Targeted expression of a pan-caspase inhibitor in tubular epithelium attenuates interstitial inflammation and fibrogenesis in nephritic but not nephrotic mice
2012; Elsevier BV; Volume: 82; Issue: 9 Linguagem: Inglês
10.1038/ki.2012.243
ISSN1523-1755
AutoresTsutomu Inoue, Hiromichi Suzuki, Hirokazu Okada,
Tópico(s)Renal Diseases and Glomerulopathies
ResumoThe caspase family of enzymes participates in apoptotic and proinflammatory reactions in any cell. Here we studied the role of caspase activation in the tubular epithelium of diseased kidneys using mice transgenic for the baculovirus pan-caspase inhibitor p35 gene held in a nonexpressed state (control mice) but target-expressed in the renal proximal tubule cells when crossed with mice expressing Cre recombinase under the control of the γ-glutamyltransferase promoter. Proinflammatory and profibrogenic parameters such as the number of monocytes and fibroblasts in the kidneys were significantly increased at 28 days in the control mice, but not in the renal tubule–targeted mice expressing p35 in a nephrotoxic serum nephritis model of disease. These cellular changes paralleled the number of apoptotic tubular cells and protein levels of active caspase-3 in the kidneys at 7 and 28 days of both the control and proximal tubule–targeted mice. Surprisingly, all of these parameters were not significantly affected at 7 and 28 days by targeted p35 expression in tubular epithelium when compared with nontargeted control mice in a model of adriamycin nephrosis. Thus, our study shows the critical role of caspase activation in the tubular epithelium in apoptosis along with proinflammatory and profibrogenic processes in nephrotoxic serum nephritis but not adriamycin nephrosis. The caspase family of enzymes participates in apoptotic and proinflammatory reactions in any cell. Here we studied the role of caspase activation in the tubular epithelium of diseased kidneys using mice transgenic for the baculovirus pan-caspase inhibitor p35 gene held in a nonexpressed state (control mice) but target-expressed in the renal proximal tubule cells when crossed with mice expressing Cre recombinase under the control of the γ-glutamyltransferase promoter. Proinflammatory and profibrogenic parameters such as the number of monocytes and fibroblasts in the kidneys were significantly increased at 28 days in the control mice, but not in the renal tubule–targeted mice expressing p35 in a nephrotoxic serum nephritis model of disease. These cellular changes paralleled the number of apoptotic tubular cells and protein levels of active caspase-3 in the kidneys at 7 and 28 days of both the control and proximal tubule–targeted mice. Surprisingly, all of these parameters were not significantly affected at 7 and 28 days by targeted p35 expression in tubular epithelium when compared with nontargeted control mice in a model of adriamycin nephrosis. Thus, our study shows the critical role of caspase activation in the tubular epithelium in apoptosis along with proinflammatory and profibrogenic processes in nephrotoxic serum nephritis but not adriamycin nephrosis. Caspases are evolutionarily conserved enzymes that execute the function of programmed cell death in normal development. They have also been implicated in a variety of pathological conditions including cerebral ischemia, myocardial infarction, and ischemia–reperfusion injury in the kidney.1.Lavrik I.N. Golks A. Krammer P.H. Caspases: pharmacological manupulation of cell death.J Clin Invest. 2005; 115: 2665-2672Crossref PubMed Scopus (498) Google Scholar, 2.Sung J.H. Zhao H. Roy M. et al.Viral caspase inhibitor p35, but not crmA, is neuroprotective in the ischemic penumbra following experimental stroke.Neurosci. 2007; 149: 804-812Crossref PubMed Scopus (11) Google Scholar, 3.Chandrashenkhar Y. Anway R. Shuros A. et al.Long-term caspase inhibition ameliorates apoptosis, reduces myocardial troponin-I cleavage, protects left ventricular function, and attenuates remodeling in rats with myocardial infarction.J Am Coll Cardiol. 2004; 43: 295-301Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 4.Daemen M.A.R.C. van't Veer C. Denecker G. et al.Inhibition of apoptosis induced by ischemia-reperfusion prevents inflammation.J Clin Invest. 1999; 104: 541-549Crossref PubMed Scopus (487) Google Scholar In the caspase-dependent apoptotic pathway, caspase-8 activation by ligation of death receptors causes direct caspase-3 activation, mitochondrial injury, and cytochrome c release, the latter of which then also triggers the activation of caspase-9 and caspase-3.5.Sanz A.B. Santamaria B. Ruiz-Ortega M. et al.Mechanisms of renal apoptosis in health and disease.J Am Soc Nephrol. 2008; 19: 1634-1642Crossref PubMed Scopus (191) Google Scholar The activation of caspase-3 results in widespread proteolysis and cell death. Caspase-1 also contributes to ischemic damage through its ability to activate inflammasome and cleave the precursor of interleukin (IL)-1β to release an active subunit, a component of innate immunity.6.Bryant C. Fitzgerald K.A. Molecular mechanisms involved in inflammasome activation.Trends Cell Biol. 2009; 19: 455-464Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar An involvement of caspases in the pathogenesis of kidney diseases was tested in in vivo experiments with systemic caspase inhibition. This revealed that treatment with caspase inhibitors significantly attenuated progression of kidney diseases such as ischemia–reperfusion injury,4.Daemen M.A.R.C. van't Veer C. Denecker G. et al.Inhibition of apoptosis induced by ischemia-reperfusion prevents inflammation.J Clin Invest. 