| Summary: | 博士 === 國立臺灣大學 === 臨床醫學研究所 === 92 === Almost all forms of kidney diseases with renal failure progression are characterized by cell proliferation, inflammation, and extracellular matrix accumulation. Whatever the initial injury, the remaining nephrons undergo adaptive hypertrophy and hyperfiltration that minimize the functional consequences of the progressive nephron loss. However, the adaptation ultimately leads to a vicious cycle in which hyperfiltration of the remaining nephrons impairs glomerular barrier, which in turn induces tubulointerstitial damage and then results in the loss of more nephrons. Angiotensin-converting enzyme inhibitor or angiotensin II receptor blocker is shown to break the vicious cycle and protect kidneys from progressive injury. However, the ultimate step of halting renal disease progression in the long term is not achieved with either drug alone, especially when the treatment is started during the late course of the renal disease. Furthermore, hyperkalemia frequently complicates the treatment. In addition to angiotensin-converting enzyme inhibitor (ACEI) and angiotensin II receptor blocker (ARB) with the well-recognized renoprotective effect, mycophenolate mofetil, a specific anti-lymphocyte agent, and cerivastatin, a hydroxymethylglutaryl-coenzyme A reductase inhibitor, have been shown to attenuate the progression of experimental chronic renal disease, suggesting a novel therapy for chronic renal disease through reducing the infiltration of inflammatory mononuclear cells.
Pentoxifylline is a phosphodiesterase inhibitor used clinically to treat patients with peripheral vascular diseases. In addition to its hemorrheological activity, many clinical studies have shown that pentoxifylline decreases the proteinuria of patients with diabetic or membranous nephropathy, improves the disease of patients with multiple sclerosis or human T lymphotropic virus type I-associated myelopathy. Pentoxifylline also inhibits the proliferation of cultured lymphocytes, fibroblasts, mesangial cells, and reduces the production of extracellular matrix proteins. Furthermore, pentoxifylline can attenuate experimental cyclosporine nephropathy and mesangial proliferative glomerulonephritis.
Thus, we hypothesized that pentoxifylline could attenuate the progression of chronic renal disease through its suppression on inflammation, cell proliferation, and fibrosis. To test this hypothesis, we first used the rat model of 5/6 subtotal nephrectomy to study the in vivo effects of pentoxifylline on renal function, pathology, the expression of proinflammatory and profibrogenic genes. Secondly, we used in vitro cultures of proximal tubular cells, mesangial cells, and interstitial fibroblasts to study the possible mechanisms for the renoprotective effect of pentoxifylline. Finally, we compared the effects of combining pentoxifylline with an angiotensin-converting enzyme inhibitor, cilazapril, versus either drug alone on the renal disease progression in this rat model.
After 5/6 subtotal nephrectomy, rats developed progressively elevated proteinuria and plasma creatinine, glomerulosclerosis, interstitial inflammation and fibrosis, all of which were attenuated by 40 to 60% by pentoxifylline. However, the elevated blood pressure was not changed by pentoxifylline. Pentoxifylline reduced the upregulation of monocyte chemoattractant protein-1 (MCP-1) gene by 60% in the cortex of remnant kidney, as well as in a dose-dependent manner in the albumin- or angiotensin II-stimulated proximal tubular cells. It also reduced the upregulation of mitogenic and profibrogenic genes by 50%, including platelet-derived growth factor (PDGF), fibroblast growth factor-2 (FGF-2), transforming growth factor-b1 (TGF-b1), connective tissue growth factor (CTGF), types I and III collagen in the cortex of remnant kidney. Furthermore, pentoxifylline was found to decrease the numbers of interstitial myofibroblasts by 60% in the cortex of remnant kidney and suppress the proliferation of cultured interstitial fibroblasts. It also reduced the angiotensin II- or TGF-b1-induced expression of connective tissue growth factor gene in cultured fibroblasts and mesangial cells. Combining pentoxifylline with an angiotensin-converting enzyme inhibitor, cilazapril, almost completely attenuated the renal disease progression in rats with remnant kidney.
