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• W2000674444 abstract "Programmed death 1 (PD-1) is a novel member of the CD28/cytotoxic T-lymphocyte-associated protein-4 superfamily, which plays an important role in the regulation of activated T cells. However, it is not clear how PD-1 is expressed in normal and diseased kidney, nor if it has a role in progression of chronic renal disease. PD-1 expression and the effect of monoclonal anti-PD-1 antibody (Ab) were examined in murine adriamycin nephropathy (AN). BALB/c mice were divided into three groups: (a) normal mice, (b) adriamycin (ADR) with control immunoglobulin (Ig)G (ADR-IgG), and (c) ADR with anti-PD-1 Ab (ADR-Ab). AN was induced by a single intravenous injection of ADR. Anti-PD-1 Ab was given by intraperitoneal injection on alternate days from day 0 to day 10, or to day 18. Animals were killed at week 4. Renal function, histological change, and cytokine expression were examined. PD-1 mRNA was detected in kidney tissue of mice with AN in a dose- and time-dependent manner. PD-1 was mainly expressed on injured tubule cells and some interstitial cells, which co-stained with α-smooth muscle actin in AN, but not in normal kidney. Anti-PD-1 treatment up to day 18, but not to day 10, worsened glomerular and tubulointerstitial injury. The ratio of urinary protein/creatinine was significantly higher in ADR-Ab mice than ADR-IgG mice. The number of macrophages was significantly increased in ADR-Ab mice compared with ADR-IgG mice. Blockade of PD-1 worsened progressive renal histopathological and functional injury in murine AN. This suggests a possible protective role for PD-1 in chronic renal disease, and its potential as a treatment to slow disease progression. Programmed death 1 (PD-1) is a novel member of the CD28/cytotoxic T-lymphocyte-associated protein-4 superfamily, which plays an important role in the regulation of activated T cells. However, it is not clear how PD-1 is expressed in normal and diseased kidney, nor if it has a role in progression of chronic renal disease. PD-1 expression and the effect of monoclonal anti-PD-1 antibody (Ab) were examined in murine adriamycin nephropathy (AN). BALB/c mice were divided into three groups: (a) normal mice, (b) adriamycin (ADR) with control immunoglobulin (Ig)G (ADR-IgG), and (c) ADR with anti-PD-1 Ab (ADR-Ab). AN was induced by a single intravenous injection of ADR. Anti-PD-1 Ab was given by intraperitoneal injection on alternate days from day 0 to day 10, or to day 18. Animals were killed at week 4. Renal function, histological change, and cytokine expression were examined. PD-1 mRNA was detected in kidney tissue of mice with AN in a dose- and time-dependent manner. PD-1 was mainly expressed on injured tubule cells and some interstitial cells, which co-stained with α-smooth muscle actin in AN, but not in normal kidney. Anti-PD-1 treatment up to day 18, but not to day 10, worsened glomerular and tubulointerstitial injury. The ratio of urinary protein/creatinine was significantly higher in ADR-Ab mice than ADR-IgG mice. The number of macrophages was significantly increased in ADR-Ab mice compared with ADR-IgG mice. Blockade of PD-1 worsened progressive renal histopathological and functional injury in murine AN. This suggests a possible protective role for PD-1 in chronic renal disease, and its potential as a treatment to slow disease progression. A universal feature of animal and human chronic renal disease of all types is infiltration with T cells and macrophages into the kidney cortex.1.Wang Y. Wang Y.P. Tay Y.C. Harris D.C. Progressive adriamycin nephropathy in mice: sequence of histologic and immunohistochemical events.Kidney Int. 2000; 58: 1797-1804Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar The effect of these mononuclear inflammatory cells on renal injury is dependent on their state of activation.2.Taal M.W. Omer S.A. Nadim M.K. Mackenzie H.S. Cellular and molecular mediators in common pathway mechanisms of chronic renal disease progression.Curr Opin Nephrol Hypertens. 2000; 9: 323-331Crossref PubMed Scopus (33) Google Scholar Costimulatory molecules provide the additional signals beyond interactions between the T-cell receptor and major histocompatibility complex–peptide complex necessary for full T-cell activation. Blockade or stimulation of costimulation pathways can regulate T-cell activation, proliferation, differentiation, and cytokine secretion,3.van Kooten C. Blockade of costimulatory pathways of T-cell activation: the solution to acute and chronic rejection?.Curr Opin Nephrol Hypertens. 1999; 8: 675-680Crossref PubMed Scopus (4) Google Scholar and therefore can be expected to influence the effect of cortical inflammatory cells on renal injury. The programmed death 1 (PD-1) receptor is a novel member of the immunoglobulin (Ig) superfamily with 23% homology to cytotoxic T-lymphocyte-associated protein-4 in its IgG variable-like domain; it functions as a negative costimulatory receptor expressed on T, B, and monocytes.4.Freeman G.J. Long A.J. Iwai Y. et al.Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation.J Exp Med. 2000; 192: 1027-1034Crossref PubMed Scopus (3777) Google Scholar B7H1 (PD-L1) and B7-DC (PD-L2) have been identified as the ligands for PD-1, which are expressed on monocytes and dendritic cells.5.Yamazaki T. Akiba H. Iwai H. et al.Expression of programmed death 1 ligands by murine T cells and APC.J Immunol. 2002; 169: 5538-5545Crossref PubMed Scopus (748) Google Scholar,6.Loke P. Allison J.P. PD-L1 and PD-L2 are differentially regulated by Th1 and Th2 cells.Proc Natl Acad Sci USA. 2003; 100: 5336-5341Crossref PubMed Scopus (487) Google Scholar PD-1 deficiency causes auto-antibody (Ab)-mediated dilated cardiomyopathy in BALB/c mice and arthritis in C57BL/6 mice.7.Nishimura H. Nose M. Hiai H. et al.Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor.Immunity. 1999; 11: 141-151Abstract Full Text Full Text PDF PubMed Scopus (2048) Google Scholar, 8.Nishimura H. Okazaki T. Tanaka Y. et al.Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice.Science. 2001; 291: 319-322Crossref PubMed Scopus (1394) Google Scholar, 9.Okazaki T. Tanaka Y. Nishio R. et al.Autoantibodies against cardiac troponin I are responsible for dilated cardiomyopathy in PD-1-deficient mice.Nat Med. 2003; 9: 1477-1483Crossref PubMed Scopus (521) Google Scholar These studies indicate that engagement of PD-1 with PD-L1 or PD-L2 contributes to maintenance of self-tolerance. PD-1 knockout mice developed auto-Ab-mediated glomerulonephritis with predominant IgG3 deposition,7.Nishimura H. Nose M. Hiai H. et al.Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor.Immunity. 1999; 11: 141-151Abstract Full Text Full Text PDF PubMed Scopus (2048) Google Scholar suggesting that PD-1 also has a role in preventing autoimmune renal disease. A single-nucleotide polymorphism in PD-1 (PDCD1 in humans) has been associated with increased risk of lupus, a major cause of autoimmune nephritis, in European and Mexican patients.10.Prokunina L. Gunnarsson I. Sturfelt G. et al.The systemic lupus erythematosus-associated PDCD1 polymorphism PD1.3A in lupus nephritis.Arthritis Rheum. 2004; 50: 327-328Crossref PubMed Scopus (89) Google Scholar It has also been shown that resting dendritic cells induce peripheral CD8 tolerance using PD-1 on the CD8 T cell.11.Probst H.C. McCoy K. Okazaki T. et al.Resting dendritic cells induce peripheral CD8+ T cell tolerance through PD-1 and CTLA-4.Nat Immunol. 2005; 6: 280-286Crossref PubMed Scopus (434) Google Scholar However, the effect of PD-1 in chronic renal disease, especially in non-immune renal disease, at present is unknown. To explore the role of PD-1 in chronic renal disease, we examined its expression in murine adriamycin nephropathy (AN) and the effect of its blockade with monoclonal Ab (mAb) against PD-1. PD-1 mRNA expression was increased in AN in a dose- and time-dependent manner. The level of PD-1 mRNA expression was increased in parallel with the severity of pathological renal injury in mild AN (9.5 mg/kg) vs severe AN (9.8 mg/kg). PD-1 expression was first seen on day 7, was moderate on day 14, and was strong on day 28 (see Figure 1). PD-1 expression paralleled the progressive renal histologic changes and infiltration with lymphocytes and macrophages of murine AN. Immunohistochemical staining revealed no expression of PD-1 within the normal kidney. In mice with AN, PD-1 staining was weak at day 14 and strong by day 28, but was not seen at day 3 and day 7 after ADR injection (control vs day 7 vs day 14 vs day 28: 0±0 vs 0±0 vs 2.6±3.5 vs 15.3±14.1, P<0.05 day 28 vs day 7). PD-1 was predominantly on injured tubule cells and some interstitial cells, but not on glomerular cells or blood vessels (Figure 2). To localize PD-1 expression in kidney, double immunofluorescent staining was used with PD-1 Ab and other Abs against CD4+ or CD8+ T cells, macrophages, and α-smooth muscle actin (α-SMA). PD-1 did not co-express with CD4, CD8, or macrophages in kidney, whereas PD-1 co-expressed with α-SMA-positive cells of dilated tubules and renal interstitium (Figure 3). All experimental animals developed histological changes typical of AN, with focal segmental glomerulosclerosis, tubular atrophy, and interstitial damage. Overt proteinuria appeared about 5 days after ADR administration and remained heavy throughout the experimental period. Mean body weight fell soon after ADR treatment and remained lower than normal controls until being killed. The surface of AN kidneys was granular and pale in color. When the anti-PD-1 Ab was given until day 10, there was no significant difference