1999; 104: 541-549Crossref PubMed Scopus (487) Google Scholar obstructive nephropathy,7.Docherty N.G. O'Sullivan O.E. Healy D.A. et al.Evidence that inhibition of tubular cell apoptosis protects against renal damage and development of fibrosis following ureteric obstruction.Am J Physiol Renal Physiol. 2006; 290: F4-F13Crossref PubMed Scopus (146) Google Scholar polycystic kidney disease,8.Tao Y. Kim J. Faubel S. et al.Caspase inhibition reduces tubular apoptosis and proliferation and slows disease progression in polycystic kidney disease.PNAS. 2005; 102: 6954-6959Crossref PubMed Scopus (93) Google Scholar lupus nephritis,9.Seery J.P. Cattell V. Watt F.M. Cutting edge: amelioration of kidney disease in a transgenic mouse model of lupus nephritis by administration of the caspase inhibitor carbobenzoxy-valyl-ananyl-aspartyl-(beta-o-methyl)-fluoromethylketone.J Immunol. 2001; 167: 2452-2455Crossref PubMed Scopus (41) Google Scholar and nephrotoxic serum nephritis (NTN).10.Yang B. Johnson T.S. Haylor J.L. et al.Effects of caspase inhibition on the progression of experimental glomerulonephritis.Kidney Int. 2003; 63: 2050-2064Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar In these studies, caspase-dependent apoptosis was considered a target in the treatment with caspase inhibitors. However, the cells principally involved in caspase activation have not been identified in the kidney diseases. Moreover, systemic administration of inhibitors that disrupt caspase activation also impair host innate immunity, resolution of inflammation, and restraining tissue hyperplasia including tumorigenesis.6.Bryant C. Fitzgerald K.A. Molecular mechanisms involved in inflammasome activation.Trends Cell Biol. 2009; 19: 455-464Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 11.Green D.R. Kroemer G. Pharmacological manipulation of cell death: clinical applications in sight?.J Clin Invest. 2005; 115: 2610-2617Crossref PubMed Scopus (218) Google Scholar, 12.Riedl S.J. Salvesen G.S. The apoptosome: signalling platform of cell death.Nat Rev Mol Cell Biol. 2007; 8: 405-413Crossref PubMed Scopus (856) Google Scholar Therefore, the ideal, preferred therapeutic modality lies in a targeted, tissue-specific inhibition of caspase activity. Thus, we aimed to determine whether tubular epithelium in a nephritic kidney could serve as an ideal target for caspase inhibition reflected in suppressing renal injuries in NTN13.Okada H. Inoue T. Kikuta T. et al.A possible anti-inflammatory role of angiotensin II type 2 receptor in immune-mediated glomerulonephritis during type 1 receptor blockade.Am J Pathol. 2006; 169: 1577-1589Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar and in a nephrotic syndrome of adriamycin nephropathy (ADR),14.Artunc F. Nasir O. Amann K. et al.Serum- and glucocorticoid-inducible kinase 1 in doxorubicin-induced nephrotic syndrome.Am J Physiol Renal Physiol. 2008; 295: F1624-F1634Crossref PubMed Scopus (30) Google Scholar which has never been tested for caspase inhibition. In this study, we used a transgenic (TG) mouse bearing the baculovirus-derived, pan-caspase inhibitor protein p35 gene separated from the universal CAG promoter by a floxed STOP sequence.15.Shibata M. Hisahara S. Hara H. et al.Caspases determine the vulnerability of oligodendrocytes in the ischemic brain.J Clin Invest. 2000; 106: 643-653Crossref PubMed Scopus (84) Google Scholar To inhibit caspase activities exclusively in the tubular cells, this p35 mouse was mated with another TG mouse carrying Cre recombinase gene under the control of a γ-glutamyl transferase (γGT) promoter that is active only in the proximal tubular epithelium in the kidney.16.Iwano M. Plieth D. Danoff T.M. et al.Evidence that fibroblasts derive from epithelium during tissue fibrosis.J Clin Invest. 2002; 110: 341-350Crossref PubMed Scopus (1724) Google Scholar In this study, NTN and ADR models were generated in p35 TG mice of SJL and 129 backgrounds, respectively. These mice were selected because they represented the mouse strains most susceptible to each disease.13.Okada H. Inoue T. Kikuta T. et al.A possible anti-inflammatory role of angiotensin II type 2 receptor in immune-mediated glomerulonephritis during type 1 receptor blockade.Am J Pathol. 2006; 169: 1577-1589Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar,14.Artunc F. Nasir O. Amann K. et al.Serum- and glucocorticoid-inducible kinase 1 in doxorubicin-induced nephrotic syndrome.Am J Physiol Renal Physiol. 