epithelial cells. Fluorescence-activated cell sorter analysis of single-cell suspensions from obstructed kidneys revealed a prominent expression of CCR2 and CCR5 by infiltrating macrophages, whereas most lymphocytes expressed CCR5 only. These data demonstrate an increased expression of MCP-1/CCL2 and RANTES/CCL5 at sites of tubulointerstitial damage and progressive fibrosis during unilateral ureteral obstruction that correlates with simultaneous accumulation of interstitial macrophages and T lymphocytes expressing the respective surface receptors CCR2 and CCR5. The chemokine receptor-mediated leukocyte influx into the tubulointerstitium could offer a new potential target for therapeutic intervention in progressive renal tubulointerstitial fibrosis.     

Chemokine receptor Ccr2 deficiency reduces renal disease and prolongs survival in MRL/lpr lupus-prone mice

MRL/MpJ-Fas(lpr)/J (MRL/lpr) mice represent a well-established mouse model of human systemic lupus erythematosus. MRL/lpr mice homozygous for the spontaneous lymphoproliferation mutation (lpr) are characterized by systemic autoimmunity, massive lymphadenopathy associated with proliferation of aberrant T cells, splenomegaly, hypergammaglobulinemia, arthritis, and fatal immune complex-mediated glomerulonephritis. It was reported previously that steady-state mRNA levels for the chemokine (C-C motif) receptor 2 (Ccr2) continuously increase in kidneys of MRL/lpr mice. For examining the role of Ccr2 for development and progression of immune complex-mediated glomerulonephritis, Ccr2-deficient mice were generated and backcrossed onto the MRL/lpr genetic background. Ccr2-deficient MRL/lpr mice developed less lymphadenopathy, had less proteinuria, had reduced lesion scores, and had less infiltration by T cells and macrophages in the glomerular and tubulointerstitial compartment. Ccr2-deficient MRL/lpr mice survived significantly longer than MRL/lpr wild-type mice despite similar levels of circulating immunoglobulins and comparable immune complex depositions in the glomeruli of both groups. Anti-dsDNA antibody levels, however, were reduced in the absence of Ccr2. The frequency of CD8+ T cells in peripheral blood was significantly lower in Ccr2-deficient MRL/lpr mice. Thus Ccr2 deficiency influenced not only monocyte/macrophage and T cell infiltration in the kidney but also the systemic T cell response in MRL/lpr mice. These data suggest an important role for Ccr2 both in the general development of autoimmunity and in the renal involvement of the lupus-like disease. These results identify Ccr2 as an additional possible target for the treatment of lupus nephritis.     

Viral double-stranded RNA aggravates lupus nephritis through Toll-like receptor 3 on glomerular mesangial cells and antigen-presenting cells

How viral infections trigger autoimmunity is poorly understood. A role for Toll-like receptor 3 (TLR3) was hypothesized in this context as viral double-stranded RNA (dsRNA) activates dendritic cells to secrete type I interferons and cytokines that are known to be associated with the disease activity in systemic lupus erythematosus (SLE). Immunostaining of nephritic kidney sections of autoimmune MRL(lpr/lpr) mice revealed TLR3 expression in infiltrating antigen-presenting cells as well as in glomerular mesangial cells. TLR3-positive cultured mesangial cells that were exposed to synthetic polyinosinic-cytidylic acid (pI:C) RNA in vitro produced CCL2 and IL-6. pI:C RNA activated macrophages and dendritic cells, both isolated from MRL(lpr/lpr) mice, to secrete multiple proinflammatory factors. In vivo, a single injection of pI:C RNA increased serum IL-12p70, IL-6, and IFN-alpha levels. A course of 50 microg of pI:C RNA given every other day from weeks 16 to 18 of age aggravated lupus nephritis in pI:C-treated MRL(lpr/lpr) mice. Serum DNA autoantibody levels were unaltered upon systemic exposure to pI:C RNA in MRL(lpr/lpr) mice, as pI:C RNA, in contrast to CpG-DNA, failed to induce B cell activation. It therefore was concluded that viral dsRNA triggers disease activity of lupus nephritis by mechanisms that are different from those of bacterial DNA. In contrast to CpG-DNA/TLR9 interaction, pI:C RNA/TLR3-mediated disease activity is B cell independent, but activated intrinsic renal cells, e.g., glomerular mesangial cells, to produce cytokines and chemokines, factors that can aggravate autoimmune tissue injury, e.g., lupus nephritis.     

