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Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that plays a pathogenic role in experimental crescentic glomerulonephritis (GN). Renal expression of MIF is also upregulated in human GN and correlates with leukocytic infiltration, histologic damage, and renal dysfunction. The study presented here examined whether MIF can be measured in urine and if so, whether the urine MIF concentration reflects the degree of renal injury. Urine and serum MIF was measured by enzyme-linked immunosorbent assay in 10 normal healthy volunteers and in a cohort of 63 patients with GN (2 thin basement membrane disease [TBM], 15 membranous GN, 10 focal segmental glomerular sclerosis, 20 IgA glomerularnephritis, 11 crescentic GN, 10 systemic lupus erythematosis World Health Organization class IV). Renal MIF expression was assessed by immunostaining of biopsy tissue. MIF was detected in urine from normal volunteers (mean+/-SD; 191+/-132 pg MIF/+/-mol creatinine). The urine MIF concentration was unchanged in patients with nonproliferative nephropathies (343 +/- 397 pg MIF/+/-mol Cr) but was increased 3.4-fold in proliferative nephropathies (645+/-527 pg MIF/mumol Cr; P < 0.05 versus normal and nonproliferative). Stratified analysis showed the greatest increase in urine MIF in crescentic GN (4.5-fold). In contrast, serum MIF levels were not different between normal patients and any patient group. Immunostaining demonstrated a significant increase in renal MIF expression in proliferative glomerulonephritides that was associated with macrophage and T cell infiltration. There was a significant correlation between the urine MIF concentration and renal MIF expression, but not with serum MIF, indicating a renal origin for the excreted urine MIF. The urine MIF concentration also correlated with the degree of renal dysfunction, histologic damage, and leukocytic infiltration, but not with the amount of proteinuria. In conclusion, this study shows that the urine MIF concentrati

Background: Low serum 25-hydroxyvitamin D (25[OH]D) levels have been associated with chronic kidney disease in cross-sectional studies. However, this association has not been studied prospectively in a large general population-based cohort. Study Design: Prospective cohort study. Setting & Participants: 6,180 adults 25 years or older participating in the baseline and 5-year follow-up phases of the Australian Diabetes, Obesity and Lifestyle (AusDiab) Study. Predictor: Serum 25(OH)D levels <15 ng/mL were considered deficient. Outcomes & Measurements: Incident chronic kidney disease was defined as being negative at baseline but positive after 5 years for (1) reduced estimated glomerular filtration rate (eGFR; <60 mL/min/1.72 m2) or (2) albuminuria (spot urine albumin-creatinine ratio >=2.5 mg/mmol [>=22.1 mg/g] for men and >=3.5 mg/mmol [>=30.9 mg/g] for women). Result(s): 623 (10.9%) participants were vitamin D deficient, 161 developed incident reduced eGFR, and 222 developed incident albuminuria. In participants with and without vitamin D deficiency, annual age-standardized incidences were 0.92% (95% CI, 0.56%-1.30%) and 0.59% (95% CI, 0.51%-0.68%), respectively, for eGFR <60 mL/min/1.72 m2 and 1.50% (95% CI, 1.06%-1.95%) and 0.66% (95% CI, 0.56%-0.76%), respectively, for albuminuria. In multivariate regression models, vitamin D deficiency was associated significantly with the 5-year incidence of albuminuria (OR, 1.71; 95% CI, 1.12-2.61; P = 0.01), but not reduced eGFR (OR, 0.93; 95% CI, 0.53-1.66; P = 0.8). Limitation(s): The observational nature of the study does not account for unmeasured confounders. Only baseline 25(OH)D level was measured and therefore may not accurately reflect lifetime levels. Differences in baseline characteristics of participants who were included compared with those excluded due to missing data or follow-up may limit the applicability of results to the original AusDiab cohort. Conclusion(s): Our prospective cohort study shows that vitamin

This study compared the measurements of total body water (TBW) by 58% body weight (TBW58%), the Watson equation (TBWWV) and bioelectric impedance (TBWBIA) with the gold standard, Deuterium oxide (TBWD2O) dilution method in twenty continuous ambulatory peritoneal dialysis (CAPD) patients. TBW volumes were highest when calculated as TBW58% (42.6 +/- 9.4 liter) and lowest when calculated from TBWWV (34.6 +/- 6.8 liter). TBWBIA underestimated TBW when compared to TBWD2O, although the difference was not statistically significant (37.1 +/- 9.8 liter and 38.8 +/- 9.3 liter, respectively). In fact, TBWBIA correlated strongly with TBWD2O (r = 0.8, P < 0.0001). These discrepancies resulted in significant differences when Kt/V week-1 derived from the four methods were compared. To determine the effect of percent fat mass on the estimation of TBW by each method, we compared TBW and Kt/V week-1 derived from the four methods in nine CAPD patients who had normal percent fat mass (Non-Obese) and 11 CAPD patients who had greater than normal % fat mass (Obese). In the Non-Obese group, there was close correlation of TBWBIA, TBWWV and TBW58% when compared with TBWD2O (r = 0.93, P < 0.001, r = 0.89, P < 0.01 and R = 0.86, P < 0.01, respectively). Also, Kt/V week-1 derived from TBWBIA, TBWWV and TBW58% correlated strongly with Kt/V week-1 from TBWD2O (r = 0.93, P < 0.0005, r = 0.83, P < 0.01 and r = 0.8, P < 0.01, respectively). In the Obese patients, the correlations between TBWD2O and TBWBIA and TBWWV were only moderate in strength (r = 0.67, P = 0.02 and r = 0.61, P = 0.04, respectively). There was no significant correlation between TBW58% and TBWD2O in the Obese group (r = 0.44, P = NS). Kt/V week-1 derived from TBWBIA; TBWWV and TBW58% had only moderate correlation with Kt/V week-1 from D2O. Similar findings in TBW measurements and Kt/V week-1 were obtained in CAPD patients (N = 9) who had normal BMI (< 26 kg/m2; termed, Normal Weight) and those who had BMI > 26 kg/m2 (N = 11; terme

Although CD44 is known to be involved in a wide array of cell to cell and cell to matrix interactions, its role in immune-mediated disease is not well understood. Therefore, using immunogold electron microscopy we have determined the precise localisation of CD44 in the rat lung in experimental Goodpasture's (GP) syndrome, a model of immune-mediated pulmonary disease. In normal rat lung CD44 was present on the surface of alveolar macrophages but was not detectable on endothelium. In GP syndrome there was strong CD44 expression on all infiltrating inflammatory leucocytes, both adherent to endothelium and within the alveolar spaces and interstitium. However the most striking finding was the progressively strong antibody staining for CD44 on pulmonary endothelium of alveolar capillaries and larger vessels over the 21 days of GP syndrome. In situ hybridisation confirmed that the endothelial CD44 staining was due to local protein synthesis. All epithelial cell surfaces, including bronchial epithelium and type I and II alveolar epithelial cells, were negative in normal rat lung and GP syndrome. De novo CD44 expression by endothelial cells during the progression of GP syndrome may contribute to leucocyte recruitment and cell-mediated lung injury.