2008; 295: F1624-F1634Crossref PubMed Scopus (30) Google Scholar At first, we tested whether p35 expression was exclusively restricted within the tubules of control and diseased kidneys of γGT.Cre;p35 F1 mice. As expected, γGT promoter–driven p35+ proximal tubular epithelia were found in the normal and diseased kidneys of γGT.Cre;p35 mice of both strains (Figure 1a and b). γGT.Cre;p35-NTN mice displayed significantly less tubular degeneration at 7 and 28 days and narrower extracellular matrix deposition at 28 days in the interstitium than in the p35-NTN mice as the positive controls (Figure 1c, e and f). In contrast, at 7 and 28 days, there were no differences in tubular degeneration and extracellular matrix deposition between γGT.Cre;p35-ADR and p35-ADR mice (Figure 1d–f). It is interesting to note that p35 expressed in the tubules did not significantly affect glomerular damages in those mice with NTN and ADR (Figure 1c, d and g). Consistent with these observations, at 28 days, serum creatinine levels were significantly lower in the γGT.Cre;p35-NTN mice than in the p35-NTN mice (serum creatinine, 0.19±0.05 vs. 0.35±0.05mg/dl, P<0.05), whereas urinary protein excretion levels were not different between them (proteinuria, 35.5±15.8 vs. 40.0±18.3mg/mg creatinine; data not shown in figure). For the nephrotic groups, no significant differences in those levels were seen between γGT.Cre;p35-ADR and p35-ADR mice (serum creatinine, 0.25±0.07 vs. 0.21±0.06mg/dl; proteinuria, 83.3±20.9 vs. 85.5±25.5mg/mg creatinine; data not shown in figure). To determine whether tubular caspase activation is involved in inflammation in the nephritic and nephrotic kidneys, immunohistochemistry and proinflammatory gene expression analysis were performed. For nephritic kidneys, at 28 days, γGT.Cre;p35-NTN mice displayed significantly less F4/80+ monocyte infiltration into the interstitium than in p35-NTN mice (Figure 2a and c). In contrast, monocyte infiltration was not attenuated in the D7 and D28 nephrotic kidneys of γGT.Cre;p35-ADR mice compared with p35-ADR mice (Figure 2b and c). Consistent with the number of monocytes, induced expression of proinflammatory genes such as monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) in the D7 and D28 nephritic kidneys were significantly lower in γGT.Cre;p35-NTN mice than in p35-NTN mice. In contrast, there were no differences in those expressions in the D7 and D28 nephrotic mice between γGT.Cre;p35-ADR and p35-ADR mice (Figure 2d). Protein levels of the active subunit of IL-1β (∼17kDa) were also significantly higher at 28 days in the p35-NTN mice than those in the γGT.Cre;p35-NTN mice (Figure 2e). At the same time points, no differences in those levels were seen in the γGT.Cre;p35-ADR and p35-ADR mice (Figure 2e). We next tested whether caspase activation in the tubules is involved in fibrogenesis in the nephritic and nephrotic kidneys. At 28 days, the γGT.Cre;p35-NTN mice displayed a significant reduction in the number of fibroblast-specific protein 1+ (FSP1+) fibroblasts in the interstitium when compared with the p35-NTN mice (Figure 3a and c). In contrast, at the same time point, the number of FSP1+ fibroblasts was not reduced in the γGT.Cre;p35-ADR mice when compared with the p35-ADR mice (Figure 3b and c). Consistent with the number of fibroblasts, at 7 and 28 days the expressions of profibrogenic genes such as plasminogen activator inhibitor-1 (PAI-1), fibronectin EIIIA isoform (FN-EIIIA), and α1 procollagen type I (α1COLI) were significantly lower in the γGT.Cre;p35-NTN mice than in the p35-NTN mice. In contrast, the expression levels of these profibrogenic genes were gradually increased in the D7 and D28 nephrotic kidneys, and there were no significant differences in those expressions between the γGT.Cre;p35-ADR and p35-ADR mice (Figure 3d). These results were also compatible with the expansion of extracellular matrix deposition shown in Figure 1c, d and f. Apoptotic cells were detected in situ by immunohistochemistry with an antibody against single-stranded DNA.17.Watanabe I. Toyoda M. Okuda J. et al.Detection of apoptotic cells in human colorectal cancer by two different in situ methods: antibody against single-stranded DNA and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method.Jpn J Cancer Res. 1999; 90: 188-193Crossref PubMed Scopus (116) Google Scholar Positively stained nuclei in tubules and glomeruli were quantitatively measured by a point-counting method described below. Very few apoptotic cells were noted in the tubules of negative controls (Figure 4a–c). At 7 and 28 days, the number of apoptotic cells in tubules was significantly higher in the p35-NTN mice than that in the γGT.Cre;p35-NTN mice, respectively, as well as in the negative controls (Figure 4a and c). In contrast, at the same time points, the increases in the number of apoptotic tubular cells were not significant in the γGT.Cre;p35-ADR mice and p35-ADR mice compared with the negative controls, and were also not different from each other (Figure 4b and c). The number of apoptotic glomerular cells was significantly increased in the nephritic and nephrotic kidneys at 7 and 28 days, which were not altered by p35 expressed in the tubules (Figure 4a and d). Immunoblotting with an anti-caspase-3 antibody showed that protein levels of an active subunit of cleaved caspase-3 (∼20kDa) were significantly higher in the D7 and D28 nephritic kidneys of p35-NTN mice than those of γGT.Cre;p35-NTN mice (Figure 4e). In contrast, no differences in those levels were seen