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The need for early nephrology referral. The incidence of end-stage renal disease is increasing. Progression to end stage can be slowed if kidney damage is detected at an early stage. Prognosis and outcomes in patients with chronic kidney disease have been related to the quality of predialysis care and the timing of referral. Many patients with chronic kidney disease are referred to a nephrologist close to commencement of renal replacement therapy. This leads to suboptimal management of complications of chronic renal insufficiency, and increased morbidity and mortality of patients on renal replacement therapy. This article addresses the evidence that examines the view that patients need to be referred early in order to avoid complications of chronic renal insufficiency. Early referral can be achieved through improved communication between primary health care givers and nephrology services. A multidisciplinary approach has a significant impact on outcomes. In the face of rising incidence of chronic kidney disease, early referral of all patients is not possible. Therefore, identification of patients at risk for rapid deterioration of renal function is important in order to rationalize and reduce health care expenditure. [ABSTRACT FROM AUTHOR]

Editorial. Focuses on the role of mast cells in causing tubulointerstitial fibrosis (TIF). Etiology of the disease; Signs and symptoms of TIF; Function of mast cells in human kidney; Change in the number of mast cells in renal interstitium during renal diseases.

Proximal tubule cells stimulated by lipopolysaccharide inhibit macrophage activation. Background. Tubule cells can produce a variety of cytokines, extracellular matrix (ECM) components, and adhesion molecules in vitro and in vivo. It is generally assumed that stimulated tubule cells are proinflammatory and at least partially responsible for interstitial inflammation. However, the overall effect of tubular cells on interstitial cells is unknown. In this study, pro- and anti-inflammatory cytokine production and net effects on macrophages of tubule cells activated by lipopolysaccharide (LPS) were examined. Methods. Tubule cells stimulated with LPS expressed tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-12, monocyte chemoattractant protein-1 (MCP-1), IL-10, and transforming growth factor-β (TGF-β). Conditioned media werecollected from confluent monolayers of rat tubule cells stimulated, or not, by LPS for 4 and 18 hours, respectively. Macrophages were cultured with conditioned media and/or LPS (0.5 μg/mL) for 18 hours. Results. TNF-α and IL-lβ mRNA of macrophages stimulated by LPS increased more than fivefold when cultured with control conditioned media from unstimulated tubule cells. Surprisingly, TNF-α and IL-lβ levels of macrophages stimulated by LPS were not increased when cultured with conditioned media from activated tubule cells. Neutralizing antibodies to IL-10 and TGF-β were used to define the inhibitory component(s) in conditioned medium. Anti-IL-10, but not anti-TGF-β, abolished partially the inhibitory effects of conditioned media on macrophages. Conclusion. Tubule cells produce both pro- and anti-inflammatory cytokines and the net effect, partially explained by IL-10, of tubule cells activated with LPS is to inhibit activity of macrophages. Thus, the net effect of activated tubule cells on interstitial pathology may in certain circumstances, be anti- rather than pro-inflammatory. [ABSTRACT FROM AUTHOR]

TGF-β1 induces epithelial-mesenchymal transition (EMT) and autophagy in a variety of cells. However, the role of autophagy in TGF-β1-induced EMT has not been clearly elucidated and the underlying mechanisms are unclear. In the present study, we found that TGF-β1 induced both autophagy and EMT in mouse tubular epithelial C1.1 cells. Inhibition of autophagy by 3-methyladenine or siRNA knockdown of Beclin 1 reduced TGF-β1-induced increase of vimentin and decreased E-cadherin expression. In contrast, rapamycin-associated enhancement of TGF-β1-induced autophagy increased EMT of C1.1 cells. Serum rescue inhibited autophagy followed by reversal of EMT. Blocking of autophagosome-lysosomal but not proteosomal degradation reduced the decrease of E-cadherin, demonstrating a role for autophagy in degradation of E-cadherin during EMT. Autophagy promoted the activation of Src and Src-associated phosphorylation of β-catenin at Y-654 leading to pY654-β-catenin/p-Smad2 complex formation. Chromatin immunoprecipitation assay demonstrated binding by the pY654-β-catenin/p-Smad2 complex to ILK promoter thus increasing ILK expression. Taken together, our results demonstrate that TGF-β1-induced autophagy links β-catenin and Smad signaling to promote EMT in C1.1 cells through a novel pY654-β-catenin/p-Smad2/ILK pathway. The pathway delineated links disruption of E-cadherin/β-catenin-mediated cell–cell contact to induction of EMT via upregulation of ILK. [ABSTRACT FROM AUTHOR]