In this experiment, we demonstrated that pentoxifylline could attenuate the chronic renal disease progression, in particular, through a renoprotective effect that was largely independent of changes in blood pressure. The renal pathology of remnant kidney is characterized with the progressive glomerulosclerosis and interstitial fibrosis as well as the preceding hyperplasia of glomerular mesangial cells and interstitial fibroblasts. These pathologic changes were ameliorated by pentoxifylline treatment in this study. Furthermore, we also demonstrated that pentoxifylline could reduce the upregulation of PDGF and FGF-2 genes in remnant kidneys, two important mitogens for mesangial cells and fibroblasts. Several lines of evidence indicate that pentoxifylline can suppress the proliferation of glomerular mesangial cells and interstitial fibroblasts in vitro or in vivo. Therefore, one of the possible mechanisms for the renoprotection may be linked to the effect of pentoxifylline against the proliferation of glomerular mesangial cells and interstitial fibroblasts.
High numbers of inflammatory mononuclear cells and cells expressing MHC class II antigen in the cortical interstitium of remnant kidney in this study were in accordance with previous studies. The pathologic significance of interstitial inflammation has been well recognized and further strengthened by the evidence that mycophenolate mofetil can attenuate the renal injury in rats with remnant kidney. In this study, we found that pentoxifylline could attenuate the infiltration of macrophages and lymphocytes in remnant kidney. Such attenuation could be in part due to the effect of pentoxifylline against the proliferation of inflammatory mononuclear cells. However, we also found that pentoxifylline could reduce the upregulation of MCP-1 gene in the remnant kidney and in the albumin- or angiotensin II-stimulated proximal tubular cells. Recent studies have shown that the increased expression of MCP-1 in proximal tubular cells and interstitial mononuclear cells of the remnant kidney is downregulated after ACEI or ARB treatment. Albumin can stimulate proximal tubular cells to produce MCP-1 through nuclear factor-k B (NFkB) activation. Furthermore, there are studies demonstrating that angiotensin II may stimulate MCP-1 to recruit inflammatory mononuclear cells through NFkB-dependent pathway. Pentoxifylline is reported to be an inhibitor of NFkB and inhibits MCP-1 production in macrophages/monocytes. Therefore, the attenuation of interstitial inflammatory cell infiltration by pentoxifylline may be also due to the downregulation of MCP-1 in remnant kidney. In addition, we also found that pentoxifylline could reduce the overexpression of MHC class II antigen in the remnant kidney. The overexpression of MHC class II antigen has been known to increase the activation of T lymphocytes by macrophages and lead to tubulointerstitial damage. The inhibitory effect of pentoxifylline on MHC class II antigen expression may be in part due to the decreased infiltration of macrophages/monocytes in the remnant kidney. However, it is also possible due to the inhibition of MHC class II antigen expression in macrophages by pentoxifylline. Therefore, the second possible mechanism to prevent the renal disease progression may be due to the effect of pentoxifylline on attenuating inflammation through reducing the overexpression of MHC class II antigen and MCP-1 in the remnant kidney.
A variety of cytokines including PDGF, FGF-2, TGF-b1 and CTGF are important growth factors for cell proliferation and extracellular matrix production in glomerular mesangial cells and interstitial fibroblasts through paracrine or autocrine stimulation. In this study, we found that pentoxifylline could downregulate the gene expression of these growth factors by 50 to 60% in the remnant kidney though it could not affect the expression of TGF-b1 gene in either glomerular mesangial cells or interstitial fibroblasts stimulated by angiotensin II. Pentoxifylline has been reported to reduce FGF-2 gene expression in serum-stimulated renal fibroblasts, but there seems no evidence that pentoxifylline can inhibit PDGF and TGF-b1 gene expression in cultured cells. We therefore suggest that pentoxifylline downregulated the gene expression of PDGF, FGF-2, and TGF-b1 in the remnant kidney in part due to the decreased numbers of cells secreting these cytokines, including infiltrating inflammatory cells, glomerular mesangial cells, and interstitial myofibroblasts. Pentoxifylline may prevent the progressive renal fibrosis through reducing the upregulation of these growth factor and collagen genes in the remnant kidney, which is the third possible mechanism for its renoprotective effect. However, the inhibition of TGF-b1 signaling and suppression of cellular immunity by pentoxifylline may impair the mechanisms for defense and healing in surgical wound. This may probably explain why we observed poor healing of surgical wound and higher mortality in rats receiving pentoxifylline from the day of surgery in pilot study.
