Proximal tubule cells stimulated by lipopolysaccharideinhibit macrophage activation

Proximal tubule cells stimulated by lipopolysaccharide inhibit macrophage activation.BackgroundTubule 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.MethodsTubule 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.ResultsTNF-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.ConclusionTubule 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

Role of CD8+ cells in the progression of murine adriamycin nephropathy

Role of CD8+ cells in the progression of murine adriamycin nephropathy.BackgroundMany studies have shown that interstitial inflammation in human and experimental renal disease is characterized by T-cell infiltration, but published data on the involvement of inflammatory cell subsets in progressive tubulointerstitial lesions are often conflicting. A previous study suggested a role for cytotoxic T lymphocytes in the damaging effect of CD4+ T-cell depletion in murine adriamycin (ADR) nephropathy, a model of focal segmental glomerulosclerosis (FSGS), and tubulointerstitial inflammation. The aim of this study was to investigate the role of CD8+ cells in this model.MethodsMale BALB/c mice were treated with five intraperitoneal injections of anti-CD8 monoclonal antibody (mAb), beginning from five days after ADR treatment, when overt proteinuria was established. Seven mice in each of groups A (ADR + mAb), B (ADR only), and C (saline treated, age matched) were sacrificed at week 6. Changes in renal function and histopathological features were assessed. Tubulointerstitial inflammation and glomerular inflammation were examined immunohistochemically.ResultsmAb treatment reduced CD8+ cell levels to <2% of normal in spleen. Proteinuria in group A was no different from that in group B at week 6, but was markedly higher than in group C. Creatinine clearance was significantly ameliorated by anti-CD8 treatment (71.8 ± 4.9 μL/min vs. 29.2 ± 2.8 in group B and 81.9 ± 3.7 in group C). Morphometric analysis showed less FSGS in group A compared with group B (6.5 ± 1.9 vs. 13.0 ± 2.8, P < 0.001), as well as less tubular atrophy (indicated by increased ratio of tubule cell height to tubular diameter, 0.25 ± 0.24 in group A vs. 0.04 ± 0.02 in group B, P < 0.05). CD8 depletion also reduced interstitial expansion (6.3 ± 2.2% vs. 16.4 ± 3.1 in group B, P < 0.001) and fibrosis (P < 0.01). Macrophage infiltration in tubulointerstitium was less in group A than in group B (P = 0.052). The number of interstitial CD4+ cells appeared to increase after anti-CD8 treatment, but was not statistically different between groups A and B.ConclusionAnti-CD8 treatment protects against renal functional and structural injury in this murine model of chronic proteinuric renal disease

Can murine diabetic nephropathy be separated from superimposed acute renal failure?

Can murine diabetic nephropathy be separated from superimposed acute renal failure?BackgroundStreptozotocin (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.MethodsMale Balb/c mice received either (1) STZ (225mg/kg by intraperitoneal injection.); or (2) two doses of STZ 5 days apart (150mg/150mg/kg; 75mg/150mg/kg; 75mg/75mg/kg; and 100mg/100mg/kg by intravenous injection). Another strain of mice, C57BL/6J, also received STZ (200mg/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.ResultsFollowing a single intraperitoneal injection of 225mg/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 (200mg/kg), at week 2 post-diabetes. At an optimal diabetogenic dose and route (75mg/150mg/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.ConclusionBy 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

HIF-1alpha expression follows microvascular loss in advanced murine adriamycin nephrosis

Cellular hypoxia has been proposed as a major factor in the pathogenesis of chronic renal injury, yet to date there has been no direct evidence to support its importance. Therefore, we examined cortical hypoxia in an animal model of chronic renal injury (murine adriamycin nephrosis; AN) by assessing nuclear localization of the oxygen-dependent alpha-subunit of hypoxia-inducible factor-1 (HIF-1alpha) in animals 7, 14, and 28 days after adriamycin. Results were assessed in conjunction with quantitation of the cortical microvasculature (by CD34 immunostaining) and cortical expression of VEGF. Cortical apoptosis was also examined by terminal deoxynucleotidyl transferase dUTP nick-end labeling staining. A dramatic and significant increase in nuclear localization of HIF-1alpha was seen 28 days after adriamycin in the context of severe glomerular and tubulointerstitial damage. Areas of nuclear HIF-1alpha staining did not colocalize with areas of cellular apoptosis. AN was also associated with a significant attenuation of the peritubular capillaries that was significant at 14 and 28 days after adriamycin. Cortical VEGF expression fell in a stepwise manner from day 7 until day 28 after adriamycin. In conclusion, these data are consistent with a significant increase in cellular hypoxia occurring in the advanced stages of murine AN. Increased cortical hypoxia was preceded by significant reductions in both the number of peritubular capillaries (i.e., oxygen supply) and the angiogenic cytokine VEGF. Apart from providing the first direct evidence for cellular hypoxia in a model of chronic renal disease, these results suggest that a primary dysregulation of angiogenesis may be the cause of increased hypoxia in this model

DNA vaccination with CCL2 DNA modified by the addition of an adjuvant epitope protects against nonimmune toxic renal injury

CC-chemokine-encoding DNA vaccine has been reported to be capable of inducing immunologic memory to corresponding pathogenic self CC-chemokines in animal models of autoimmune disease. This study investigated whether introduction of a foreign T helper epitope into monocyte chemoattractant protein 1 (CCL2) DNA vaccine could boost its immunogenicity by inducing strong neutralizing autoantibody against the pathogenic chemokine CCL2 sufficiently to be protective in a classically nonimmune model of disease, Adriamycin nephropathy (AN). Modification of the CCL2 DNA vaccine by replacing a surface loop region of CCL2 sequence with tetanus toxoid T helper epitope P30 elicited a strong self-specific CCL2 autoantibody production, as well as an IFN-gamma-producing T cell cellular response. The increased immunogenicity of modified CCL2 DNA vaccination but not unmodified CCL2 DNA vaccination was protective against functional and structural renal injury in rat AN. The protective effect of the modified CCL2 DNA vaccine was associated with blockade of glomerular and interstitial macrophage recruitment by neutralizing autoantibody against CCL2, which plays a critical role in eliciting renal injury in AN. Therefore, modification with a foreign T helper epitope breaks self-tolerance by inducing a cellular and humoral response against self-protein and provides a strategy to increase the potency of DNA vaccination sufficiently to afford protection in toxin-induced chronic renal disease