in any aspect of renal injury, including glomerular sclerosis, tubular atrophy, and interstitial change, between the ADR-Ab group and ADR-IgG group (Table 1). Levels of urine protein and serum albumin were similar in these two groups.Table 1Morphology and renal function in normal mice and mice with AN, treated with anti-PD-1 mAb (ADR-Ab) or IgG control antibody (ADR-IgG) until day 10Normal (n=4)ADR-IgG (n=8)ADR-Ab (n=8)Glomerulosclerosis (0–3)01.44±0.781.29±0.70Tubular atrophy (0–3)01.94±0.901.86±0.99Interstitial infiltrates (0–3)01.31±0.750.86±0.56Urinary protein (mg/12 h)0.29±0.152.49±0.962.08±1.00Serum albumin (g/l)24.5±1.925.9±3.126.3±2.9Urinary protein to creatinine ratio (g/mmol)0.16±0.052.19±0.842.15±0.96AN, adriamycin nephrosis; ADR-Ab, adriamycin with anti-PD-1 antibody; ADR-IgG, ADR with control immunoglobulin G; PD-1, programmed death 1.There was no significant difference between ADR-IgG and ADR-Ab groups. Open table in a new tab AN, adriamycin nephrosis; ADR-Ab, adriamycin with anti-PD-1 antibody; ADR-IgG, ADR with control immunoglobulin G; PD-1, programmed death 1. There was no significant difference between ADR-IgG and ADR-Ab groups. When the anti-PD-1 Ab was given until day 18, renal cortex was more severely damaged in ADR-Ab group than ADR-IgG group (Table 2 and Figure 4). In ADR-Ab mice, glomerulosclerosis was significantly increased (mean±s.d. of glomerular sclerosis index of ADR-Ab vs ADR-IgG: 0.77±0.29 vs 0.49±0.16, P<0.05). In ADR-Ab mice, glomerulosclerosis was significantly increased in association with reduction in glomerular surface area; relative interstitial volume was expanded accompanied by a marked increase in inflammation; and the degree of tubular atrophy as characterized by a decrease in the height of tubular epithelial cells, loss of brush border, and vacuolization was aggravated in the ADR-Ab group as compared to ADR-IgG controls. The ratio of urinary protein to creatinine was significantly higher in ADR-Ab mice compared with ADR-IgG mice (5.7±1.5 vs 3.7±0.9 g/mmol, P<0.05). Urinary protein was increased and serum albumin decreased in the ADR-Ab group compared to the ADR-IgG group, but the differences did not reach statistical significance.Table 2Morphology and renal function in normal mice and mice with AN, treated with anti-PD-1 mAb (ADR-Ab) or IgG control antibody (ADR-IgG) until day 18Normal (n=4)ADR-IgG (n=8)ADR-Ab (n=8)Glomerulosclerosis (0–3)01.13±0.901.81±0.60*P<0.05 vs ADR-IgG.Tubular atrophy (0–3)01.13±1.002.00±0.90Interstitial infiltrates (0–3)00.69±0.601.50±0.70*P<0.05 vs ADR-IgG.Urinary protein (mg/12 h)0.44±0.192.90±1.964.55±5.61Serum albumin (g/l)24.5±1.922.6±5.818.7±7.7Urinary protein to creatinine ratio (g/mmol)0.19±0.053.69±0.915.69±1.47*P<0.05 vs ADR-IgG.AN, adriamycin nephrosis; ADR-Ab, adriamycin with anti-PD-1 antibody; ADR-IgG, ADR with control immunoglobulin G; PD-1, programmed death 1.* P<0.05 vs ADR-IgG. Open table in a new tab AN, adriamycin nephrosis; ADR-Ab, adriamycin with anti-PD-1 antibody; ADR-IgG, ADR with control immunoglobulin G; PD-1, programmed death 1. In normal mice, a few macrophages and rare lymphocytes were found within the kidney interstitium. The number of CD4+ and CD8+ lymphocytes and macrophages was significantly increased in the renal interstitium in AN. When the anti-PD-1 Ab was given until day 18, but not when given until day 10, the number of infiltrating CD4+ and CD8+ lymphocytes was increased mildly in the ADR-Ab mice compared to ADR-IgG mice, but this result did not reach statistical significance (number of CD4+ cells per mm2: 21.7±17.6 vs 12.5±10.6; number of CD8+ cells per mm2: 9.3±2.1 vs 7.0±3.0). The ratio of CD4 to CD8 lymphocytes was higher in Ab-treated animals (ADR+Ab group: CD4:CD8 ratio 2.7±2.8 vs 1.1±1.0 in ADR+IgG group), but this result was not statistically significant. In contrast, the number of infiltrating macrophages was significantly higher in ADR-Ab mice compared to ADR-IgG mice (Figures 5 and 6).Figure 6Representative immunostaining for renal macrophages at day 28 after ADR. Original magnification × 600. (a) Normal control, (b) ADR with IgG, and (c) ADR with anti-PD-1 Ab administered for 18 days. The number of interstitial macrophages was significantly increased in the group receiving Ab treatment until day 18 compared to the IgG control group.View Large Image Figure ViewerDownload (PPT) Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to semiquantify monocyte chemoattractant protein-1 (MCP-1) gene expression in renal tissue. MCP-1 mRNA expression was higher in the ADR-Ab group compared to the ADR-IgG group and control group (MCP-1:18S ratio, 0.49±0. 