Cilazapril alone prevented renal disease progression remarkably, but the therapy combining cilazapril with pentoxifylline provided more pronounced protection that halted the renal disease progression almost completely. The result suggests that such a combined therapy may have additional effects. Our observation was similar to the outcome of rats with remnant kidney receiving the therapy combining ACEI with mycophenolate mofetil. However, pentoxifylline is a drug with few side effects clinically, especially sparing bone marrow suppression.
One of the possible mechanisms for the renoprotection may be linked to the effect of pentoxifylline against the proliferation of glomerular mesangial cells, which occurs early in remant kidney after 5/6 subtotal nephrectomy and is responsible for the glomerular hypertrophy and subsequent glomerulosclerosis. The expression of PDGF increases before the glomerular mesangial cell proliferation, and the source of PDGF is initially derived from the influxed platelets and then maintained by the mesangial cells and the other inflammatory cells. Although PDGF is a crucial growth factor for mesangial cell proliferation, however, it also plays an important role in the pathogenesis of mesangial proliferative glomerular disease. Specific antagonism of PDGF reduces mesangial cell proliferation and glomerulosclerosis in experimental mesangial proliferative glomerular disease. Therefore, pentoxifylline-induced inhibition of PDGF-stimulated mesangial cell proliferation represents an excellent strategy for ameliorating the renal disease. Because pentoxifylline alters neither PDGF binding to receptor nor receptor phosphorylation, a possible mechanism for its inhibitory effect on PDGF-stimulated proliferation is the inhibition of PDGF postreceptor signaling. Indeed, pentoxifylline has been found to inhibit PDGF-induced activation of mitogen-activated protein kinase (MAPK) pathway.
Both MAPK and phosphatidylinositol 3-kinase (PI3K) pathways transmit PDGF signaling to regulate the expression of cyclins and cyclin-dependent kinase (Cdk) inhibitors, thereby activating Cdk to promote cell cycle progression. Accordingly, activation of mesangial cell proliferation has been shown to be associated with upregulation of cyclin D1 and downregulation of p27Kip1. PI3K and its downstream effector Akt have been demonstrated to mediate p27Kip1 downregulation induced by PDGF. The pathway that is responsible for cyclin D1 induction in mesangial cells, however, has not been defined. Whether pentoxifylline inhibits PDGF postreceptor signaling pathways, thereby inhibiting mesangial cell proliferation, is not clear.
Thus, we hypothesized that pentoxifylline could inhibit mesangial cell proliferation through the inhibition of PDGF postreceptor signaling. To test this hypothesis, we first used the flow cytometry to analyze the cell cycle profiles of PDGF-stimulated mesangial cells after pentoxifylline or chemical inhibition of MAPK, or PI3K. Secondly, we studied the target cell cycle protein which was responsible for the growth-inhibitory effect of pentoxifylline. We then delineated the signaling pathway by which PDGF regulated the cell cycle protein and mesangial cell proliferation. Finally, we studies the mechanisms and possible secondary messengers by which pentoxifylline inhibited the signaling pathway by western blot analysis and transient transfection of constitutively active mutant of MAPK or PI3K/Akt.