in the D7 and D28 nephrotic kidneys of γGT.Cre;p35-ADR and p35-ADR mice (Figure 4e). The results of caspase-3 activation in Figure 4e were compatible with those of apoptotic cells in the tubules in Figure 4a–c. Apoptosis, a form of programmed cell death, is believed to delete excessive, damaged, or nonfunctioning cells and infiltrating inflammatory cells for the resolution of glomerulonephritis.18.Savill J. Regulation of glomerular cell number by apoptosis.Kidney Int. 1999; 56: 1216-1222Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar In contrast, continuous and inappropriate cell loss by disregulated apoptosis may be harmful, as suggested in various models of progressive kidney diseases such as NTN,10.Yang B. Johnson T.S. Haylor J.L. et al.Effects of caspase inhibition on the progression of experimental glomerulonephritis.Kidney Int. 2003; 63: 2050-2064Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar polycystic kidney disease,8.Tao Y. Kim J. Faubel S. et al.Caspase inhibition reduces tubular apoptosis and proliferation and slows disease progression in polycystic kidney disease.PNAS. 2005; 102: 6954-6959Crossref PubMed Scopus (93) Google Scholar and subtotal nephrectomy models.19.Yang B. Johnson T.S. Thomas G.L. et al.Apoptosis and caspase-3 in experimental anti-glomerular basement membrane nephritis.J Am Soc Nephrol. 2001; 12: 485-495PubMed Google Scholar Therefore, timely and spatially dependent roles of apoptosis in the progressive kidney diseases should be identified before application of an antiapoptosis strategy into clinical nephrology. In the NTN model, we demonstrated that inhibition of caspase activation in proximal tubules from the onset of disease significantly attenuated interstitial inflammatory and fibrotic changes, which slowed deterioration of renal function. One of the most striking upregulated proinflammatory cytokines in the NTN kidneys is TNF-α following activation of the nuclear factor-κB pathway.20.Kim J.H. Ha I.S. Hwang C.I. et al.Gene expression profiling of anti-GBM glomerulonephritis model: the role of NF-kB in immune complex kidney disease.Kidney Int. 2004; 66: 1826-1837Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar TNF-α increases the expressions of TNF-like weak inducer of apoptosis (TWEAK) receptor, Fas, Bax, and Smac/DIABLO while decreasing BclxL in tubular epithelial cells.21.Sanz A.B. Justo P. Sanchez-Nino M.D. et al.The cytokine TWEAK modulates renal tubulointerstitial inflammation.J Am Soc Nephrol. 2008; 19: 695-703Crossref PubMed Scopus (150) Google Scholar TWEAK, in the presence of TNF-α and interferon-γ, induces apoptosis in proliferating tubular cells. Cells within the damaged glomerulus in the NTN model are thought to produce TNF-α, which in combination with other inflammatory substances and urinary proteins subsequently causes apoptosis and proinflammatory and profibrogenic processes in the tubular epithelium.21.Sanz A.B. Justo P. Sanchez-Nino M.D. et al.The cytokine TWEAK modulates renal tubulointerstitial inflammation.J Am Soc Nephrol. 2008; 19: 695-703Crossref PubMed Scopus (150) Google Scholar, 22.Theilig F. Spread of glomerular to tubulointerstitial disease with a focus on proteinuria.Ann Anat. 2010; 192: 125-132Crossref PubMed Scopus (30) Google Scholar, 23.Erkan E. Garcia C.D. Patterson L.T. et al.Induction of renal tubular cell apoptosis in focal segmental glomerulosclerosis: roles of proteinuria and Fas-dependent pathways.J Am Soc Nephrol. 2005; 16: 398-407Crossref PubMed Scopus (58) Google Scholar, 24.Zeisberg M. Neilson E.G. Mechanisms of tubulointerstitial fibrosis.J Am Soc Nephrol. 2010; 21: 1819-1834Crossref PubMed Scopus (669) Google Scholar, 25.Rodriguez-Iturbe B. Garcia G.G. The role of tubulointerstitial inflammation in the progression of chronic renal failure.Nephron Clin Pract. 2010; 116: c81-c88Crossref PubMed Scopus (75) Google Scholar In this study, p35 expressed in the tubules significantly reduced the number of apoptotic cells in tubules, but not in glomeruli, in the NTN model. Conceivably, this could be due to the inhibition of apoptosis-promoting caspase-3 activated by TNF-α, which in turn contributed to attenuation of tubular degeneration in the D7 and D28 nephritic kidneys of γGT.Cre;p35-NTN mice. The activation of the initiator caspase-1 takes place in a complex that was labeled as an inflammasome.6.Bryant C. Fitzgerald K.A. Molecular mechanisms involved in inflammasome activation.Trends Cell Biol. 2009; 19: 455-464Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar The inflammasome is an intracellular, multiprotein complex containing caspase-1. Caspase-1 regulates the release of proinflammatory cytokines such as IL-1β and IL-18 in response to exogenous pathogens (pathogen-associated molecular patterns) and endogenous danger signals (danger-associated molecular patterns) from damaged or dying cells.6.Bryant C. Fitzgerald K.A. Molecular mechanisms involved in inflammasome activation.Trends Cell Biol. 2009; 19: 455-464Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar Recently, the role of the inflammasome in renal injury has also been studied.26.Anders H.J. Muruve D.A. The inflammasomes in kidney disease.J Am Soc Nephrol. 