36 vs 0.14±0.09 vs 0.07±0.06, P<0.05 comparing ADR-Ab to ADR-IgG). To investigate if the exacerbation in renal injury was owing to increased T helper (Th)1 cytokine expression, RT-PCR was used to semiquantify expression of Th1 and Th2 cytokines in renal tissue. There was no significant difference in Th1 cytokine (interleukin (IL)-2, IL-12, interferon-γ) nor Th2 cytokine (IL-4, IL-10) mRNA expression in the ADR-Ab group compared to ADR-IgG. Deposition of C3, as expected, was seen in the peritubular capillaries and tubular cells in all AN-treated groups,12.Rangan G.K. Pippin J.W. Couser W.G. C5b-9 regulates peritubular myofibroblast accumulation in experimental focal segmental glomerulosclerosis.Kidney Int. 2004; 66: 1838-1848Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar but was not seen in glomeruli of Ab-treated animals. There was no evidence of glomerular tuft deposition of mouse Ig or hamster Ig. Furthermore, using enzyme-linked immunosorbent assay, Abs against hamster Ig were not detected in the sera of mice treated with anti-PD-1 Ab. The present study was designed to investigate the possible role of PD-1 in the pathogenesis of chronic renal disease. PD-1 was significantly upregulated on tubule cells and interstitial cells in the later stages of a robust model of focal segmental glomerulosclerosis, murine AN. Interestingly, PD-1 was expressed predominantly on α-SMA-positive cells in the tubulointerstitium. Most importantly, blockade of PD-1 by a neutralizing mAb worsened the renal injury of AN. These results indicate that PD-1 plays an important role in protecting against renal injury in this model of chronic renal disease. By both RT-PCR and immunohistochemistry, we examined PD-1 expression in renal cortex in AN. The expression of PD-1 was time- and dose-dependent, and the level of PD-1 expression correlated with severity of renal injury. These findings suggest that PD-1 may exert its most important effect at a later stage of renal disease rather than early after the onset of disease. This suggestion is strengthened by the fact that early blockade of PD-1 did not affect the severity of renal injury. Although PD-L1 expression has been reported on renal tubular cells following interferon-γ stimulation in vitro and kidney transplants in vivo,13.Schoop R. Wahl P. Le Hir M. et al.Suppressed T-cell activation by IFN-gamma-induced expression of PD-L1 on renal tubular epithelial cells.Nephrol Dial Transplant. 2004; 19: 2713-2720Crossref PubMed Scopus (76) Google Scholar PD-1 expression in the kidney is largely unexplored. In the present study, we found that PD-1 was predominantly expressed on injured tubule cells as well as on some interstitial cells. However, PD-1 was not expressed on glomerular cells or normal tubule cells. To determine which cells express PD-1, we characterized the cells by double immunofluroescence staining with other cell markers. Surprisingly, PD-1 was not expressed on T cells or macrophages, and was only expressed on α-SMA-positive cells, specifically injured tubule cells, and some interstitial cells. This suggests that PD-1-expressing tubule cells are undergoing epithelial mesenchymal transition. It is possible that PD-1 may be a useful marker for epithelial mesenchymal transition in tubule cells. This is in contrast to the findings by Ding et al.14.Ding H. Wu X. Gao W. PD-L1 is expressed by human renal tubular epithelial cells and suppresses T cell cytokine synthesis.Clin Immunol. 2005; 115: 184-191Crossref PubMed Scopus (65) Google Scholar – in normal human kidneys there was no PD-1 expression, but in renal biopsies of human type 4 lupus nephritis, PD-1 was expressed on mononuclear cells in the vicinity of proximal tubules. The most important finding of this study is that administration of anti-PD-1 mAb worsened renal injury in AN. These results support a role for PD-1 as a negative regulator of immune response15.Okazaki T. Maeda A. Nishimura H. et al.PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine.Proc Natl Acad Sci USA. 2001; 98: 13866-13871Crossref PubMed Scopus (646) Google Scholar,16.Ozkaynak E. Wang L. Goodearl A. et al.Programmed death-1 targeting can promote allograft survival.J Immunol. 2002; 169: 6546-6553Crossref PubMed Scopus (201) Google Scholar and inhibitor of renal injury. We did not find any deposition of C3, mouse Ig in glomeruli of PD-1 Ab-treated animals, hence the worsening of renal injury was unlikely to be owing to immune complex nephritis induced by PD-1 Ab. However, administration of anti-PD-1 mAb only during the early stage of disease did not affect the degree of injury as compared to the IgG control group. The reason for this may relate to the fact that PD-1 was expressed strongly on renal tubule cells and interstitial cells only during later stages of the disease. Thus, it is suggested that PD-1/PD-L interaction could be more important in later rather than early stages of chronic renal disease. As a negative regulator of T-cell responses, PD-1 has been associated with decreased expression of pathogenic T cells and downregulated Th1 cytokine production.17.Carter L. Fouser L.A. Jussif J. et al.PD-1:PD-L inhibitory pathway affects both CD4(+) and CD8(+) T cells and is overcome by IL-2.Eur J Immunol. 