Background: Urine dipsticks, an inexpensive accessible test for proteinuria, are widely advocated for mass screening; however, their diagnostic accuracy in the general community is largely unknown. Study Design: Evaluation of diagnostic test accuracy in a cross-sectional cohort. Setting & Participants: AusDiab, a representative survey of Australian adults 25 years and older (conducted in 1999/2000). Stratified cluster random sampling from 11,247 individuals participating in the biomedical examination; complete urinalysis data available for 10,944. Index Test: Urine dipsticks (Bayer Multistix), with a positive result defined as <1+ or trace or higher protein. Reference Test: Albumin-creatinine ratio (ACR), measured on a random spot urine sample. Reference test positivity was defined as ACR <30 mg/g or ACR <300 mg/g. Result(s): Numbers of participants with ACR <30, 30-300, and <300 mg/g were 10,219 (93.4%), 634 (5.8%), and 91 (0.8%), respectively. The area under the receiver operating characteristic curve (AUC) for dipstick detection of ACR <30 mg/g was 0.8451 +/- 0.0129 (SE) in men and 0.7775 +/- 0.0131 in women (P < 0.001). The AUROC for dipstick detection of ACR <300 mg/g was 0.9904 +/- 0.0030 in men and 0.9950 +/- 0.0016 in women (P = 0.02). Dipstick result <1+ identified ACR <30 mg/g with 57.8% sensitivity (95% CI, 54.1%-61.4%) and 95.4% specificity (95% CI, 95.0%-95.8%) and identified ACR <300 mg/g with 98.9% sensitivity (99% CI, 92.1%-100%) and 92.6% specificity (99% CI, 92.0%-93.3%). A dipstick result of trace or higher identified ACR <30 mg/g with 69.4% sensitivity (95% CI, 65.9%-72.7%) and 86.8% specificity (95% CI, 86.1%-87.4%) and identified ACR <300 mg/g with 100% sensitivity (99% CI, 94.3%-100%) and 83.7% specificity (99% CI, 82.8%-84.6%). A negative dipstick result (less than trace) had a negative predictive value of 97.6% (95% CI, 97.2%-97.9%) for ACR <30 mg/g and a negative predictive value of 100% (99% CI, 99.9%-100%) for ACR <300 mg/g. The probabili

Renal fibrosis and tubular apoptosis are common mechanisms of progressive kidney disease. In vitro studies have implicated the c-Jun amino-terminal kinase (JNK) pathway in these processes. Both of the major JNK isoforms, JNK1 and JNK2, are expressed in the kidney, but their relative contribution to JNK signaling is unknown. This study investigated the role of JNK signaling in renal fibrosis and tubular apoptosis in the unilateral ureteral obstruction model using two different approaches: (1) Mice that were deficient in either JNK1 or JNK2 and (2) a specific inhibitor of all JNK isoforms, CC-401. Western blotting and immunostaining identified a marked increase in JNK signaling in the obstructed kidney, with substantial redundancy between JNK1 and JNK2 isoforms. Administration of CC-401 blocked JNK signaling in the rat obstructed kidney and significantly inhibited renal fibrosis in terms of interstitial myofibroblast accumulation and collagen IV deposition. This effect was attributed to suppression of gene transcription for the profibrotic molecules TGF-beta1 and connective tissue growth factor. CC-401 treatment also significantly reduced tubular apoptosis in the obstructed kidney. Genetic deletion of JNK1 or JNK2 did not protect mice from renal fibrosis in the unilateral ureteral obstruction model, but JNK1 deletion did result in a significant reduction in tubular cell apoptosis. In conclusion, this is the first study to demonstrate that JNK signaling plays a pathogenic role in renal fibrosis and tubular apoptosis. Furthermore, JNK1 plays a nonredundant role in tubular cell apoptosis. These studies identify the JNK pathway as a potential therapeutic target in progressive kidney disease. Copyright © 2007 by the American Society of Nephrology.

Background. Macrophages constitute 38% to 60% of infiltrating cells during acute renal allograft rejection. Their contribution to tissue damage during acute rejection was examined by depleting macrophages in a rat model. Methods. Lewis rats underwent bilateral nephrectomy and then received a Dark Agouti renal allograft and liposomal-clodronate, control phosphate-buffered saline liposomes, or saline intravenously (n=7 per group) on days 1 and 3 postsurgery. Grafts were harvested on day 5. Results. Liposomal-clodronate treatment resulted in a 70% reduction in blood ED1+ monocytes and 60% reduction in intragraft ED1+ macrophages (both P < 0.01). Half of all remaining interstitial ED1+ cells were undergoing apoptosis (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling +/ED1+), and thus functional depletion of more than 75% of macrophages was achieved. Histologic and functional parameters of acute rejection were attenuated: interstitial infiltrate, tubulitis, and glomerulitis (P < 0.01); tubular cell apoptosis (P < 0.001); tubular cell proliferation (P < 0.001); and serum creatinine (P < 0.01). Production of inducible nitric oxide synthase by infiltrating cells and urinary nitric oxide excretion was reduced by 90% (P < 0.001). In contrast, no reduction in the number of other leukocytes was seen (CD3+, CD4+, CD8 +, and natural killer cells). Activation of lymphocytes (CD25 +) and production of lymphocyte effector molecules (granzyme B) were unaltered. Conclusion. This study demonstrates that macrophages contribute to tissue damage during acute rejection.