2011; 22: 1007-1018Crossref PubMed Scopus (285) Google Scholar Experiments with mice deficient for NLRP3, another component of the inflammasome, demonstrated that the NLRP3 inflammasome contributes to renal inflammation in ischemia–reperfusion injury and obstructive nephropathy.27.Iyer S.S. Pulskens W.P. Sadler J.J. et al.Necrotic cells trigger a sterile inflammatory response through the Nlrp3 inflammasome.PNAS. 2009; 106: 20388-20393Crossref PubMed Scopus (539) Google Scholar,28.Vilaysane A. Chun J. Seamone M.E. et al.The NLRP3 inflammasome promotes renal inflammation and contributes to CKD.J Am Soc Nephrol. 2010; 21: 1732-1744Crossref PubMed Scopus (392) Google Scholar Although we did not directly measure inflammasome formation in this study, p35 expressed in the tubules significantly inhibited the formation of an active subunit of IL-1β at 28 days in the γGT.Cre;p35-NTN mice and attenuated interstitial inflammation. This suggests that tubular caspase-1 activated by danger-associated molecular patterns such as adenosine triphosphate, uric acid crystals, biglycan, heat shock protein 60, and reactive oxygen species, some of which operate through Toll-like receptors, in the nephritic kidneys significantly contributes to interstitial inflammation.26.Anders H.J. Muruve D.A. The inflammasomes in kidney disease.J Am Soc Nephrol. 2011; 22: 1007-1018Crossref PubMed Scopus (285) Google Scholar, 29.Lang A. Benke D. Eitner F. et al.Heat shock protein 60 is released in immune-mediated glomerulonephritis and aggravates disease: in vivo evidence for an immunologic danger signal.J Am Soc Nephrol. 2005; 16: 383-391Crossref PubMed Scopus (47) Google Scholar, 30.Fu Y. Xie C. Chen J. et al.Innate stimuli accentuate end-organ damage by nephrotoxic antibodies via Fc receptor and TLR stimulation and IL-1/TNF-alpha production.J Immunol. 2006; 176: 632-639Crossref PubMed Scopus (49) Google Scholar This was compatible with the results of a previous study in which treatment with soluble IL-1 receptor, an antagonist against active IL-1β, significantly attenuated tissue inflammation in the NTN model.31.Karkar A.M. Tam F.W. Steinkasserer A. et al.Modulation of antibody-mediated glomerular injury in vivo by IL-1ra, soluble IL-1 receptor, and soluble TNF receptor.Kidney Int. 1995; 48: 1738-1746Abstract Full Text PDF PubMed Scopus (66) Google Scholar Apoptosis itself was revealed to be a crucial event that can initiate ischemia–reperfusion-induced inflammation in the kidney via posttranslational processing of the endothelial monocyte–activating polypeptide II and subsequent influx of leukocytes into the kidney.4.Daemen M.A.R.C. van't Veer C. Denecker G. et al.Inhibition of apoptosis induced by ischemia-reperfusion prevents inflammation.J Clin Invest. 1999; 104: 541-549Crossref PubMed Scopus (487) Google Scholar In addition, apoptotic cells release MCP-1 to promote the recruitment of monocytes at the site of apoptosis.32.Cailhier J. Laplante P. Hebert M. Endothelial apoptosis and chronic transplant vasculopathy: recent results, novel mechanisms.Am J Transplant. 2006; 6: 247-253Crossref PubMed Scopus (73) Google Scholar Thus, p35 expression in the tubules reduced the number of apoptotic tubular cells in the D7 and D28 nephritic kidneys of γGT.Cre;p35-NTN mice, which might also contribute to attenuation of interstitial inflammation. Inflammatory cells infiltrating into the peritubular interstitium further promoted cell influx and interstitial inflammation directly via the production of proinflammatory substances, and indirectly via enhancing the proinflammatory process including pan-caspase activation in the tubular epithelium, resulting in a vicious cycle.25.Rodriguez-Iturbe B. Garcia G.G. The role of tubulointerstitial inflammation in the progression of chronic renal failure.Nephron Clin Pract. 2010; 116: c81-c88Crossref PubMed Scopus (75) Google Scholar Therefore, p35 expressed in the tubules could significantly attenuate interstitial inflammation in the nephritic kidneys by terminating this vicious cycle via pan-caspase inhibition. In this study, p35 expressed in the tubules significantly attenuated not only inflammation but also fibrogenesis in the interstitium of kidneys of γGT.Cre;p35-NTN mice. This may be accounted for by the simple fact that attenuation of early interstitial inflammation resulted in the reduction of subsequent fibrogenesis, as fibrosis develops secondarily because of inflammation.24.Zeisberg M. Neilson E.G. Mechanisms of tubulointerstitial fibrosis.J Am Soc Nephrol. 2010; 21: 1819-1834Crossref PubMed Scopus (669) Google Scholar,25.Rodriguez-Iturbe B. Garcia G.G. The role of tubulointerstitial inflammation in the progression of chronic renal failure.Nephron Clin Pract. 2010; 116: c81-c88Crossref PubMed Scopus (75) Google Scholar Recently, apoptosis has been identified to be directly involved in organ fibrogenesis.33.Canbay A. Taimr P. Torok N. et al.Apoptotic body engulfment by a human stellate cell line is profibrogenic.Lab Invest. 2003; 83: 655-663Crossref PubMed Scopus (329) Google Scholar, 34.Kodama T. Takehara T. Hikita H. et al.Increases in p53 expression induce CTGF synthesis by mouse and human hepatocytes and result in liver fibrosis in mice.J Clin Invest. 