2002; 32: 634-643Crossref PubMed Scopus (560) Google Scholar, 18.Mazanet M.M. Hughes C.C. B7-H1 is expressed by human endothelial cells and suppresses T cell cytokine synthesis.J Immunol. 2002; 169: 3581-3588Crossref PubMed Scopus (279) Google Scholar, 19.Kishimoto K. Sandner S. Imitola J. et al.Th1 cytokines, programmed cell death, and alloreactive T cell clone size in transplant tolerance.J Clin Invest. 2002; 109: 1471-1479Crossref PubMed Scopus (74) Google Scholar As PD-1 is weakly homologous to cytotoxic T-lymphocyte-associated protein-4, it may possess an inhibitory function during T-cell activation. Supporting this, experiments have shown that the ligation of PD-1 by PD-L1 inhibited proliferation and cytokine production by activated T cells.20.Cai G. Karni A. Oliveira E.M. et al.PD-1 ligands, negative regulators for activation of naive, memory, and recently activated human CD4+ T cells.Cell Immunol. 2004; 230: 89-98Crossref PubMed Scopus (66) Google Scholar, 21.Bennett F. Luxenberg D. Ling V. et al.Program death-1 engagement upon TCR activation has distinct effects on costimulation and cytokine-driven proliferation: attenuation of ICOS, IL-4, and IL-21, but not CD28, IL-7, and IL-15 responses.J Immunol. 2003; 170: 711-718Crossref PubMed Scopus (242) Google Scholar, 22.Brown J.A. Dorfman D.M. Ma F.R. et al.Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production.J Immunol. 2003; 170: 1257-1266Crossref PubMed Scopus (789) Google Scholar In addition, PD-1 knockout mice develop a lupus-like arthritis and glomerulonephritis. However, in the current study, there was no difference in number of infiltrating T cells or the ratio of CD4+ to CD8+ T cells within renal cortex between the PD-1 blockade group and the IgG control group. Moreover, IL-2, IL-12, and interferon-γ mRNA expression within renal cortex was not significantly increased in the PD-1 blockade group compared to IgG controls. Therefore, mechanisms underlying the exacerbation of renal injury by anti-PD-1 are not related to an increase in the number of T cells nor upregulation of Th1 cytokines. Most studies have focused on the role of PD-1 on inhibition of T-cell activation through PD-1 on the surface of T cells, with PDL-1 and PDL-2 expressed on antigen-presenting cells or on parenchymal cells initiating this. Little attention has been paid to the role of PD-1 influencing macrophages or dendritic cells through their surface expression of PDL-1 and PDL-2 through inside-out signalling. This has recently been shown to occur in the cytotoxic T-lymphocyte-associated protein-4 B7 system leading to IDO production in the macrophage. Consistent with PD-1 playing a role in downregulating macrophages was the striking finding in this study of an increase in the number of macrophages in the PD-1 blockade group in comparison to IgG controls. We found that PD-1 blockade was associated with increased macrophage infiltration in treated animals. This may be owing to increased production of chemotactic peptides as we found increased MCP-1 expression in treated animals. Tubular cells secrete MCP-1, so in renal disease PD-1 may inhibit tubular cell MCP-1 production. It is also possible that the effect of PD-1 and its blockade in chronic renal disease may relate to an effect on regulatory cells.23.Baecher-Allan C. Brown J.A. Freeman G.J. Hafler D.A. CD4+CD25+ regulatory cells from human peripheral blood express very high levels of CD25 ex vivo.Novartis Found Symp. 2003; 252 (discussion 88–91, 106–114): 67-88Crossref PubMed Google Scholar,24.Sandner S.E. Clarkson M.R. Salama A.D. et al.Role of the programmed death-1 pathway in regulation of alloimmune responses in vivo.J Immunol. 2005; 174: 3408-3415Crossref PubMed Scopus (157) Google Scholar Aramaki et al.25.Aramaki O. Shirasugi N. Takayama T. et al.Programmed death-1-programmed death-L1 interaction is essential for induction of regulatory cells by intratracheal delivery of alloantigen.Transplantation. 