Background. Many antibody labelling studies have suggested that there are few or no glomerular macrophages in mouse models of glomerulonephritis, despite the presence of a prominent interstitial macrophage infiltrate. These findings conflict with studies of human and rat glomerulonephritis. Therefore, we examined whether heterogeneity of macrophage antigen expression could explain this apparent discrepancy. Methods. Kidneys were collected from normal mice and mice killed at 2 and 10 days after induction of accelerated anti-glomerular basement membrane (GBM) glomerulonephritis. Following fixation, macrophages were detected by immunoperoxidase staining in serial kidney sections using antibodies recognising CD11b, F4/80 and CD68. Results. Induction of anti-GBM nephritis caused a progressive increase in glomerular and interstitial leukocytes. At days 2 and 10, there were more CD68+ macrophages in glomeruli than macrophages expressing CD11b or F4/80. At day 10, CD68+ macrophages accounted for almost all glomerular CD45+ total leukocytes. In contrast, CD11b+ and F4/80+ macrophages at day 10 accounted for only 65 and 13% of glomerular leukocytes, respectively. However, in the interstitium the number of macrophages expressing CD68, CD11b and F4/80 were not different. Conclusion. Antibody detection of mouse CD68 identifies all glomerular macrophages in mouse anti-GBM nephritis, indicating a similar infiltrate to that seen in human and rat anti-GBM nephritis. Our finding of substantial heterogeneity in glomerular macrophage antigen expression in this model suggests that previous studies of mouse glomerulonephritis may have under-estimated glomerular macrophages and their role in glomerular injury.

Macrophage accumulation has long been recognized as a feature of allograft rejection, yet the role of macrophages in rejection remains underappreciated. Macrophages contribute to both the innate and acquired arms of the alloimmune response and thus may be involved in all aspects of acute and chronic allograft rejection. Recent advances in macrophage biology have allowed a better understanding of the mechanisms of macrophage accumulation, their state of activation and the pleuripotent roles they play in allograft rejection. Therapeutic attention to macrophages, in addition to T lymphocytes, may lead to improved outcomes in organ transplantation. Copyright © 2005 by Lippincott Williams & Wilkins.

Production of macrophage-colony stimulating factor (M-CSF), the major macrophage growth factor, is increased in tissues during inflammation. Therefore, we determined whether M-CSF, acting through its receptor c-fms, contributes to macrophage accumulation at a site of tissue injury. Daily treatment with anti-c-fms or control antibody was given to mice with renal inflammation resulting from unilateral ureteric obstruction (UUO). Following UUO, kidney M-CSF mRNA increased in association with macrophage accumulation (days 1, 5, and 10) and local macrophage proliferation (days 5 and 10). Anti-c-fms treatment caused a minor inhibition of monocyte recruitment at day 1, reduced macrophage accumulation by 75% at day 10, but did not affect blood monocyte counts or the CD4 and CD8 lymphocytic infiltrate. Prevention of macrophage accumulation by anti-c-fins treatment was associated with a 90% reduction in local macrophage proliferation at days 5 and 10 without evidence of increased macrophage apoptosis. Therefore, M-CSF/c-fms signaling plays a key role in macrophage accumulation during tissue injury.

Background. Advances in immunosuppression have made renal transplantation an effective therapy for end stage renal failure; with low rejection rates and long graft survival times. However, the major adverse consequences, infection and malignancy have not diminished. To predict this risk a score of immune competence has been developed from the simultaneous laboratory assessment of multiple parameters of immune function. Methods. The immune status of 152 transplant recipients (138 renal and 14 pancreas/renal) was assessed by measurement of lymphocyte subsets, mitogen-induced T-cell proliferative responses, neutrophil phagocytic capacity and reactive oxygen species (ROS) generation. A scoring system was devised based on the average number of these parameters below 10th percentile of normal. Results. The most common abnormality was B-cell lymphopenia (85%) followed by reduced neutrophil ROS production (63% of patients), NK cell lymphopenia (50%), lymphocyte mitogen response (49%) and CD4 number (23%). The abnormalities were unrelated to the duration of immunosuppression (up to 15 years), and variable combinations of cyclosporine A, azathioprine, prednisolone and mycophenolate mofetil (MMF) (except for a consistent reduction in lymphocyte mitogen response in MMF treated patients). Retrospective comparison of infective episodes showed a significantly greater index of infections in patients with the worst score compared with a normal score. Conclusions. The data suggests that this quantification of immune function may allow assessment of the level of host immune defence reflecting the level of drug-induced immunosuppression and thus risks of immunosuppressive complications.

Interleukin-1 is a proinflammatory cytokine produced in glomerulonephritis. Blocking the action of interleukin-1 by the administration of the interleukin-1 receptor antagonist (IL-1ra) has been shown to prevent renal function impairment, reduce glomerular injury, inhibit leukocyte infiltration, and suppress tubulointerstitial damage in experimental antiglomerular basement membrane disease. A key mechanism in the entry of leukocytes into the kidney is the interaction between the interleukin-1 inducible intercellular adhesion molecule-1 (ICAM-1; CD54) and lymphocyte function-associated antigen-1 (CD11a/CD18). Therefore, this study investigated whether the inhibition of this mechanism was the means by which IL-1ra suppressed leukocyte infiltration in rat accelerated antiglomerular basement membrane glomerulonephritis. Disease was induced in two groups of six rats; animals were treated by constant sc infusion of recombinant human IL-1ra or saline from the initiation of disease until being euthanized 14 days later. In saline-treated animals, there was marked up-regulation of ICAM-1 in the glomerulus and interstitium, In which was associated with leukocyte infiltration. In particular, focal accumulation of CD11a+ and CD18+ cells was apparent in areas of tubulointerstitial damage exhibiting intense ICAM-1 expression. IL-1ra treatment partially reduced glomerular ICAM-1 expression and leukocyte infiltration. However, IL-1ra treatment resulted in a dramatic inhibition of interstitial ICAM-1 expression, interstitial leukocyte infiltration, and tubulointerstitial damage. In conclusion, this study has shown that interleukin-1 is a major inducer of ICAM-1 expression within the renal tubulo-interstitium--a process associated with focal leukocyte infiltration and tubulointerstitial damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor alpha production by renal tumors, acting through the epidermal growth factor receptor, has been implicated in malignant transformation by studies which compared gene expression in neoplastic and normal human tissue. We sought confirmation of this hypothesis by measuring the growth responses of a human renal tumor cell line to the addition of epidermal growth factor and transforming growth factor alpha. Surprisingly, it was found that both growth factors could induce either mitogenic or inhibitory signals depending on the growth status of the cultures. Confluent cultures were stimulated by both growth factors, and nonconfluent cultures were inhibited, as determined by thymidine incorporation, cell cycle analysis, and direct cell counting. These signals appear to use different transduction pathways, as growth factor induced inhibition was reversed by Bordetella pertussis toxin (which affects G protein signaling), whereas the stimulatory effects were not reversed. Two clones isolated from these cells responded in the same manner as the main cell isolate. These data show that the same cell may display opposite responses to equivalent concentrations of the same growth factor, depending on the transduction pathway used after triggering by receptor occupancy of either ligand (epidermal growth factor or transforming growth factor alpha).