Platelet activating factor receptor blockade ameliorates murine systemic lupus erythematosus

Untreated 16-week-old MRL/MpJ-lpr/lpr (lpr) mice, when compared to congenic MRL/MpJ-+/+ (+/+) mice, are characterized by a systemic lupus erythematosus syndrome, including severe glomerulonephritis, proteinuria and reduction of renal function. We hypothesized that platelet activating factor (PAF), a potent chemotactic and proinflammatory phospholipid mediator synthesized and released by circulating cells, glomerular mesangial and renal medullary interstitial cells, may play a role in the development of renal injury in lupus mice. We assessed renal PAF synthesis in lpr as well as +/+ mice and the effect of treatment with a PAF receptor blocking agent. Treatment with the PAF receptor antagonist L659,989 for four weeks, starting at 12 weeks of age, significantly reduced acute glomerular infiltration and proliferation, and prevented chronic glomerular histological changes; proteinuria and serum creatinine levels were also significantly reduced in treated mice. Renal PAF production was increased in lpr when compared to +/+ mice, and treatment with L659,989 restored renal PAF synthesis to the control levels. Our results support the hypothesis that PAF can be one of the mediators of glomerular injury characteristic of murine lupus nephritis, and indicate the possible therapeutic utility of PAF receptor antagonists in immunologic renal diseases.     

CCR1 blockade reduces interstitial inflammation and fibrosis in mice with glomerulosclerosis and nephrotic syndrome

BACKGROUND: CC chemokines mediate leukocyte infiltration into inflamed tissue. We have recently shown that blockade of the CC chemokine receptor CCR1 reduces interstitial inflammation and fibrosis in murine obstructive nephropathy. However, it is not known whether CCR 1 blockade is protective in progressive renal injury associated with severe proteinuria. We therefore studied the effect of the small-molecule CCR1 antagonist BX471 in a murine model of adriamycin-induced focal segmental glomerulosclerosis (FSGS) with nephrotic syndrome and progressive interstitial inflammation and fibrosis. METHODS: Adriamycin nephropathy with persistent proteinuria was induced in male BALB/c mice by two intravenous injections of adriamycin (13 mg/kg) at day 0 and 14. BX471 treatment was started at day 14 when proteinuria and interstitial inflammation had developed. At 6 weeks, renal histology was studied by morphometry and immunohistochemistry. RESULTS: At week 6, adriamycin-treated mice showed FSGS, associated with tubulointerstitial injury consisting of tubular dilation and atrophy, interstitial leukocyte infiltration, and fibrosis. The mRNA expression of CCR1 and CC chemokines, including the CCR1 ligands CCL3 (MIP-1alpha) and CCL5 (RANTES), was up-regulated in diseased kidneys, with a prominent interstitial expression of CCL5. Compared to vehicle-treated controls BX471 significantly reduced the amount of macrophages and T lymphocytes in interstitial lesions by 51% and 22%, respectively. Markers of renal fibrosis such as interstitial fibroblasts (48%) and interstitial volume (23%) were significantly reduced by BX471 treatment. In contrast, the extent of proteinuria and glomerular sclerosis was not affected by BX471 treatment. CONCLUSION: Blockade of CCR1 substantially reduced interstitial leukocyte accumulation and the subsequent renal fibrosis in a murine model of nephrotic syndrome and FSGS. These findings support a role for CCR1 in interstitial leukocyte recruitment and suggest that CCR1 blockade might be a new therapeutic strategy in progressive nephropathies such as FSGS.     