We demonstrated that, in PDGF-stimulated mesangial cells, pentoxifylline caused G1 arrest by downregulation of cyclin D1 expression, which subsequently attenuated Cdk4 activity. In vivo, pentoxifylline similarly reduced cyclin D1 expression in mesangial cells of rats with acute Thy1 glomerulonephritis. The mechanism by which pentoxifylline reduced cyclin D1 was also investigated. Pentoxifylline blocked Akt, but not PI3K activation in response to PDGF and abrogated cyclin D1 induction by PI3K, suggesting an effect of pentoxifylline on Akt itself. Indeed, pentoxifylline was capable of blocking the membrane translocation of Akt, and enforced targeting of Akt to cell membrane prevented the inhibition of Akt and cyclin D1 by pentoxifylline. Since pentoxifylline has been known to increase intracellular cyclin adenosine monophosphate (cAMP) levels by inhibiting phosphodiesterase, the role of PKA in these events was investigated. PKA antagonist H89 abolished cell proliferation effects of pentoxifylline, restored cyclin D1 expression as well as Akt membrane translocation and activation by PDGF, whereas dibutyryl cAMP and forskolin recapitulated the functions of pentoxifylline in mesangial cells.
In this experiment, we demonstrated that pentoxifylline reduced Akt phosphorylation triggered by constitutive activation of PI3K in mesangial cells. We further demonstrated that pentoxifylline inhibited Akt activation through blocking its membrane translocation, and enforced membrane targeting of Akt could overcome the inhibition of Akt kinase activity by pentoxifylline. The membrane translocation of Akt is crucial for phosphorylation and activation by phosphoinositide-dependent kinase, which is usually constitutively active in various cell types, including mesangial cells. How pentoxifylline blocks Akt membrane translocation is currently unclear. Nevertheless, the blockage of Akt membrane targeting without affecting PI3K activity has been reported for ceramide. Additional experiments are required for determining whether pentoxifylline and ceramide block Akt membrane translocation through the same mode.
The PI3K/Akt pathway mediates the mitogenic effect of PDGF through regulating G1 Cdk activity by promoting the synthesis of cyclin subunits, as well as decreasing the levels of Cdk inhibitors. In this study, we first confirmed that the PI3K/Akt pathway mediated PDGF signaling to cyclin D1 expression and Cdk4 activation in mesangial cells, which is in agreement with reports in the other cell types. We further demonstrated that cyclin D1 induced in mesangial cells overexpressing a constitutively membrane-localized Akt was refractory to pentoxifylline, whereas that induced in cells overexpressing constitutively active PI3K remained sensitive. This novel finding indicates that blockage of Akt membrane translocation is one of the mechanisms by which pentoxifylline downregulates PDGF-induced cyclin D1. Although Akt has been well documented as a pro-survival protein, we found that neither dominant-negative interference nor pentoxifylline-induced inhibition of its activity caused apoptosis in rat mesangial cells, which is in consistent with a previous report. Among the in vivo substrates of Akt, glycogen synthase 3b is probably the most important in regulating its cell cycle effects. Inhibitory phosphorylation of glycogen synthase 3b by Akt prevents its phosphorylation of cyclin D1, thereby suppressing cyclin D1 proteolytic cleavage. Accordingly, we observed that pentoxifylline attenuated PDGF-induced glycogen synthase 3b phosphorylation, which will cause cyclin D1 phosphorylation and degradation. In addition to the inhibitory effect on glycogen synthase 3b -induced cyclin D1 degradation, Akt has been shown to induce cyclin D1 transcription by phosphorylating and repressing FKHR, a member of forkhead transcription factors. Whether regulation of cyclin D1 expression by Akt is due to phosphorylation of glycogen synthase 3b and forkhead transcription factors is yet to be established in mesangial cells.
In addition to PI3K/Akt pathway, we also confirmed that MAPK pathway mediated the regulation of cyclin D1 expression in PDGF-stimulated mesangial cells, which has well been demonstrated in the other cell types. Pentoxifylline, indeed, has been shown to inhibit Raf-1/extracellular signal-regulated kinase and Jun N-terminal kinase pathways, thereby suppressing cell proliferation. Accordingly, pentoxifylline is a potent inhibitor of mesangial cell proliferation by blocking the multiple postreceptor signaling pathways of PDGF.