We examined the functional role of interleukin (IL)-1 in mesangial cell proliferation during rat anti-Thy-1 nephritis by blocking its action with IL- 1 receptor antagonist (IL-1ra). Anti-Thy-1 nephritis was induced by intravenous injection of 5 mg/kg OX-7 IgG (day 0) into inbred Wistar rats. Groups of animals (n = 9) were implanted with a micro-osmotic pump on day - 1, which delivered 25 mug hour human recombinant IL-1ra or saline continuously until the rats were killed at day 6, the peak of mesangial cell proliferation. Immunostaining showed that IL-1 was expressed by mesangial cells during disease. IL-1ra treatment did not affect the mild, but significant, proteinuria seen after OX-7 injection. Compared with saline treatment, IL-1ra treatment reduced mesangial cell proliferation ( 249%; P < 0.05), glomerular hypercellularity ( 29%; P < 0.05), and glomerular macrophage accumulation ( 20%; P < 0.05). However, IL-1ra treatment had no effect on glomerular IL-1 beta mRNA expression and caused only a small reduction in the high levels of glomerular expression of platelet-derived growth factor-beta protein ( 6%; P < 0.05). IL-1ra caused a modest reduction in the marked up-regulation of glomerular transforming growth factor-beta1 mRNA expression on day 6 ( 26%; P < 0.05), although urinary, excretion of this factor was unaffected. Interestingly, IL-1ra treatment had relatively little effect upon glomerular deposition of laminin, fibronectin, and collagen type IV seen in this acute disease. In conclusion, this study has 1) demonstrated that IL-1 is expressed by mesangial cells in vivo, 2) demonstrated that IL-1 is a mesangial cell growth factor in experimental mesangioproliferative nephritis, and 3) suggests that IL-1 has little or no fibrogenic activity in mesangial matrix deposition.

Objective: To assess the usefulness of fat-free mass (FFM) as an index of total body protein (TBPr) status in continuous ambulatory peritoneal dialysis (CAPD) patients. Design(s): TBPr was measured by in vivo neutron activation analysis (IVNAA) and expressed as a standardised protein index (PI). FFM was estimated by dual energy X-ray absorptiometry (DXA), whole body counting of total body potassium (TBK), and creatinine kinetics (CK), and expressed as a standardised FFM index (FFMI). FFM was also determined by a criterion method based on a four compartment model (4CM) which is defined as the sum of total body water determined by D2O dilution, TBPr determined by IVNAA, bone mineral determined by DXA, and glycogen estimated to be 4.4% of TBPr. Each patient was measured within a four hour period by all methods. Setting(s): Body Composition Laboratory, Monash Medical Centre. Subjects: Six male and twelve female CAPD patients (33-77 years). Result(s): FFMI assessed by DXA and by TBK agreed with measurements of PI on identifying the mean TBPr status of the CAPD group as significantly below a comparable normal reference population (mean Z score: PI = -1.01 (P < 0.05); FFMI by DXA = -0.50 (P < 0.05); FFMI by TBK = - 1.24 (P < 0.05)). In contrast, FFMI assessed by CK did not reveal a significantly reduced TBPr status (mean Z score: -0.70 (NS)). Furthermore, significant linear correlations were noted between PI and FFMI estimated by DXA and by TBK (r = 0.57 (P < 0.05) vs r = 0.69 (P < 0.005)) however no significant correlation was observed between PI and FFMI estimated by CK (r = 0.36 (NS)). Moderate variation in FFM hydration did not compromise the ability of DXA, TBK or CK to differentiate between protein depleted, normal and enriched patients. Comparison of FFM estimates between the criterion method and either DXA, TBK or CK revealed no significant bias (+ 1.8 kg vs -2.0 kg vs + 0.8 kg) and respective SEE values of 3.8 kg (8.3%), 5.9kg (14.3%) and 9.6 kg (21.7%). Conclusio

Background. Studies of infiltrating cells from acutely rejecting renal allografts show that a high proportion of these cells are macrophages, and early macrophage infiltration is a poor prognostic sign for transplant survival. Macrophage colony-stimulating factor (M-CSF), produced by tubular and mesangial cells, has been associated with macrophage infiltration and proliferation in experimental and human kidney diseases. We investigated the expression of M-CSF in a model of acute rejection. Methods. Lewis rats underwent bilateral nephrectomies and received an orthotopic Dark Agouti allograft or Lewis isograft. Animals received cyclosporine (10 mg/kg/day) from day 0 to day 3 and were killed at days 4, 8, or 14 after transplantation. Macrophages (ED1+) and T cells (W3-13+) were identified by immunohistochemistry, and M-CSF expression was identified by Northern blotting and in situ hybridization. Results. Isografts had normal renal function without histological evidence of rejection. Allografts exhibited a moderate infiltrate at day 4 but progressed to severe rejection at day 14, with elevated serum creatinine level and severe tubulointerstitial damage. Macrophages and T cells were present in equal proportion in the infiltrate at day 4. At day 14, the number of macrophages increased fivefold (2580/mm2), although T cells were unchanged (380/mm2). Proliferating macrophages (ED1+, BrdU+) increased from day 4 (4%) to day 14 (10%). M-CSF mRNA expression was strongly up-regulated in allografts compared with isografts and normal rat. In situ hybridization demonstrated M-CSF expression by resident and infiltrating cells. Renal tubular expression was minimally increased at day 4 but strongly up-regulated at day 14 (more than 50% of tubules positive), particularly in areas of tubular damage. Tubular M-CSF expression colocalized with areas of intense macrophage infiltration and proliferation. Serial sections with double labeling demonstrated that T cells were the dominant source o

Osteopontin (OPN) is a secreted acidic glycoprotein that has potent monocyte chemoattractant and adhesive properties. Up-regulation of tubular OPN expression is thought to promote interstitial macrophage infiltration in experimental nephritis; however, the role of OPN in glomerular lesions, particularly crescent formation, is unknown. The present study used Northern blotting, in situ hybridization and immunohistochemistry to examine OPN expression in a rat model of accelerated anti-GBM glomerulonephritis. Osteopontin mRNA and protein is expressed by some parietal epithelial cells, thick ascending limbs of Henle and medullary tubules and collecting ducts in normal rat kidney. De novo OPN mRNA and protein expression was evident in glomerular visceral and parietal epithelial cells in anti-GBM glomerulonephritis. Glomerular OPN expression preceded and correlated with macrophage infiltration in the development of hypercellularity, focal and segmental lesions and, notably, crescent formation. There was marked up- regulation of OPN expression by tubular epithelial cells that also preceded and correlated with interstitial macrophage (r = 0.93, P < 0.001) and T-cell infiltration (r = 0.85, P < 0.001). Both glomerular and tubular OPN expression correlated significantly with proteinuria (P < 0.001) and a reduction in creatinine clearance (P < 0.01). In addition, double immunohistochemistry showed co-expression of osteopontin and one of its ligands, CD44, in intrinsic renal cells. CD44 and OPN expression by parietal epithelial cells was evident in crescent formation, while virtually all OPN- positive tubules expressed CD44. Infiltrating macrophages and T-cells were CD44-positive, but only a small proportion of T-cells and few macrophages showed OPN expression. Interestingly, strong OPN mRNA and protein expression was seen in macrophage multinucleated giant cells. In summary, this study suggests that OPN promotes macrophage and T-cell infiltration in the development of renal les