2004; 77: 6-12Crossref PubMed Scopus (34) Google Scholar have assessed the development of regulatory cells in adoptive transfer experiments, and suggested that PD-1–PDL1 interaction was essential for the induction of regulatory cells and subsequent prolongation of allograft survival. In recent studies on the effect of CD4+CD25+ T cells in AN, we have demonstrated that CD4+CD25+ T cells directly inhibit macrophage function (unpublished observations). Therefore, interruption of PD-1–PDL could impair regulatory T-cell function, eventually leading to more activated macrophages in renal cortex. In summary, the present study showed that PD-1 is expressed on injured tubule cells, possibly those undergoing epithelial mesenchymal transition in later stages of AN. Blockade of PD-1 increased renal injury, suggesting that the PD-1–PDL costimulatory pathway has a protective role in chronic renal disease. Amplifying the inhibitory action of PD-1 could be used as a novel therapeutic approach for preventing progression in chronic renal disease. Blocking mAb specific for murine PD-1 (clone J43, Armenian hamster IgG) was purchased from Bioexpress Cell Culture Services (West Lebanon, NH, USA). This Ab has been previously shown to neutralize binding of PD-1 to its ligands PD-L1 and PD-L2.26.Ansari M.J. Salama A.D. Chitnis T. et al.The programmed death-1 (PD-1) pathway regulates autoimmune diabetes in nonobese diabetic (NOD) mice.J Exp Med. 2003; 198: 63-69Crossref PubMed Scopus (617) Google Scholar The control hamster IgG was purified by protein G (Amersham Pharmacia Biotech, Piscataway, NJ, USA) affinity chromatography according to the manufacturer's instructions from the hamster serum obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). mAbs specific for murine Abs CD4 (clone RM4-5) and CD8 (clone 53-6.7) were purchased from BD Bioscience (San Diego, CA, USA) and for macrophage (clone F4/80) from Serotec (Oxford, UK). We obtained fluorescein isothiocynate (FITC)-labelled anti-rat C3, which crossreacts with mouse C3 (MP Biomedicals, Seven Hills, NSW, Australia), as well as FITC-labelled goat anti-mouse Ig (Serotec, USA) and FITC-labelled goat anti-Armenian hamster Ig (eBioscience, USA). Biotinylated rabbit anti-rat Ig was obtained from Dako Corporation (Carpinteria, CA, USA) and the biotin-conjugated anti-hamster Ab was obtained from eBioscience (San Diego, CA, USA). BALB/c mice, weighing 20–25 g, were supplied by the Animal Care Facility (Westmead Hospital, NSW, Australia), and bred and housed under standard conditions. To examine PD-1 expression, mice were divided into three groups: normal, low dose (9.5 mg/kg) of ADR, and high dose (9.8 mg/kg) of ADR. Mice were killed at day 3, day 7, day 14, and day 28. For the Ab-blocking experiment, mice were divided into the following groups: (a) normal control; (b) ADR+hamster IgG (ADR-IgG), and (c) ADR+hamster anti-PD-1 Ab (ADR-Ab). Murine AN was induced by single tail vein injection of 9.6 mg/kg ADR (doxorubicin hydrochloride; Pharmacia & Upjohn Pty Ltd, Perth, Australia). All mice were housed individually. Body weight was measured daily. In two different Ab-blocking strategies, hamster anti-PD-1 Ab was administrated intraperitoneally at 500 μg/dose on day 0, then at 200 μg/dose on alternate days from day 2 to day 10 or to day 18 after ADR treatment. An equivalent amount of hamster IgG was given according to the same protocol to the ADR-IgG groups. The dose of Ab is similar to other studies using anti-PD-1 (clone J43) to block its in vivo effects in experimental murine models of autoimmune type I diabetes,26.Ansari M.J. Salama A.D. Chitnis T. et al.The programmed death-1 (PD-1) pathway regulates autoimmune diabetes in nonobese diabetic (NOD) mice.J Exp Med. 2003; 198: 63-69Crossref PubMed Scopus (617) Google Scholar graft-versus-host disease,27.Blazar B.R. Carreno B.M. Panoskaltsis-Mortari A. et al.Blockade of programmed death-1 engagement accelerates graft-versus-host disease lethality by an IFN-gamma-dependent mechanism.J Immunol. 2003; 171: 1272-1277Crossref PubMed Scopus (268) Google Scholar and contact hypersensitivity.28.Tsushima F. Iwai H. Otsuki N. et al.Preferential contribution of B7-H1 to programmed death-1-mediated regulation of hapten-specific allergic inflammatory responses.Eur J Immunol. 2003; 33: 2773-2782Crossref PubMed Scopus (112) Google Scholar We used two different durations of therapy because of the possibility of different effects on disease, as shown in experimental autoimmune encephalitis.29.Salama A.D. Chitnis T. Imitola J. et al.Critical role of the programmed death-1 (PD-1) pathway in regulation of experimental autoimmune encephalomyelitis.J Exp Med. 2003; 198: 71-78Crossref PubMed Scopus (406) Google Scholar On day 28 after injection of ADR, mice were killed. Blood samples were collected by cardiac puncture. Each kidney was removed, weighed immediately, and divided into four pieces for histology, immunohistology, and RNA extraction. Mice were placed in metabolic cages for 12 h before the killing to collect urine for the determination of urinary protein and creatinine. Urinary volume was measured. Blood samples for serum albumin were collected at the time of killing. Serum albumin, urine creatinine, and urine protein were assayed in the Institute of Clinical Pathology and Medical Research, Westmead Hospital. The ratio of urine protein to creatinine was determined. Kidney slices were fixed in 10% neutral-buffered formalin for 24 h and