2011; 121: 3343-3356Crossref PubMed Scopus (129) Google Scholar, 35.Laplante P. Sirois I. Raymond M. et al.Caspase-3-mediated secretion of connective tissue growth factor by apoptotic endothelial cells promotes fibrosis.Cell Death Differ. 2010; 17: 291-303Crossref PubMed Scopus (60) Google Scholar By engulfing apoptotic cells, reticuloendothelial system cells such as hepatic stellate cells can produce transforming growth factor-β1, an efficient profibrogenic growth factor, and likely contribute to tissue fibrogenesis.33.Canbay A. Taimr P. Torok N. et al.Apoptotic body engulfment by a human stellate cell line is profibrogenic.Lab Invest. 2003; 83: 655-663Crossref PubMed Scopus (329) Google Scholar In addition, Kodama et al.34.Kodama T. Takehara T. Hikita H. et al.Increases in p53 expression induce CTGF synthesis by mouse and human hepatocytes and result in liver fibrosis in mice.J Clin Invest. 2011; 121: 3343-3356Crossref PubMed Scopus (129) Google Scholar reported that some hepatocytes expressing p53 underwent apoptosis and were phagocytosed by hepatic stellate cells, whereas others produced profibrogenic connective tissue growth factor (CCN2/CTGF), jointly resulting in liver fibrogenesis in TG mice in which albumin promoter–driven p53 expression localized to the hepatocytes. In the nephritic kidneys, apoptotic tubular cells are likely phagocytosed by infiltrating monocytes, as well as adjacent, kidney injury molecule-1+ (KIM-1+) tubular cells,36.Ichimura T. Asseldonk E.J. Humphreys B.D. et al.Kindney injury molecule-1 is a phosphatidylserine receptor that confers a phagocytic phenotype on epithelial cells.J Clin Invest. 2008; 118: 1657-1668Crossref PubMed Scopus (555) Google Scholar which likely turn to be profibrogenic and contribute to interstitial fibrogenesis. Caspase-3 activation in apoptotic endothelial cells also triggers the export of CCN2, which in turn promotes tissue fibrogenesis.35.Laplante P. Sirois I. Raymond M. et al.Caspase-3-mediated secretion of connective tissue growth factor by apoptotic endothelial cells promotes fibrosis.Cell Death Differ. 2010; 17: 291-303Crossref PubMed Scopus (60) Google Scholar Therefore, p35 expressed in the tubules reduced tubular cell apoptosis/phagocytosis and subsequent interstitial fibrosis in the γGT.Cre;p35-NTN mice. In contrast to the NTN model, pan-caspase activation in the tubular epithelium seemed to contribute little to interstitial inflammation and fibrogenesis in the ADR model, as p35 expressed in the tubules did not affect such interstitial alterations in the D7 and D28 nephrotic kidneys of γGT.Cre;p35-ADR mice compared with those of p35-ADR mice. Incidence of apoptosis in the tubular cells was significantly lower in the ADR nephrotic kidneys than in the NTN nephritic kidneys in this study, which could partially be accounted for by mild increases in TNF-α expression in the nephrotic kidneys. In addition, protein levels of the active IL-1β subunit were not altered in the nephrotic kidneys, suggesting that active danger-associated molecular patterns were not involved in the nephrotic kidneys to activate caspase-1. Pan-caspase inhibition may affect the disease process in another pathway via acceleration of necrosis, as caspase-8 was recently discovered to cleave receptor-interacting protein kinase proteins to suppress their execution of necrosis.37.Peter M. Apoptosis meets necrosis.Nature. 2011; 471: 310-312Crossref PubMed Scopus (138) Google Scholar If it was true in the ADR nephrotic kidneys, accelerated necrosis might cancel out the beneficial effects of p35 in this study. However, as the number of necrotic cells was not counted, this scenario remains speculative. Regardless, all of these issues lead us to a notion that pan-caspase inhibition from the onset of disease is unlikely to result in preventive/therapeutic effects on the mild inflammation and steadily progressive fibrosis in the interstitium of ADR nephrotic kidneys. In addition, pathways other than caspase activation in the tubules were suggested to have a role in the mediation of glomerular damage to the tubulointerstitial changes in the ADR. Different mouse strains were used to generate NTN and ADR models in this study by selecting strains most susceptible to each disease. We previously demonstrated that the genetic background of the mouse significantly affects renal fibrogenesis.38.Kato N. Watanabe Y. Ohno Y. et al.Mapping quantitative trait loci for proteinuria-induced renal collagen deposition.Kidney Int. 2008; 73: 1017-1023Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar The possibility remains that this strain difference influenced the contribution of the tubular epithelium of apoptosis/caspase activities to renal fibrogenesis between NTN (in SJL mice) and ADR (in 129 mice). The present study suggests that early pan-caspase activation in the tubular epithelium is a crucial event in the process that initiates interstitial inflammation and subsequent fibrogenesis in the rapidly progressive, nephritic diseases such as NTN, but not in the constant, monotonous advancement of nephrotic diseases such as ADR. This puts forth new and important opportunities to prevent acute exacerbations in chronic kidney diseases. Chronic glomerulonephritis as in immunoglobulin A nephropathy, and even nephrotic syndrome as in membranous nephropathy, can be transiently exacerbated during episodes of systemic inflammation such as viral infection.39.Gluba A. Banach M. Hannam S. et al.The role of Toll-like receptors in renal diseases.Nat Rev Nephrol. 