Abstract: CCL2 DNA vaccines are directed against the host chemoattractant molecule CCL2 (MCP-1), a key chemokine in recruiting macrophages to sites of inflammation. Macrophages recruited by CCL2 lead to progressive renal injury. In rat models of disease unmodified CCL2 DNA vaccine in combination with a CCL5 (RANTES) DNA vaccine can protect against chronic renal disease. The mechanism of protection involves the induction of auto-antibodies to the CCL2. Introduction of the adjuvant p-tet into the DNA structure of the CCL2 vaccine leads to enhanced potency with the induction of specific Th1 cellular immunity. The strategies outlined here demonstrate a model for developing potent vaccines against highly restricted self targets. [Copyright &y& Elsevier]

Can murine diabetic nephropathy be separated from superimposed acute renal failure? Background. Streptozotocin (STZ) is commonly used to induce diabetes in experimental animal models, but not without accompanying cytotoxic effects. This study was undertaken to ( 1) determine an optimal dose and administration route of STZ to induce diabetic nephropathy in wild-type mice but without the concurrent acute renal injury resulting from cytotoxic effects of STZ and ( 2) evaluate the pattern of tubular injury and interstitial inflammation in this model. Methods. Male Balb/c mice received either ( 1) STZ (225 mg/kg by intraperitoneal injection.); or ( 2) two doses of STZ 5 days apart (150 mg/150 mg/kg; 75 mg/150 mg/kg; 75 mg/75 mg/kg; and 100 mg/100 mg/kg by intravenous injection). Another strain of mice, C57BL/6J, also received STZ (200 mg/kg intravenously or intraperitoneally). Renal function and histology were examined at weeks 1, 2, 4, and 8 after induction of diabetes. In initial optimization studies, animals were sacrificed at week 1 or week 2 and histology examined for acute renal injury. Results. Following a single intraperitoneal injection of 225 mg/kg of STZ, only two thirds of animals developed hyperglycemia, yet the model was associated with focal areas of acute tubular necrosis (ATN) at week 2. ATN was also observed in C57BL/6J mice given a single intravenous or intraperitoneal dose of STZ (200 mg/kg), at week 2 post-diabetes. At an optimal diabetogenic dose and route (75 mg/150 mg/kg by intravenous injection 5 days apart), all mice developed diabetes and no ATN was observed histologically. However, even with this regimen, glomerular filtration rate (GFR) was significantly impaired from week 2. This regimen was accompanied by progressive histologic changes, including tubular and glomerular hypertrophy, mesangial area expansion, as well as interstitial macrophage, CD4+ and CD8+ T-cell accumulation. Conclusion. By careful optimization of STZ dose, a stable and reproducible diabetic murine model was established. However, even in this optimized model, renal functional impairment was observed. The frequency of ATN and functional impairment casts doubt on conclusions about experimental diabetic nephropathy drawn from reports in which ATN has not been excluded rigorously. [ABSTRACT FROM AUTHOR]

DNA vaccination with naked DNA encoding MCP-1 and RANTES protects against renal injury in adriamycin nephropathy.Background.We have previously shown that monocyte chemoattractant protein-1 (MCP-1) and regulated upon activation, normal T-cell expressed and secreted (RANTES) are significantly increased in renal cortex in adriamycin nephropathy. In this study, we tested the effect of DNA vaccination encoding the C-C chemokines MCP-1 and RANTES in a rat model of adriamycin nephropathy.Methods.Both reverse transcription-polymerase chain reaction (RT-PCR) products of MCP-1 and RANTES used as constructs were cloned into a pTarget vector for naked DNA vaccination. Two hundred micrograms of DNA was injected into the tibialis anterior muscle four times at weekly intervals. One week after the last DNA vaccination, rats received adriamycin. All animals were sacrificed 4 weeks after adriamycin administration. Changes in renal function and histologic features were assessed. Enzyme-linked immunosorbent assay (ELISA) and Western blot were used for autoantibody determination. Antibody specificity was assessed in in vitro transmigration assays.Results.Chemokine DNA vaccination significantly reduced proteinuria (P<0.05) and ameliorated creatinine clearance (P<0.05) at 2, 3, and 4 weeks after adriamycin administration. Morphometric analysis showed less glomerular sclerosis (P<0.001) and interstitial infiltrates (P<0.005) in chemokine DNA vaccination group compared with control groups. Anti-MCP-1 and RANTES autoantibodies were detected in higher concentrations in chemokine DNA vaccinated rats than in control rats (P<0.001) and serum from vaccinated rats blocked T-cell transmigration to MCP-1 and RANTES.Conclusion.In this study, we have shown that naked DNA vaccination against MCP-1 and RANTES ameliorates the progression of renal disease in the rat adriamycin nephropathy model of chronic proteinuric renal disease. The protective mechanism may involve the production of autoantibodies against MCP-1 and RANTES. [ABSTRACT FROM AUTHOR]