Proteinuria: detection and role in native renal disease progression

The presence of albuminuria or proteinuria constitutes a sign of kidney damage and, together with the estimation of glomerular filtration rate, is based on the evaluation of chronic kidney disease. Proteinuria is a strong marker for progression of chronic kidney disease, and it is also a marker of increased cardiovascular morbimortality.Filtration of albumin by the glomerulus is followed by tubular reabsorption, and thus, the resulting albuminuria reflects the combined contribution of these 2 processes. Dysfunction of both processes may result in increased excretion of albumin, and both glomerular injury and tubular impairment have been involved in the initial events leading to proteinuria.Independently of the underlying causes, chronic proteinuric glomerulopathies have in common the sustained or permanent loss of selectivity of the glomerular barrier to protein filtration. The integrity of the glomerular filtration barrier depends on its 3-layer structure (the endothelium, the glomerular basement membrane, and the podocytes). Increased intraglomerular hydraulic pressure or damage to glomerular filtration barrier may elicit glomerular or overload proteinuria. The mechanisms underlying glomerular disease are very variable and include infiltration of inflammatory cells, proliferation of glomerular cells, and malfunction of podocyte-associated molecules such as nephrin or podocin.Albumin is filtered by the glomeruli and reabsorbed by the proximal tubular cells by receptor-mediated endocytosis. Internalization by endocytosis is followed by transport into lysosomes for degradation. The multiligand receptors megalin and cubilin are responsible for the constitutive uptake in this mechanism. Albumin and its ligands induce expression of inflammatory and fibrogenic mediators resulting in inflammation and fibrosis resulting in the loss of renal function as a result of tubular proteinuria. TGF-β, which may be induced by albumin exposure, may also act in a feedback mechanism increasing albumin filtration and at the same time inhibiting megalin- and cubilin-mediated albumin endocytosis, leading to increased albuminuria.Urinary proteins themselves may elicit proinflammatory and profibrotic effects that directly contribute to chronic tubulointerstitial damage. Multiple pathways are involved, including induction of tubular chemokine expression, cytokines, monocyte chemotactic proteins, different growth factors, and complement activation, which lead to inflammatory cell infiltration in the interstitium and sustained fibrogenesis. This tubulointerstitial injury is one of the key factors that induce the renal damage progression.Therefore, high-grade proteinuria is an independent mediator of progressive kidney damage. Glomerular lesions and their effects on the renal tubules appear to provide a critical link between proteinuria and tubulointerstitial injury, although several other mechanisms have also been involved. Injury is transmitted to the interstitium favoring the self-destruction of nephrons and finally of the kidney structure.     

Chemokines in renal injury

The main function of chemokines is to guide inflammatory cells in their migration to sites of inflammation. During the last 2 decades, an expanding number of chemokines and their receptors have driven broad inquiry into how inflammatory cells are recruited in a variety of diseases. Although this review focuses on chemokines and their receptors in renal injury, proinflammatory IL-17, TGFβ, and TWEAK signaling pathways also play a critical role in their expression. Recent studies in transgenic mice as well as blockade of chemokine signaling by neutralizing ligands or receptor antagonists now allow direct interrogation of chemokine action. The emerging role of regulatory T cells and Th17 cells during renal injury also forges tight relationships between chemokines and T cell infiltration in the development of kidney disease. As chemokine receptor blockade inches toward clinical use, the field remains an attractive area with potential for unexpected opportunity in the future.     