then dehydrated in graded alcohols and embedded in paraffin. Tissues were cut at 5 μm and stained with periodic acid-Schiff. A semiquantitative score from two blinded trained observers was used to evaluate the degree of renal injury and a minimum of eight consecutive fields at a magnification of × 400 were assessed and scored in each section. The degree of renal injury was estimated by evaluating the percentage of renal injury per field and was graded on a scale of 0 to 4: 0, normal glomeruli, tubules, and interstitial volume; 0.5, small focal area of glomerular and tubular injury and interstitial infiltration; 1, involvement of <10% of the cortex; 2, involvement of up to 25% of the cortex; 3, 50–75% of the cortex; and 4, extensive damage involving >75% of the cortex. Quantitative analysis of glomerulosclerosis was performed using a modification of a technique described by Saito et al.30.Saito T. Sumithran E. Glasgow E.F. Atkins R.C. The enhancement of aminonucleoside nephrosis by the co-administration of protamine.Kidney Int. 1987; 32: 691-699Abstract Full Text PDF PubMed Scopus (102) Google Scholar The degree of sclerosis was scored from 0 to 4 in each of 20 glomeruli from periodic acid-Schiff-stained sections from each mouse. The average of the degree of sclerosis was calculated as the index of glomerulosclerosis. These analyses were performed with image analysis software (ImageJ, NIH). Coronal slices of kidneys were embedded in OCT compound (Tissue-Tek; Sakura Finetek, Torrance, CA, USA), frozen in liquid nitrogen, and stored at -70°C. Frozen sections were cut at 5 μm and fixed with acetone at -20°C for 8 min. Endogenous peroxidase activity was blocked by incubating sections for 15 min in 0.3% (vol/vol) H2O2 solution in phosphate-buffered saline; and endogenous avidin-binding activity was blocked by incubating the sections with Biotin Blocking System (Dako Corporation, Carpinteria, CA, USA); then, nonspecific Ab binding was minimized with normal serum. Sections were incubated with primary Abs to macrophages, CD4+ cells and CD8+ cells for 60 min at room temperature or PD-1 Ab overnight at 4°C. After rinsing twice in phosphate-buffered saline for 5 min each, sections were incubated with secondary Abs for 30 min at room temperature and then in avidin–biotin–horseradish peroxidase complex for 30 min each. After phosphate-buffered saline washing, the reaction was visualized by the addition of freshly prepared 3, 3-diaminobenzidine tetrahydrochloride. Slides were counterstained with hematoxylin (Sigma-Aldrich Pty Ltd, Castle Hill, NSW, Australia), dehydrated, cover-slipped, and examined by light microscopy. The number of macrophages, and CD4+ and CD8+ cells was counted in eight non-overlapping cortical fields (× 400). The number of PD-1-positive cells was counted in eight non-overlapping cortical fields (× 200). The mean number of positive cells per field was calculated for each section. To exclude the possibility that worsening of AN by anti-PD-1 was owing to immune complex nephritis, we examined for the presence of immune complex deposition by performing immunofluorescence for C3, mouse Ig, and hamster Ig deposition. Frozen sections of 8 μm thickness were fixed in acetone for 8 min at -20°C. Background staining was blocked with avidin, biotin, and Background Buster. Sections were incubated with FITC-labelled anti-C3 (1:40 dilution) (MP Biomedicals, USA), FITC-labelled goat anti-mouse Ig (1:50 dilution) (Serotec, USA), or FITC-labelled goat anti-Armenian hamster Ig (eBioscience, USA) (1:50 dilution) for 1 h and then photographed. To localize PD-1 expression in kidney tissue, double immunofluorescence staining was used with mAbs against PD-1 and Abs against CD4+ or CD8+ T cells, macrophage, and α-SMA on the same sections. Frozen sections (5-μm) were fixed in acetone for 10 min at -20°C and blocked with superblock solution for 30 min. The sections were incubated with primary hamster anti-mouse mAb against PD-1 followed by secondary FITC-conjugated anti-hamster IgG and then incubated with the primary anti-mouse mAb against CD4, CD8, or macrophage followed by rhodamine-conjugated rabbit-anti-rat IgG, or incubated with primary cy3-conjugated anti-mouse α-SMA. After mounting, the slides were examined with a microscope equipped for fluorescence. The CD4+, CD8+, macrophage, or α-SMA-positive cells were visualized with rhodamine or cy3 (red) fluorescence and the PD-1-positive cells with FITC (green) fluorescence on the same sections. The presence of serum Abs against hamster Ig was determined by a direct enzyme-linked immunosorbent assay.31.Wu H. Wang Y. Tay Y.C. et al.DNA vaccination with naked DNA encoding MCP-1 and RANTES protects against renal injury in adriamycin nephropathy.Kidney Int. 2005; 67: 2178-2186Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar Blood was collected from anti-PD-1-treated, IgG-treated, and normal mice at days 10, 18, and 28, and sera stored at -20°C. Wells of an enzyme-linked immunosorbent assay microtiter plate (Sarstedt, SA, Australia) were coated with J43 Ab at a concentration of 5 μg/well in 100 μl of coating buffer and reacted sequentially with mouse sera, alkaline phosphatase-conjugated goat-anti-mouse polyclonal IgG (Abcam, Cambridge, MA, USA), and substrate solution (0.5% p-nitrophenyl phosphate (Sigma Chemical Co.) in carbonate buffer, pH 9.6). Absorbance was read at 405 nm on an enzyme-linked immunsorbent assay reader (Thermo Multiskan Ex). As a positive control, mouse anti-hamster Ig (BD Pharmingen, San Diego, CA, USA) was substituted for mouse serum. Total RNA was extracted using TRIZOL (Gibco BRL, Grand Island, NY, USA). Frozen kidney tissue was homogenized in a volume of 0.7 ml of TRIZOL and the extracted RNA was precipitated according to the manufacturer's instructions and suspended in 30 μl of RNase-free water. The yield and the purity of RNA were measured spectrophotometrically by absorption at 260/280 nm. First-strand cDNA was transcribed from total RNA using a Supertranscript RT-PCR kit (Invitrogen, Branchburg, NJ, USA) with 10 μg of total RNA in a final volume of 40 μl as per the manufacturer's instructions. Two units of RNase-free DNase I per microgram of RNA (Boehringer Mannheim, Indianapolis, IN, USA) was added to the reaction mixture, which was incubated for 20 min at 37°C. cDNA then was amplified by PCR in the presence of PCR buffer, 3 mmol/l MgCl2, 0.3 U Taq DNA polymerase, 0.2 mM deoxynucleoside triphosphates, and 400 pmol of primers. Primers used in PCR reactions were as follows: PD-1 forward, 5′-TCAAGGCATGGTCATTGGTA-3′; PD-1 reverse, 5′-TAGGCCACACTAGGGACAGG-3′, amplifying a 177 bp (base pair) gene fragment; IL-2 forward, 5′-AACCTGAAACTCCCCAGGAT-3′; IL-2 reverse, 5′-TCCACCACAGTTGCTGACTC-3′ (254 bp); MCP-1 forward, 5′-GCTGGAGAGCTACAAGAGGATCA-3′; MCP-1 reverse, 5′-CTCTCTCTTGAGCTTGGTGACAAA-3′; IL-10 forward, CCAGTTTTACCTGGTAGAAGTGATG-3′; IL-10 reverse, TGTCTAGGTCCTGGAGTCCAGCAGACTCAA-3′; IL-4 (396 bp), IL-12 (211 bp), and interferon-γ (384 bp) primer pairs (R&D Systems, Minneapolis, MN, USA); 18s forward, 5′-GTAACCCGTTGAACCCCATT-3’; and 18s reverse, 5’-CCATCCAATCGGTAGTAGCG-3’ (166 bp). After an initial 4-min period at 95°C, all PCR reaction mixtures were incubated for 35 cycles at 94°C for 45 s, 55°C (except for IL-10, which used 60°C) for 45 s, 72°C for 45 s, and a final extension of 72°C for 10 min 18s served as an internal control for RNA semiquantification and standardization of the amplified RNA products; PCR profile for 18s was 28 cycles for 30 s at 94°C and 100 s at 56°C followed by 5 min at 72°C. PCR products were analyzed with a 1.2% or 1.5% gel electrophoresis. The bands were visualized with ethidium bromide, photographed, and analyzed using image analysis software (ImageJ, NIH). Statistical analysis was performed by one-way analysis of variance for multiple comparisons of parametric data, and Wilcoxon's rank-sum test for non-parametric data. Results are expressed as the group mean±s.d. Two group differences were analyzed by Student's t-test for parametric data, and Wilcoxon's rank-sum test for non-parametric data. A P-value of less than 0.05 was considered statistically significant. This project was supported by funding from the National Health & Medical Research Council of Australia (Project Grant No. 307621 and Medical Postgraduate Scholarship No. 357412)." @default.
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• W2000674444 title "A protective role for programmed death 1 in progression of murine adriamycin nephropathy" @default.
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• W2000674444 cites W2044272039 @default.
• W2000674444 cites W2051605675 @default.
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• W2000674444 cites W2072361327 @default.
• W2000674444 cites W2079062617 @default.
• W2000674444 cites W2081394793 @default.
• W2000674444 cites W2081447339 @default.
• W2000674444 cites W2086275112 @default.
• W2000674444 cites W2086397613 @default.
• W2000674444 cites W2098853737 @default.
• W2000674444 cites W2118471749 @default.
• W2000674444 cites W2138905083 @default.
• W2000674444 cites W2140419562 @default.
• W2000674444 cites W2144953100 @default.
• W2000674444 cites W2147579197 @default.
• W2000674444 cites W2154610258 @default.
• W2000674444 cites W2154685147 @default.
• W2000674444 cites W2158840473 @default.
• W2000674444 cites W2162889226 @default.
• W2000674444 cites W2168233301 @default.
• W2000674444 cites W2168557022 @default.
• W2000674444 cites W2323513256 @default.
• W2000674444 cites W2408729906 @default.
• W2000674444 cites W4242440453 @default.
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