The role of tubulointerstitial inflammation. Exploration of the role of tubulointerstitial inflammation in experimental chronic renal disease (CRD) is an essential step to understanding and finding new treatments for human CRD. Adriamycin nephrosis (AN) is an experimental analogue of human focal glomerular sclerosis and tubulointerstitial inflammation. Using murine and rat AN, we have systematically investigated the pathogenic roles of chemokines, costimulatory molecules, and inflammatory cells, such as macrophages and effector and regulatory T lymphocytes. The profile of humoral and cellular mediators was studied in vitro and in vivo. The pathogenic significance of various factors was investigated by DNA vaccination, leukocyte reconstitution and depletion, retroviral transduction, and blockade with monoclonal antibodies. Renal cortical and tubular cell CC-chemokines, including MCP-1, RANTES, and MIP-1α, were up-regulated via mediation of NFκB, and contributed to disease by attracting inflammatory cells into the interstitium. The role of these chemokines was confirmed by DNA vaccination. CD40-CD40L costimulation signals were involved in expansion and activation of the inflammatory infiltrate, whereas PD-1 signals were inhibitory, and CD28-B7 appeared to have a neutral effect. Macrophage and CD8+ T cells were shown to be effectors of injury, whereas CD4+CD25+ and γδ T cells acted as regulatory cells. FoxP3 transduction was able to convert naïve T cells to CD4+CD25+ regulatory T cells. There is a broad range of humoral and cellular factors involved in the pathogenesis of experimental CRD, some of which are potential targets for treatment of human CRD. [ABSTRACT FROM AUTHOR]

Transcriptome analysis using microarray technology represents a powerful unbiased approach for delineating pathogenic mechanisms in disease. Here molecular mechanisms of renal tubulointerstitial fibrosis (TIF) were probed by monitoring changes in the renal transcriptome in a glomerular disease-dependent model of TIF (adriamycin nephropathy) using Affymetrix (mu74av2) microarray coupled with sequential primary biological function-focused and secondary "baited"-global cluster analysis of gene expression profiles. Primary cluster analysis focused on mRNAs encoding matrix proteins and modulators of matrix turnover as classified by Onto-Compare and Gene Ontology and identified both molecules and pathways already implicated in the pathogenesis of TIF (e.g. transforming growth factor β1CTGF-fibronectin-1 pathway) and novel TIF-associated genes (e.g. SPARC and Matrilin-2). Specific gene expression patterns identified by primary extracellular matrix-focused cluster analysis were then used as bioinformatic bait in secondary global clustering, with which to search the renal transcriptome for novel modulators of TIF. Among the genes clustering with ECM proteins in the latter analysis were endoglin, clusterin, and gelsolin. In several notable cases (e.g. claudin-1 and meprin1β) the pattern of gene expression identified in adriamycin nephropathy in vivo was replicated during transdifferentiation of renal tubule epithelial cells to a fibroblast-like phenotype in vitro on exposure to transforming growth factor-β and epidermal growth factor suggesting a role in fibrogenesis. The further exploration of these complex gene networks should shed light on the core molecular pathways that underpin TIF in renal disease. [ABSTRACT FROM AUTHOR]