Flow cytometry has recently become a useful technique for the quantitative analysis of cytoplasmic and nuclear antigens. We report here a rapid, simple, reproducible, and sensitive method for the simultaneous detection of cytoplasmic and nuclear antigens by flow cytometry. This technique involves the treatment of cell suspensions with 60 s of microwave oven heating after fixation with 2% paraformaldehyde. Following this treatment, a number of cytoplasmic and nuclear antigens were detected on the human myelomonocytic cell line U937 (CD68, PCNA and Ki-67), peripheral blood leukocytes from both normal donors and leukemia patients (CD68, lipocortin-1 and PCNA) and a rat mesangial cell line 1097 (desmin, alpha-smooth muscle actin) using a standard indirect immunofluorescent staining with mouse monoclonal antibodies (mAbs). There are several advantages of this technique over the routinely used methods currently available. Firstly, microwave treatment is a rapid, simple, and reproducible method, which largely reduces both time and cost expenditure, and,makes this technique widely available for flow cytometric analysis in many areas of diagnostic and research purposes. Secondly, microwave treatment produces optimal results for simultaneous detection of both cytoplasmic (CD68, lipocortin-1, desmin, alpha-smooth actin) and nuclear (PCNA, Ki-67) antigens. Thirdly, microwave treatment also produces a discrete, profile for DNA content. analysis. Finally, microwaving retains a clear discrimination between cells and debris as measured by light scatter. This study demonstrates that microwave treatment is a powerful technique which will be particularly applicable to flow cytometric analysis in the detection of many cytoplasmic and nuclear antigens.

Macrophage accumulation is a prominent feature in most forms of human glomerulonephritis and correlates with renal dysfunction. Macrophages can directly mediate acute renal injury in animal models, but the mechanisms of macrophage activation required for mediating renal injury are unknown. This study examined whether activation of the Jun amino terminal kinase (JNK) signaling pathway is necessary for macrophage-mediated renal injury. An adoptive transfer model was used in which rats were immunized with sheep IgG (day -5), made leukopenic by administration of cyclophosphamide (CyPh) (day -2), and then injected with sheep anti-glomerular basement membrane (GBM) serum (day 0). Animals were then given an intravenous injection of bone marrow-derived macrophages (BMM) (day 1) and killed 24 h later (day 2). The induction of proteinuria and glomerular cell proliferation (PCNA+ cells) in CyPh-treated anti-GBM disease was dependent on transfer of BMM. Exposure of BMM to the specific JNK inhibitor, SP600125, for 3 h before adoptive transfer had no effect on glomerular accumulation of BMM in CyPh-treated anti-GBM disease. However, SP600125 treatment of BMM caused a 75% reduction in proteinuria and a 70% reduction in glomerular cell proliferation (P < 0.01 versus vehicle or untreated BMM). In conclusion, this study has defined a critical role for the JNK signaling pathway in macrophage-mediated renal injury.

Background. Glomerular cellular crescents consist of epithelial cells and macrophages, which can undergo an irreversible process of fibrous organization. However, the origin of the fibroblast-type cells that mediate this fibrous organization is unclear. Methods. This study examined glomerular epithelial-myofibroblast transdifferentiation (GEMT) in the formation and evolution of glomerular crescents in two distinct rat models of glomerulonephritis: 5/6 nephrectomy and antiglomerular basement membrane (GBM) disease. Results. Early in the course of both disease models, and prior to crescent formation, immunohistochemistry staining and in-situ hybridization demonstrated de novo expression of alpha-smooth-muscle actin (alpha-SMA), a marker of smooth muscle cells and myofibroblasts, by glomerular parietal epithelial cells (GPEC). The expression of alpha-SMA by GPEC was accompanied by a loss of E-cadherin staining, a marker of epithelial cells. At this early stage of GEMT, ultrastructural studies identified the presence of characteristic actin microfilaments and dense bodies within GPEC which retained a normal epithelial morphology with apical-basal polarity and microvilli. A late stage of transdifferentiation was seen in fibrocellular crescents. In this case, GPEC attached to intact segments of the capsular basement membrane contained large bundles of actin microfilaments throughout the cell, and this was accompanied by a loss of polarity, microvilli, and tight junctions. There was a significant correlation between the presence of alpha-SMA+ GPEC and glomerular crescent formation. Cellular crescents contained small numbers of alpha-SMA+ myofibroblasts. These cells become the dominant population in fibrocellular crescents, which was associated with marked local proliferation. Relatively few alpha-SMA+ myofibroblasts remained in fibrotic/organizing crescents. Most cells within cellular and fibrocellular crescents expressed transforming growth factor-beta (TGF-beta) and basi

Background. Most cases of pauci-immune crescentic glomerulonephritis (PICGN) are associated with serum anti-neutrophil cytoplasmic antibodies (ANCA). This article studied the sensitivity and specificity of serum ANCA, determined by flow cytometry and indirect immunofluorescence (IIF), to identify patients with acute PICGN. Methods. 577 adults presenting for first biopsy of their native kidneys with serum taken for ANCA (flow cytometry and IIF) determination were studied. A positive ANCA was defined using a flow cytometric ANCA assay as a screening test, followed by a slide-based indirect IIF technique. Pathological confirmation of acute PICGN was used to assess the sensitivity and specificity of this combined approach and its positive predictive value (PPV) and negative predictive value (NPV) in patients presenting for renal biopsy due to abnormal urinary sediment. Results. Forty-nine patients were found to have acute PICGN on renal biopsy. Of these 47 were ANCA positive (sensitivity 95.9%). Overall 93 of the renal biopsy patients were ANCA positive, (specificity 91.3%). A further seven patients (two ANCA positive) had advanced sclerosing disease consistent with PICGN but without evidence of current disease activity. The PPV and NPV of ANCA, assessed by flow cytometry and slide IIF, in predicting that patients presenting with undifferentiated renal disease would have acute PICGN was 50.5 and 99.8%, respectively. Conclusions. Flow cytometric screening of serum for ANCA in patients undergoing renal biopsy has a high NPV for determining those with acute PICGN. It may provide a rapid, simple screening test for this lesion in laboratories using diagnostic flow cytometry and may complement IIF/ELISA in evaluating ANCA positive patients.