In this study, we found that the antiproliferative effect of pentoxifylline on PDGF-stimulated mesangial cells was protein kinase A-dependent, which is in agreement with findings observed from other cell types. In addition to pentoxifylline, protein kinase A is also involved in adrenomedullin-induced antiproliferative effect on PDGF-stimulated mesangial cells, indicating a cross talk between protein kinase A and PDGF signaling pathways. We thus dissected the mechanism of this cross talk, and found that the inhibitory effect of pentoxifylline on Akt activation and cyclin D1 expression in PDGF-stimulated mesangial cells was again protein kinase A -dependent. These findings, in conjunction with the demonstration that pentoxifylline interferes with Akt membrane translocation, strongly suggest that protein kinase A blocks PDGF signaling by inhibiting Akt membrane translocation. In accordance with our findings, stimulation of protein kinase A was previously found to mediate the inhibitory effect of cAMP on Akt activation in HEK 293 cells. Therefore, blockage of Akt membrane translocation by protein kinase A might not be restricted to the mesangial cell system used in this study. However, the effect of cAMP on cell proliferation and Akt activation has been shown to be cell type-specific. Multiple cAMP-mediated pathways exist and only some are protein kinase A dependent. For instance, cAMP has been reported to inhibit PI3K/Akt activation in C6 glioma cells through Rap1 inhibition, a mechanism independent of protein kinase A. In addition to the inhibitory effect of protein kinase A on Akt activation, there is evidence that protein kinase A can prevent Raf from activation by Ras through phosphorylating Raf and promoting the binding of protein 14-3-3.
These studies have shown that pentoxifylline has antiproliferative effect on glomerular mesangial cells and interstitial fibroblasts, effectively reduces inflammatory cytokine expression, inflammatory mononuclear cell infiltration, and extracellular matrix accumulation. Among the renoprotective effects, we have first demonstrated the pentoxifylline inhibits PDGF-stimulated cyclin D1 expression in mesangial cells by blocking Akt membrane translocation. However, the detail mechanism by which pentoxifylline blocks the Akt membrane translocation is unkown. We have also not disclosed whether the downregulation of cyclin D1 is totally responsible for the growth-inhibitory effect of pentoxifylline on mesangial cells because overexpression of cyclin D1 is found to cause mesangial cell apoptosis, which makes it impossible to examine whether overexpression of cyclin D1 overcomes the growth-inhibitory effect of pentoxifylline. The detail mechanism by which pentoxifylline inhibits inflammation and fibrosis remains to be determined, expecially which one of the TGF-b1-activated SMAD and MAPK pathways is inhibited by pentoxifylline, and whether pentoxifylline downregulates MCP-1 through inhibiting albumin- or angiotensin II-stimulated NF-kB, MAPK pathways.
Pentoxifylline has been shown to cause indiscernible cytotoxicity in these studies as well as little adverse effect in clinical application, indicating that pentoxifylline is a safe drug for kidney disease treatment. Because pentoxifylline is demonstrated to attenuate the remnant kidney disease progression through improving multiple pathogenic mechanisms as well as to inhibit mesangial cell proliferation through blocking both MAPK and PI3K/Akt pathways, it should be a potent drug for kidney disease treatment. Clinical trial for pentoxifylline treatment in chronic kidney disease patients will be conducted to evaluate its efficacy in preventing human chronic kidney disease progression. In the first set of clinical trial, we will first examine whether pentoxifylline can prevent the chonic kidney disease progression in patients with severe renal insufficiency (serum creatinine beyond 3.0 mg/dl) under low protein diet and targeted blood pressure (diastolic blood pressure below 80 mmHg and/or systolic blood pressure below 130 mmHg). In the second set of clinical trial, we will examine whether pentoxifylline has additive renoprotective effect to ARB in patients with mild to moderate renal insufficiency (serum creatinine between 1.3 and 3.0 mg/dl)。 We believe that the two clinical trials will answer the questions whether pentoxifylline can attenuate the chonic kidney disease progression of human.
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