2010; 6: 224-235Crossref PubMed Scopus (160) Google Scholar The abrogation of pan-caspase activation exclusively in the tubular epithelium during such transient exacerbation periods could prevent further deterioration of renal function after the episode. New anti-pan-caspase substances currently under investigation1.Lavrik I.N. Golks A. Krammer P.H. Caspases: pharmacological manupulation of cell death.J Clin Invest. 2005; 115: 2665-2672Crossref PubMed Scopus (498) Google Scholar,40.Linton S.D. Caspase inhibitors: a pharmaceutical industry perspective.Curr Top Med Chem. 2005; 5: 1697-1717Crossref PubMed Scopus (69) Google Scholar may provide new therapeutic means to treat these conditions. Two sets of TG mice were used in this study. The γGT.Cre mice express Cre recombinase under the control of the γGT promoter mainly in tubular epithelia. The p35 mice were a generous gift from Professor M. Miura (Department of Genetics, Graduate School of Pharmaceutical Sciences, University of Tokyo).15.Shibata M. Hisahara S. Hara H. et al.Caspases determine the vulnerability of oligodendrocytes in the ischemic brain.J Clin Invest. 2000; 106: 643-653Crossref PubMed Scopus (84) Google Scholar In the p35 mice, the p35 gene coding a baculovirus-derived, pan-caspase inhibitor protein p35 was separated from the universal CAG promoter by a floxed STOP sequence. All of these mice were back-crossed to SJL and 129 mice more than 10 times before experiments. TG protocols were approved by the Institutional Animal Care and Use Committee at the Saitama Medical University. DNA was extracted from tail biopsies from γGT.Cre, p35, and γGT.Cre;p35 mice, and genotyping PCR was performed with an Extract-N-Amp Tissue PCR Kit (XNAT2; Sigma, St. Louis, MO) according to the manufacturer's instructions. The following PCR primers and annealing temperatures were used. γGT.Cre: Cre-FW 5′-AGGTGTAGAGAAGGCACTTAGC-3′, Cre-RV 5′-CTAATCGCCATCTTCCCAGCAGG-3′, 63°C; p35: p35-FW 5′-TGGATGGATTCCACGATAGC-3′, p35-RV 5′-TGCACACTCTCCACGTAAGC-3′, 55°C. Cycle programs were performed in a thermocycler (iCycler; Bio-Rad Japan, Tokyo, Japan). All primers were started with 4min at 95°C, followed by 35 cycles for 1min at 95°C, 1min at the primer's appropriate annealing temperature, and 1min at 72°C, finishing with 5min at 72°C. Products were analyzed by electrophoresis in 1% agarose using tris–acetate–EDTA buffer. Six male, 5- to 6-week-old mice from each group of TG mice such as γGT.Cre;p35 and p35 mice were used to generate the NTN and ADR models. TG mice in the SJL background were used for the NTN model, whereas those in the 129 background were for the ADR model as the mouse strains most susceptible to each disease. Generation of the NTN and ADR models was reported in detail previously.13.Okada H. Inoue T. Kikuta T. et al.A possible anti-inflammatory role of angiotensin II type 2 receptor in immune-mediated glomerulonephritis during type 1 receptor blockade.Am J Pathol. 2006; 169: 1577-1589Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar,14.Artunc F. Nasir O. Amann K. et al.Serum- and glucocorticoid-inducible kinase 1 in doxorubicin-induced nephrotic syndrome.Am J Physiol Renal Physiol. 2008; 295: F1624-F1634Crossref PubMed Scopus (30) Google Scholar The p35 mice with or without such manipulation were used as the positive and the negative controls, respectively. At 7 and 28 days after NTN and ADR generation, we confirmed the development of early and advanced pathological changes in those diseased kidneys in preliminary experiments, and thus we used these protocols in this study. At these time points, mice were killed after collection of serum and urine samples, and kidney tissues were harvested for RNA and protein extraction, and paraformaldehyde fixation for paraffin blocks. Animal care and treatment were conducted in accordance with the institutional guidelines that adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Serum creatinine levels were measured with an auto-analyzer (Dri-Chem 3000; Fujifilm, Tokyo, Japan). Urinary protein and creatinine concentrations were measured by using a protein assay kit (Thermo Fisher Scientific, Waltham, MA) and a creatinine HA test kit (Wako Pure Chemical, Osaka, Japan), respectively. Sections (4μm) cut from paraffin-embedded kidneys were processed for Masson's trichrome staining. The number of cortical tubules suffering from degeneration including cast formation, atrophy, dilatation, and vacuolar change was counted among 10 tubules in 10 high-power ( × 200) cortical fields, and the tubular degeneration indices were expressed as the percentage of affected tubules in each kidney section. Interstitial fibrosis was quantitatively determined with the Mac SCOPE software version 2.5 (Mitani, Fukui, Japan) in 10 high-power ( × 200) cortical fields, and the interstitial fibrosis indices were expressed as the mean percentage area in blue per cortical field in Masson's trichrome–stained sections.13.Okada H. Inoue T. Kikuta T. et al.A possible anti-inflammatory role of angiotensin II type 2 receptor in immune-mediated glomerulonephritis during type 1 receptor blockade.Am J Pathol. 