CD44 is a widely expressed cell surface glycoprotein which is involved in both cell-matrix and cell-cell interactions which regulate a variety of processes, including leucocyte migration and activation. Therefore, we examined the expression of CD44, and its major ligand hyaluronan, during the induction and progression of experimental glomerulonephritis. Antibody staining of normal rat kidney showed constitutive CD44 expression by resident glomerular macrophages, parietal epithelial cells, medullary and occasional cortical tubules. There was a marked increase in CD44 expression over days 1, 7 and 21 of rat crescentic anti-glomerular basement membrane (GBM) glomerulonephritis. Infiltrating monocytes and lymphocytes were CD44+, with ultrastructural studies showing high levels of CD44 expressed on the surface of lymphocytes adherent to activated endothelium. Marked hyaluronan deposition was seen in areas of fibrosis on days 7 and 21, such as glomerular crescents and the periglomerular area. Hyaluronan deposition was accompanied by the presence of many CD44+ cells. Double immunohistochemistry showed that both CD44+ED1+ macrophages and CD44+ myofibroblasts (identified by expression of alpha-smooth muscle actin) were present in areas of fibrosis. There was also a dramatic increase in cortical tubular CD44 expression, which was most evident in areas of tubular damage. Although tubular epithelial cells expressed CD44 upon both the basolateral and luminal surface, CD44 expression was most prominent within tight junctions, suggesting a role for CD44-CD44 interactions in cell-cell adhesion within the tubule. Analysis of CD44 isoforms by reverse transcriptase-polymerase chain reaction (RT-PCR) showed that the standard form of CD44 predominated in both normal and diseased kidney. However, a series of alternatively spliced CD44 isoforms was also detected, whose expression was markedly increased during disease. At least seven isoforms containing the v6 domain were identified, with

There are no accurate noninvasive methods to distinguish renal allograft dysfunction due to rejection or cyclosporine nephrotoxicity. We have studied the value of Tc-99M-DTPA renal scanning in 90 episodes of renal allograft dysfunction occurring in 44 patients subjected to 57 renal biopsies in whom a clear diagnosis could be established. Renal scintigrams were assessed qualitatively and quantitatively by a blinded observer. Rejection was diagnosed when deterioration in perfusion occurred in the presence of maintained or declining radionuclide excretion. The diagnosis of cyclosporine nephrotoxicity was made by exclusion. The final diagnosis was based on the clinical response to therapy and/or the findings on renal biopsy. The scintigraphic diagnosis of rejection had a specificity of 87.9% and significantly contributed to the exclusion of cyclosporine nephrotoxicity (negative predictive value of 90.6%). Furthermore, a scintigraphic diagnosis compatible with cyclosporine nephrotoxicity, in the presence of a drug level above the therapeutic range, indicated a 90.4% probability of true nephrotoxicity. We conclude that, even in cyclosporine-treated renal transplant patients, Tc-99M-DTPA scintigraphy is of clinical value and can be incorporated into an effective diagnostic algorithm for allograft dysfunction.

Background. There is limited population-based epidemiological data on renal disease. An insight into the spectrum of clinically significant glomerulonephritis can be obtained from renal biopsy diagnoses. This is a descriptive report of biopsy-proven glomerulonephritis within a defined population. Methods. A retrospective review of the pathology reports of all native renal biopsies performed in the Australian state of Victoria in 1995 and 1997 was undertaken. Trends in the average annual age- and sex-specific incidence rates for biopsy-proven glomerulonephritis were calculated. Comparisons were made with the incidence rates of end-stage renal disease due to glomerulonephritis confirmed on renal biopsy. Results. The most common glomerulonephritides in adults are IgA disease, focal glomerulosclerosis, lupus nephritis and vasculitis, and in children are lupus nephritis focal glomerulosclerosis, IgA disease and minimal change disease. A male predominance is seen for all glomerulonephritides, except lupus nephritis, in both adults and children. An increase in incidence of disease with age, particularly in males, is seen for vasculitis and focal glomerulosclerosis. The most common glomerulonephritides on renal biopys are reflected in the most common causes of end-stage renal disease due to glomerulonephritis. Conclusions. This review has provided population-based descriptive epidemiological data on clinically significant glomerulonephritis. This data provides important clues for further studies relating to the identification of risk factors for the various types of glomerulonephritis.

Studies on the effect of rat testicular interstitial fluid (IF) on T- cell function have reported both stimulatory and inhibitory actions. Specific cytokines produced within the testis, particularly interleukin-1 (IL-1) and transforming growth factor beta (TGFbeta), may contribute to these apparently conflicting observations. In proliferation assays employing lectin- or antibody-activated thymocytes or mature T cells in vitro, adult rat testicular IF stimulated T-cell activation and/or proliferation at low assay doses and was inhibitory at higher doses. The stimulatory activity was blocked by recombinant IL-1 receptor antagonist. The inhibitory activity was not affected by a polyspecific TGFbeta antiserum. The biological characteristics of the inhibitor were distinct from those of a similar, but considerably less potent, activity in platelet-depleted serum. These data demonstrate that rat testicular IF contains biologically significant concentrations of IL-1 but has a predominantly inhibitory action on T-cell responses. The factor predominantly responsible for this inhibitory activity displays a relatively large apparent molecular weight, is protease sensitive and partially heat labile, but does not appear to be one of the known mammalian TGFbeta isoforms.

The fate of macrophages at the site of inflammation is unknown. We investigated this question in a macrophage-mediated model of crescentic glomerulonephritis in which macrophage accumulation is relatively stable despite the presence of high levels of local macrophage proliferation. Accelerated anti-glomerular basement membrane glomerulonephritis was induced in groups of six rats that were killed on day 1, 7, 14, or 21. Macrophage apoptosis was demonstrated in kidney sections by three methods: in situ terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) combined with ED1 antibody immunostaining of macrophages, ED1 immunostaining combined with classical nuclear morphology, and electron microscopy. Substantial macrophage apoptosis became evident on day 14 of the disease, following the appearance of high levels of macrophage proliferation. The parallel relationship between proliferation and apoptosis is the likely explanation for the stabilization of macrophage numbers within the inflamed kidney. A striking feature was that macrophage proliferation and apoptosis was largely restricted to areas of focal damage, such as in the development of glomerular crescents. Increasing levels of macrophage proliferation and apoptosis were evident as crescents developed from a cellular to a fibrocellular phenotype, with a dramatic reduction in both of these processes in the progression to a fibrotic phenotype, suggesting an important role for macrophage apoptosis in the resolution of fibrocellular crescents to an acellular fibrotic structure. In conclusion, this study has identified apoptosis as an important mechanism counterbalancing local proliferation in the regulation of macrophage accumulation at sites of inflammation. Indeed, apoptosis may be a central regulator of the progression and resolution of macrophage-mediated tissue injury.