Blockade of CD40-CD40 ligand protects against renal injury in chronic proteinuric renal disease. Background. Interaction between CD40 and CD40 ligand (CD40L) is involved in both cognate and innate immune responses. Blockade of CD40-CD40L interactions reduces severity of renal injury in murine lupus nephritis and membranous nephropathy. We hypothesized that CD40-CD40L could contribute to renal injury in models that are not antibody-dependent, and that anti-CD40L could diminish inflammation and fibrosis in murine adriamycin nephropathy. Methods. Male BALB/c mice were divided into three groups (N = 6 per group): (1 ) saline-treated, age-matched control; (2 ) adriamycin only; and (3 ) MR1 + adriamycin. In group 3, mice were treated with intraperitoneal injections of anti-CD40L antibody (clone MR1, 0.4 mg per mouse) after the onset of proteinuria at days 5, 7, 9, and 11 after adriamycin treatment. Animal subgroups were compared at 14 and 42 days after induction of adriamycin nephropathy. Functional and pathologic markers of disease severity, cellular components of interstitial inflammation, and the degree of CD40 expression were assessed. Relative cortical RNA expression of the chemokine monocyte-chemoattractant protein-1 (MCP-1) and regulated on activation normal T cell expressed and secreted (RANTES) was also compared between animal groups. Results. CD40 was weakly expressed in tubules of normal mice but was expressed in tubules, interstitium, and glomeruli of mice with adriamycin nephropathy in a time-dependent manner. MR1 treatment resulted in a significant attenuation of the severity of adriamycin nephropathy at day 42 [e.g., glomerular sclerosis (%), group 3, 20.1 ± 4.7 vs. group 2, 30.2 ± 7.2, P < 0.001]. CD40L blockade significantly reduced tubulointerstitial injury as well [tubular diameter (μm), group 3, 42.5 ± 6.9 vs. group 2, 66.3 ± 13.7, P < 0.001; and group 1, 37.3 ± 5.7, P < 0.01; tubular cell height (μm), group 3, 16.3 ± 1.7 vs. group 2, 11 ± 1.8, P < 0.01; and group 1, 18.2 ± 1.9, P < 0.01; interstitial volume (%), group 3, 13.9 ± 5.1 vs. group 2, 26.2 ± 4.9, P < 0.001; and group 1, 1.3 ± 0.7, P < 0.001; proteinuria (mg/24 hours), group 3, 1.8 ± 0.6 vs. group 2, 4.3 ± 0.8, P < 0.001; and group 1, 0.7 ± 0.2, P < 0.05; and creatinine clearance (μL/min), group 3, 75 ± 4 vs. group 2, 35 ± 2, P < 0.001; and group 1, 82 ± 4, P < 0.01] were also improved by MR1. MR1 treatment also resulted in a significant reduction in the number of cortical macrophages at both 14 and 42 days after adriamycin (P < 0.01). Cortical expression of MCP-1 and RANTES was significantly reduced by MR1 treatment at 42 days after adriamycin (P < 0.01 and P < 0.05, respectively). Conclusion. Blockade of CD40-CD40L interaction protects against renal structural and functional injury in this murine model of chronic proteinuric renal disease. [ABSTRACT FROM AUTHOR]

Background: Since the Fas/Fas Ligand (FasL) interaction is recognized as a major pathway of apoptosis in immune cells, we hypothesized that selective expression of FasL by tubular cells (TC) may promote the resolution of interstitial inflammation by inducing apoptosis of infiltrating immune cells. In this study, the effect of FasL transfection of rat TC on apoptosis of leukocytes was examined.Methods: Rat tubule cells (NRK52E) were transfected with plasmids constructed using human and rat FasL (hFasL and rFasL). The propensity of activated, transfected TC to undergo apoptosis was examined. Similarly, the effects of FasL transfection on apoptosis of Jurkat cells and activated leukocytes were assessed directly following co-culture for 12 hours and in a cell insert system intended to assess the effects of soluble FasL. Fas and FasL expression was assessed by flow cytometry and apoptosis was examined using Annexin V staining and the TUNEL method.Results: Expression of FasL in TC was increased after FasL transfection. Transfected TC showed no detectable increase in apoptosis following lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha) activation. Jurkat cell apoptosis was increased ninefold and eightfold after co-culture with TC transfected with hFasL and rFasL, respectively (67.0 +/- 12.1% and 60.1 +/- 8.8% vs. 6.7 +/- 1.8% with un-transfected TC, P < 0.01). Similarly, apoptosis of activated leukocytes was increased fourfold by co-culture (26.8 +/- 4.9% vs. 6.7 +/- 2.0% with untransfected TC, P < 0.01). Leukocyte apoptosis also was increased in an insert culture system (18.2 +/- 4.4% vs. 5.8 +/- 2.3% with un- transfected TC, P < 0.01). No increase of TC apoptosis was detected in any of the co-culture experiments.Conclusion: Enhanced expression of FasL by TC is capable of inducing apoptosis of activated leukocytes, without evidence for increased susceptibility to apoptosis of the transfected cells themselves. This suggests a potential role for this approach in the limitation and resolution of renal tubulointerstitial inflammation. [ABSTRACT FROM AUTHOR]

Examines the role of CD8[sup+] cells in the progression of murine adriamycin nephropathy. Effects of antibody treatment to CD8[sup+] cell levels; Increase of creatinine clearance; Reduction of interstitial expansion by CD8 depletion.