2006; 169: 1577-1589Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar Glomerular damage such as sclerosis and crescent formation was quantitatively determined with the Mac SCOPE software in 30 glomeruli, and the glomerular damage indices were expressed as the mean percentage of sclerotic/crescentic area per glomerulus.41.Okada H. Moriwaki K. Kalluri R. et al.Inhibition of monocyte chemoattractant protein-1 expression in tubular epithelium attenuates tubulointerstitial alteration in rat Goodpasture syndrome.Kidney Int. 2000; 57: 927-936Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar For immunohistochemistry, the antibodies against p3515.Shibata M. Hisahara S. Hara H. et al.Caspases determine the vulnerability of oligodendrocytes in the ischemic brain.J Clin Invest. 2000; 106: 643-653Crossref PubMed Scopus (84) Google Scholar (a generous gift from Professor M. Miura), single-stranded DNA17.Watanabe I. Toyoda M. Okuda J. et al.Detection of apoptotic cells in human colorectal cancer by two different in situ methods: antibody against single-stranded DNA and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method.Jpn J Cancer Res. 1999; 90: 188-193Crossref PubMed Scopus (116) Google Scholar (18731; Immuno-Biological Laboratories, Fujioka, Japan), mouse monocytes (F4/80; AbD Serotec, Oxford, UK), and fibroblasts (FSP1)42.Strutz F. Okada H. Lo C.W. et al.Identification and characterization of a fibroblast marker: FSP1.J Cell Biol. 1995; 130: 393-405Crossref PubMed Scopus (889) Google Scholar were diluted (1:400) into 1% bovine serum albumin in phosphate-buffered saline as the blocking buffer, and then applied on the sections for 8h. Signals were amplified using the TSA kit (Molecular Probe/Invitrogen, Carlsbad, CA) and labeled using Alexa Fluor 555 (Molecular Probe) according to a conventional indirect method. Negative-control sections were treated as described above, but the primary antibody was omitted. YO-PRO-1 (Molecular Probe) was used to detect the nuclei. A confocal laser scanning microscope (FV1000; Olympus, Tokyo, Japan) was used for data acquisition. The numbers of single-stranded DNA+ nuclei in the tubules and in the glomeruli, and F4/80+ monocytes and FSP1+ fibroblasts in the interstitium, were counted in 10 microscopic fields using a × 40 objective lens, and their indices were expressed as the mean number per field. We performed RNA preparation and quantitative PCR as described previously.13.Okada H. Inoue T. Kikuta T. et al.A possible anti-inflammatory role of angiotensin II type 2 receptor in immune-mediated glomerulonephritis during type 1 receptor blockade.Am J Pathol. 2006; 169: 1577-1589Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar The primers used were as follows: p35 primer, FW 5′-TGTCCCAGACGATTATTCGAG-3′, RV 5′-GTTTTGTCAATTGCGTGTTCA-3′; MCP-1 primer, FW 5′-CTCTCTTCCTCCACCACCAT-3′, RV 5′-ACTGCATCTGGCTGAGCCA-3′; TNF-α primer, FW 5′-TCTTCTCATTCCTGCTTGTGG-3′, RV 5′-GGTCTGGGCCATAGAAACTGA-3′; transforming growth factor-β1 primer, FW 5′-CAGTGGCTGAACCAAGGAGAC-3′, RV 5′-ATCCCGTTGATTTCCACGTG-3′; CCN2 primer, FW 5′-GTGGAATATTGCCGGTGCA-3′, RV 5′-CCATTGAAGCATCTTGGTTCG-3′; PAI-1 primer, FW 5′-AGGATCGAGGTAAACGAGAGC-3′, RV 5′-GCGGGCTGAGATGACAAA-3′; FN-EIIIA primer, FW 5′-ATCCGGGAGCTTTTCCCTG-3′, RV 5′-TGCAAGGCAACCACACTGAC-3′; COLI primer, FW 5′-TGTAAACTCCCTCCACCCCA-3′, RV 5′-TCGTCTGTTTCCAGGGTTGG-3′; and GAPDH primer, FW 5′-TGCAGTGGCAAAGTGGAGATT-3′, RV 5′-TTGAATTTGCCGTGAGTGGA-3′. All of these oligodeoxynucleotides were designed using the Primer Express software (Perkin Elmer, Foster City, CA). Preliminary real-time PCR experiments in which these primer sets were used yielded appropriately sized, single products. Immunoblotting was performed using protein samples extracted from kidney tissues, as described previously.43.Okada H. Inoue T. Kikuta T. et al.Poly(ADP-ribose) polymerase-1 (PARP-1) and PARP-1 binding element enhance murine CCN2 gene transcription in renal tubular epithelial cells.J Am Soc Nephrol. 2008; 19: 933-942Crossref PubMed Scopus (23) Google ScholarAnti-IL-1β (NIBSC, Potters Bar, UK), anti-caspase-3 (BD Transduction, Franklin Lakes, NJ), and anti-actin (sc-8432; Santa Cruz Biotechnology, Santa Cruz, CA) were used as the primary antibodies in this study. The immunoreactive proteins were detected by enhanced chemiluminescence and captured on X-ray film. Molecular weight standards (LC5602; Invitrogen) were run with each blot. The intensity of each band was measured using the Mac SCOPE software. Values are presented as the mean±s.d. Statistical differences between groups were evaluated by analysis of variance, followed by a Bonferroni/Dunnett's test. P-values that were ±0.05 were considered to be statistically significant. We thank M Funabashi for her technical assistance. This research was supported in part by research grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology, and Asubio Pharma.
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