The cytokine TWEAK modulates renal tubulointerstitial inflammation

TNF-like weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily of cytokines. In addition to binding and activating the fibroblast growth factor-inducible 14 receptor, TWEAK may regulate apoptosis, proliferation, and inflammation; however, the role of this system in kidney injury is unknown. In vitro, it was found that TWEAK induced the sustained activation of NF-kappaB in a murine tubular epithelial cell line (MCT). NF-kappaB activation was associated with degradation of IkappaB-alpha; translocation of RelA to the nucleus; and increased mRNA and protein expression of monocyte chemoattractant protein-1, RANTES, and IL-6. Similarly, in vivo, the systemic administration of TWEAK induced renal NF-kappaB activation, chemokine and IL-6 expression, and interstitial inflammation in mice. Parthenolide, which prevents IkappaB-alpha degradation, inhibited TWEAK-induced NF-kappaB activation and prevented the aforementioned changes in vitro and in vivo. After folic acid-induced acute kidney injury, fibroblast growth factor-inducible 14 expression increased in mouse tubular epithelium. Neutralization of TWEAK decreased the expression of chemokines in tubular cells and reduced interstitial inflammation. In conclusion, TWEAK has NF-kappaB-dependent proinflammatory effects on tubular epithelial cells in vitro and in vivo. Moreover, blockade of TWEAK reduces tubular chemokine expression and macrophage infiltration, suggesting that TWEAK modulates acute kidney injury by regulating the inflammatory response.     

Renin-angiotensin system activation and interstitial inflammation in human diabetic nephropathy

BACKGROUND: The molecular mechanisms of renal injury in diabetic nephropathy (DN) are not completely understood, although inflammatory cells play a key role. The renin-angiotensin system (RAS) is involved in kidney damage; however, few studies have examined the localization of RAS components in human DN. Our aim was to investigate in renal biopsies the expression of RAS and their correlation with proinflammatory parameters and renal injury. METHODS: The biopsies from 10 patients with type 2 diabetes mellitus and overt nephropathy were studied for the expression of RAS components by immunohistochemistry (IH). In addition, by Southwestern histochemistry we studied the in situ detection of the activated nuclear factor kappa B (NFkappaB), and by IH and/or in situ hybridization (ISH), the expression of monocyte chemoattractant protein-1 (MCP-1) and regulated upon activation, normal T cell expressed and secreted (RANTES), whose genes are regulated by NFkappaB. RESULTS: Angiotensin-converting enzyme (ACE) immunostaining was elevated in tubular cells and appeared in interstitial cells. Elevated levels of angiotensin II (Ang II) immunostaining were observed in tubular and infiltrating interstitial cells. There was also a down-regulation of AT1 and up-regulation of AT2 receptors. An activation of NFkappaB and a marked up-regulation of NFkappaB-dependent chemokines mainly in tubular cells was observed. Elevated levels of NFkappaB, chemokines, and Ang II in tubules were correlated with proteinuria and interstitial cell infiltration. CONCLUSIONS: Our results show that in human DN, RAS components are modified in renal compartments, showing elevated local Ang II production, activation of tubular cells, and induction of proinflammatory parameters. These data suggest that Ang II contributes to the renal inflammatory process, and may explain the molecular mechanisms of the beneficial effect of RAS blockade.     

Progression of kidney disease: blocking leukocyte recruitment with chemokine receptor CCR1 antagonists

Chronic kidney disease (CKD) is usually associated with interstitial leukocytic cell infiltrates, which may contribute to disease progression by production of proinflammatory, proapoptotic, and profibrotic mediators. Recruiting leukocytes into the kidney involves local expression of chemotactic cytokines, that is, chemokines, that interact with respective chemokine receptors on the leukocyte's outer surface. Thus, specific chemokine receptor antagonists may represent an attractive therapeutic concept to interfere with renal leukocyte recruitment. Among the proinflammatory chemokine receptors, chemokine receptor (CCR)-1 has nonredundant roles for leukocyte adhesion to activated vascular endothelium and for transendothelial migration. In fact, blocking CCR-1 with specific small-molecule antagonists was shown to retard progression in various types of rodent CKD models. Here we discuss the perspective of CCR-1 as a new potential target for the treatment of CKD.     