Examines the role of CD4[sup+] T cells to disease progression and loss of renal function in chronic proteinuric renal disease (CPRD). Combination of adriamycin and CD4[sup+] depletion mice; Effects of CD4 depletion on recruitment of inflammatory cells; Number of inflammatory infiltrates.

Investigates the sequence of histologic and immunohistochemical events of progressive adriamycin nephropathy in mice. Characterization of progressive renal disease; Analysis of leukocytic inflammatory pattern; Detection of overt proteinuria.

Lipopolysaccharide-induced MCP-1 gene expression in rat tubular epithelial cells is nuclear factor-κB dependent. Background. Endotoxin is an important factor in the development of acute renal failure related to infection and in acceleration of chronic nephritis. Lipopolysaccharide (LPS; the major component of endotoxin) is one of the most potent triggers for renal cells to produce monocyte chemoattractant protein-1 (MCP-1), a key cytokine involved in immune cell recruitment into the renal interstitium in acute and chronic renal diseases. Knowledge about the transcriptional regulation of MCP-1 in renal tubular epithelial cells in response to LPS is incomplete. Methods. Transcriptional regulation of MCP-1 was investigated in rat proximal tubule cells (PTCs) in primary culture and was exposed to LPS using electromobility shift assay and supershift analysis for nuclear factor-κB (NF-κB) and Western blot for the NF-κB inhibitory protein IκB. To prove the role for NF-κB, activator protein (AP-1), and sequence-specific transcription factor (Sp1), mutation and deletion analysis was performed using a 3.5 kb fragment of rat MCP-1 5′-flanking region inserted into a luciferase reporter construct transfected into tubular epithelial cell line (NRK-52E). Results. LPS increased NF-κB in a dose- and time-dependent manner, which paralleled that of MCP-1 mRNA expression. IκBα decreased within 30 minutes of LPS treatment, but returned to basal levels by two hours. IκBβ levels were depressed within one hour and remained low throughout the culture period after LPS stimulation. The activity of the transfected 5′-flanking region of the MCP-1 gene increased nearly threefold after LPS stimulation. Mutation or deletion of NF-κB binding sites, located in the enhancer region of the 5′-flanking region, resulted in a total loss of LPS-induced increase in luciferase activity. Mutation of putative AP-1 and Sp1... [ABSTRACT FROM AUTHOR]

Inhibition of nuclear factor-κB activation reduces cortical tubulointerstitial injury in proteinuric rats. Background.Protein-induced chemokine expression in proximal tubular cells is mediated by the transcription factor nuclear factor-kappa B (NF-κB). We hypothesized that in vivo inhibition of renal NF-κB activation would reduce interstitial monocyte infiltration in a rat model of nonimmune proteinuric tubulointerstitial inflammation. Methods.Male Wistar rats received a single intravenous injection of doxorubicin hydrochloride [adriamycin (ADR), 7.5 mg/kg] and were studied 7, 14, 21, and 28 days later. In a second study, inhibitors of NF-κB [N-acetylcysteine (NAC; 150 mg/kg, b.i.d., i.p.), pyrrolidine dithiocarbamate (PDTC, 50 mg/kg, b.i.d., i.p.)] or vehicle were commenced on day 14 after the onset of proteinuria and were continued until day 30. Results.Rats injected with ADR had increased proteinuria (UpV, day 28, 474 ± 57; control, 18 ± 2 mg/day; P < 0.01) and cortical tubulointerstitial injury [tubule cell atrophy, interstitial volume, and monocyte/macrophage (ED-1) infiltration]. Electrophoretic mobility shift assay of nuclear extracts from whole cortex of ADR rats demonstrated that NF-κB activation (p50/65, p50/c-Rel) increased from day 7 (4.7 ± 0.2 fold-increase above control; P < 0.01) was maximal at day 28 (6.2 ± 0.7; P < 0.01) and correlated with UpV (r = 0.63; P < 0.05) and interstitial ED-1 infiltration (r = 0.67; P < 0.01). Chronic treatment of ADR rats with PDTC suppressed NF-κB activation (by 73%; P < 0.05) without any effect on UpV. NF-κB inhibition with PDTC was accompanied by a reduction in tubule cell atrophy (59%; P < 0.01), interstitial volume (49%; P < 0.05) and ED-1 infiltration (48%; P < 0.01), and cortical lipid peroxidation (41%; P < 0.05) compared with vehicle-treated ADR rats. In contrast NAC had no effect on NF-κB activation,... [ABSTRACT FROM AUTHOR]