There are relatively few monoclonal antibodies (MAbs) to rat monocyte/macrophages available. We describe here 2 new such antibodies. The first, 109.2, recognizes most rat monocyte/ macrophages and all polymorphs. The antigen recognized by this antibody is upregulated by 15 mins exposure to PMA (Phorbol myristate acetate) but down regulated by overnight exposure to LPS (lipopolysaccharide). It is probably an adhesion molecule and is likely to represent the rat equivalent of CD11 b. The second antibody, 112.1, recognizes lysosyme in rat macrophages, particularly alveolar macrophages. In addition it also recognizes lysosyme in hen, rabbit and human macrophages. It also recognizes lysosyme in other tissues such as Paneth cells and proximal renal tubular cells.

The world's disease profile is changing, and chronic diseases now account for the majority of global morbidity and mortality, rather than infectious diseases. The causes of chronic kidney diseases reflect this change and diabetes, together with hypertension, is now the major cause of end-stage renal failure worldwide, not only within the developed world, but also increasingly within the emerging world. Diabetes is of epidemic proportions, and its prevalence will double in the next 25 years, particularly in the developing countries. This will place an enormous financial burden on countries, including the cost of the management of end-stage renal failure. Thus, it is medically and economically imperative for awareness, detection, and prevention programs to be introduced across the world, particularly in the developing countries. This will require concerted action from global institutions, governments, health service providers, and medical practitioners. © 2005 by the International Society of Nephrology.

Chronic diseases are now in pandemic proportions and are the major cause of morbidity and mortality worldwide. Long-term predictions show that the burden will fall increasingly on developing countries. Type II diabetes is now the major global cause of chronic kidney disease. However, chronic kidney disease should not be viewed in isolation, because it is also a major risk factor for cardiovascular deaths. It is both a medical and economic imperative to introduce screening and management programs for chronic kidney disease, diabetes, hypertension, and cardiovascular disease worldwide but particularly in the emerging world. Such programs should be tailored to the needs of the individual country, involve local health providers, be assessable as to their efficacy, and become self-sufficient within 5 years. © 2005 by the International Society of Nephrology.

Background: Control of serum phosphate remains a difficult clinical issue in most hemodialysis (HD) patients. This study examines 2 nonpharmacological approaches to improving phosphate control in HD patients. Method(s): First, 9 stable HD patients underwent dialysis in random fashion for either 4 hours 3 times weekly or 5 hours 3 times weekly. Adjustments were made to blood flow rates such that Kt/V was the same for all sessions, thus allowing independent assessment of the influence of time. The primary end point was weekly dialysate phosphate removal. In the second study, 12 different patients underwent an exercise program in which they pedaled a bicycle ergometer either immediately before or during dialysis. Again, weekly dialysate phosphate removal was measured. Result(s): In the time study, urea reduction ratio (69% +/- 0.02% versus 68% +/- 0.07, 4 versus 5 hours) and weekly urea removal were no different between the 2 groups. However, weekly phosphate removal (3,007 +/- 641 versus 3,400 +/- 647 mg; P < 0.02) significantly improved with longer dialysis duration. Serum phosphate levels improved, but did not reach statistical significance in this short-term study. In the exercise study, weekly phosphate removal improved with exercise, but did not reach significance (2,741 +/- 715 [no exercise] versus 2,917 +/- 833 [exercise predialysis] versus 2,992 +/- 852 mg [exercise during dialysis]; P = 0.055), although comparing only exercise during dialysis with no exercise reached significance (P = 0.02). There was no significant difference in serum phosphate levels. Conclusion(s): Both increased dialysis time and exercise result in increased dialytic removal of phosphate and could be expected in the long term to improve phosphate control. © 2004 by the National Kidney Foundation, Inc.

Background: Microalbuminuria is an independent risk factor for cardiovascular morbidity and mortality in the general population. Standard immunochemical urinary albumin assays detect immunoreactive albumin, whereas high-performance liquid chromatography (HPLC) detects both immunoreactive and immunounreactive albumin. Method(s): Using data from the Australian Diabetes, Obesity, and Lifestyle cohort study of randomly selected community-based Australian adults, spot urine samples were tested for albuminuria (spot urine albumin-creatinine ratio [ACR]: normal, <30 mg/g; microalbuminuria, 30 to 300 mg/g; and macroalbuminuria, >300 mg/g) by using both immunonephelometry (IN) and HPLC (n = 10,010). Result(s): Bland-Altman analysis showed significant bias, with a greater ACR by means of HPLC, particularly at lower levels of ACR. Mean ACR was 15.8 mg/g (95% confidence interval [CI], 12.3 to 19.2) by means of IN compared with 30.0 mg/g (95% CI, 27.0 to 35.0) by means of HPLC. The prevalence of microalbuminuria was 4 times greater by means of HPLC compared with IN (20% versus 5.5%). In all demographic and comorbid subgroups associated with microalbuminuria, the prevalence of microalbuminuria increased by 2 to 4 times. A total of 1,743 subjects (17.4%) classified as normoalbuminuric by means of IN were reclassified as microalbuminuric by means of HPLC. Using multivariate logistic regression, women, patients with untreated and treated hypertension, and those with impaired glucose tolerance or diabetes were associated significantly with a change in category from normoalbuminuric to microalbuminuria by means of HPLC. Conclusion(s): HPLC measures significantly more urinary albumin within the normoalbuminuria and microalbuminuria range, resulting in a significant increase in prevalence of microalbuminuria. Longitudinal studies are needed to determine whether the extra individuals identified by means of HPLC are at increased risk for developing hard clinical outcomes (renal and cardio

Macrophage accumulation is a prominent feature in most types of human glomerulonephritis. In particular, tubulointerstitial macrophage accumulation correlates with the degree of renal dysfunction and is predictive of disease progression. Depletion studies have shown that macrophages can induce glomerular injury in experimental glomerulonephritis. Moreover, recent studies targeting chemokines and adhesion molecules have shown that inhibiting macrophage accumulation can suppress progressive renal injury in animal models of glomerulonephritis. Macrophages can produce many molecules with the potential to cause renal damage, although the precise mechanism(s) of macrophage-mediated renal injury have yet to be determined. It is now evident that tubules-a major source of chemokines and adhesion molecules-play an active role in promoting interstitial macrophage infiltration and activation. Thus, targeting pro-inflammatory functions of tubular epithelial cells may be an effective means to inhibit macrophage-mediated tubulointerstitial injury without causing systemic immunosuppression.