The Duffy antigen receptor for chemokines is up-regulated during acute renal transplant rejection and crescentic glomerulonephritis

BACKGROUND: Recruitment of leukocytes during immune responses requires the coordinate expression of adhesion molecules in concert with chemokines and their receptors. The Duffy antigen receptor for chemokines (DARC) binds multiple chemokines and is expressed on postcapillary venules in the normal kidney. The chemokine receptor CCR5, which shares the ligand regulated upon activation, normal T-cell expressed and secreted (RANTES) with DARC, is expressed by infiltrating T cells in the renal interstitium. As DARC might be involved in the attraction of CCR5-positive cells, we studied the distribution of DARC and CCR5 in two forms of cell-mediated renal injury: renal allograft rejection and crescentic glomerulonephritis (cGN). METHODS: A total of 87 renal specimens, including 12 pretransplant biopsies, 47 transplant biopsies (Banff 1, N = 10; Banff 2, N = 19; and various other lesions N = 18), and 28 biopsies from patients with cGN, was analyzed. Immunohistochemistry for CCR5 and DARC was performed on serial sections of formalin-fixed and paraffin-embedded tissue. RESULTS: Compared with pretransplant biopsies, the mean number of DARC-positive interstitial venules was significantly increased during both transplant rejection and cGN. This was accompanied by an infiltration of CCR5-positive leukocytes. During transplant rejection, the number and distribution of CCR5-positive cells correlated with DARC-positive venules. Infiltrating CCR5-positive leukocytes were found mainly in the interstitium, often clustering around Bowman's capsules in biopsies from cGN. The number of glomerular CCR5 positive cells is low, but they are common in a subset of crescents. CONCLUSIONS: We hypothesize that the increased number of DARC-positive venules in areas of interstitial injury and the colocalization with CCR5-positive infiltrating leukocytes may indicate a role for endothelial DARC expression during leukocyte adhesion and interstitial infiltration.     

Overexpression of chemokines, fibrogenic cytokines, and myofibroblasts in human membranous nephropathy

Overexpression of chemokines, fibrogenic cytokines, and myofibroblasts in human membranous nephropathy. BACKGROUND: Proteinuria plays a central role in the progression of glomerular disease, and there is growing evidence suggesting that it may determine tubular cell activation with release of chemokines and fibrogenic factors, leading to interstitial inflammatory reaction. However, most studies on this subject have been performed in experimental models, and the experience in human kidney biopsies has been scarce. We analyzed the tissue sections of patients with idiopathic membranous nephropathy (IMN), a noninflammatory glomerular disease that may follow a progressive disease with heavy persistent proteinuria, interstitial cell infiltration, and decline of renal function. METHODS: Paraffin-embedded biopsy specimens from 25 patients with IMN (13 progressive and 12 nonprogressive) were retrospectively studied by immunohistochemistry [monocyte chemoattractant protein-1 (MCP-1), regulated on activation normal T-cell expressed and secreted chemokine (RANTES), osteopontin (OPN), platelet-derived growth factor-BB (PD-GF-BB)] and in situ hybridization [MCP-1, RANTES, PDGF-BB, transforming growth factor-beta1 (TGF-beta1)]. Moreover, we studied the presence of myofibroblasts, which were identified by the expression of alpha-smooth muscle actin (alpha-SMA), the monocytes/macrophages (CD68-positive cells), and T-cell infiltration (CD4+ and CD8+ cells). All of the patients were nephrotic and without treatment at time of the biopsy. RESULTS: A strong up-regulation of MCP-1, RANTES, and OPN expression was observed, mainly in tubular epithelial cells, with a significant major intensity in the progressive IMN patients. A strong correlation between the mRNA expression and the corresponding protein was noted. The presence of these chemokines and OPN was associated with interstitial cell infiltration. TGF-beta and PDGF were also up-regulated, mainly in tubular epithelial cells, with a stronger expression in the progressive IMN, and an association with the presence of myofibroblasts was found. CONCLUSIONS: Patients with severe proteinuria and progressive IMN have an overexpression in tubular epithelial cells of the chemokines MCP-1, RANTES, and OPN and the profibrogenic cytokines PDGF-BB and TGF-beta. Because this up-regulation was associated with an interstitial accumulation of mononuclear cells and an increase in myofibroblastic activity, it is suggested that those mediators are potential predictors of progression in IMN. Finally, based on experimental data and the findings of this article, we speculate that severe proteinuria is the main factor responsible for the up-regulation of these factors in tubular epithelial cells.