OBJECTIVES: The Atherosclerosis and Folic Acid Supplementation Trial (ASFAST) aimed to establish whether high-dose folic acid would slow the progression of atherosclerosis and reduce cardiovascular events in patients with chronic renal failure (CRF). BACKGROUND: Hyperhomocysteinemia is a potential contributor to the high rates of cardiovascular morbidity and mortality in patients with CRF. METHOD(S): A total of 315 subjects with CRF, mean age 57 years (range 24 to 79 years) were randomized to 15 mg folic acid daily or placebo and followed for a median of 3.6 years. The primary intima-media thickness (IMT) and clinical end points were: rate of progression of mean maximum carotid IMT and a composite of myocardial infarction (MI), stroke, and cardiovascular death. Secondary end points included all cardiovascular events and change in pulse wave velocity, systemic arterial compliance and augmentation index. Data were analyzed by intention-to-treat. RESULT(S): Plasma total homocysteine was reduced by 19% in the folic acid group. There was no significant difference between the treatment groups in rate of change of IMT or any measure of artery function. Seventy-seven events occurred in the folic acid group (14.9 per 100 patient-years) as compared with 86 in the placebo group (16.3 per 100 patient-years). The rates of the primary and secondary clinical end points at five years were not significantly different after adjustment for baseline differences between the groups (adjusted hazard ratio for MI, stroke, and cardiovascular death: 0.98 [95% confidence interval: 0.66 to 1.47]; p = 0.94; for all cardiovascular events: 0.95 [95% confidence interval: 0.69 to 1.30]; p = 0.75). CONCLUSION(S): High-dose folic acid does not slow atheroma progression or improve cardiovascular morbidity or mortality in patients with CRF. © 2006 by the American College of Cardiology Foundation.

Background: Determinants of native arteriovenous fistula (AVF) placement have been well studied. Little is known on whether these factors impact on subsequent blood flow (Qa) in the mature AVF. Method(s): Arteriovenous fistula Qa and cardiac index (CI) were determined by ultrasound dilution. Multiple linear regression was used to assess independent predictors of AVF Qa. Result(s): Of the 148 patients available for the analysis, 68% were male, with 61% using a radiocephalic AVF. Aetiology of renal disease was: 38% glomerulonephritis (GN), 22% diabetes mellitus (DM), 9% hypertension/ischaemic (HTN) and 31% other. Thirty per cent had coronary artery disease (CAD), 10% cerebrovascular disease and 11% peripheral vascular disease (PVD). Median (iqr) Qa was 1185 mL/min (790-1650) and CI was 3.15 L/min per 1.73 m2 (2.60-3.93). On univariable analysis, log CI (0.98, P< 0.001), age (-0.1 per 10 years, P = 0.002), access position (upper vs lower 0.26, P= 0.003, PVD (-0.35, P= 0.015), CAD (-0.25, P = 0.008), and primary renal disease (DM vs GN, -0.35, P= 0.003, HTN vs GN, -0.34, P= 0.04) were associated with Qa. On multivariable analysis, CI (0.84, P < 0.001), access position (upper vs lower, 0.17, P = 0.018) and primary renal disease (DM vs GN, -0.26, P = 0.005, and HTN vs GN, -0.26, P = 0.038) remained significant predictors of AVF Qa. Conclusion(s): Once established, CI, AVF position and primary renal disease (hypertension/ ischaemic and diabetes) are the major determinants of AVF Qa while female gender, CAD, PVD and body mass index were not significant determinants of Qa in this cohort.

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory molecule involved in cell-mediated immunity and delayed-type hypersensitivity (DTH). We inhibited systemic and local MIF production to determine its contribution to acute rejection (AR). Skin DTH response and acute rejection of skin and kidney allografts were examined using MIF gene knockout (MIF -/-) and wild-type mice (MIF +/+) with anti-MIF or control antibody. MIF-Ab reduced skin DTH by 60% (p<0.01), but absence of the MIF gene (MIF -/-) had no effect. Local absence of MIF had no effect on the survival of skin grafted onto BALB/c recipients. Similarly MIF +/+ and MIF -/- kidneys transplanted into BALB/c recipients showed a similar degree of histological rejection, graft dysfunction and cellular infiltrate suggesting that AR is not dependent on local MIF production. To investigate the influence of systemic MIF, BALB/c donor skin was grafted onto MIF +/+ and MIF -/- mice. The tempo of AR was not altered by systemic absence of MIF (MIF-Ab or MIF -/-). BALB/c kidneys transplanted into MIF +/+ (with or without MIF-Ab) and MIF -/- mice showed similar parameters of rejection. MIF blockade reduces the DTH response; however, neither local nor systemic MIF are required for the rejection of fully mismatched skin and renal allografts.

Background and Aim: The progression of renal injury, initiated by either an immune or non-immune insult, is closely associated with the accumulation of leucocytes and fibroblasts in the damaged kidney. Macrophage migration inhibitory factor (MIF) regulates leucocyte activation and fibroblast proliferation in vitro. Studies have identified a pathological role for MIF in immune-initiated renal injury in the rat. In this study, we examined the role of MIF in obstructive nephropathy, where renal injury is initiated by a non-immune insult. Methods and Results: Unilateral ureteric ligation was performed on MIF wildtype (+/+) and MIF deficient (-/-) mice. Groups of five mice were killed at days 0, 1, 5 or 10 after obstruction, and kidneys were examined via immunohistochemistry and northern blotting. In MIF +/+ mice, expression of the MIF protein increased in obstructed kidneys compared to normal control kidneys. Interstitial macrophage and T cell accumulation was significantly increased in obstructed kidneys at day 5 and 10, but was unaffected by MIF deficiency. Osteopontin and macrophage colony stimulating factor (M-CSF) mRNA expression in obstructed kidneys were equally increased in both genotypes, indicating that expression of these chemokines is not influenced by MIF. No difference was detected in the development of renal fibrosis in obstructed MIF +/+ and MIF -/- kidneys, as assessed by myofibroblast accumulation and proliferation and expression of profibrotic molecules (transforming growth factor-beta 1(TGF-beta1) and collagen). Conclusion(s): These results demonstrate that MIF expression is increased in obstructive nephropathy without affecting kidney leucocyte accumulation or the development of renal fibrosis. This suggests that the progression of renal injury in obstructive nephropathy is independent of MIF.