Cross-communication between glomerular cells and infiltrating mononuclear cells plays an importan... more Cross-communication between glomerular cells and infiltrating mononuclear cells plays an important role in the generation of or recovery from glomerular diseases. We found that cultured mesangial cells secrete a factor that inhibits production of proinflammatory cytokines by activated macrophages. Treatment of J774.2 macrophages with conditioned media from rat mesangial cells blunted the transcriptional induction of IL-1 beta, IL-6, and TNF-alpha by LPS. None of the media conditioned by other fibroblastic, epithelial, or endothelial cell lines exhibited the inhibitory effect. Media conditioned by normal rat glomeruli contained a similar inhibitory activity, which was enhanced in an acute model of mesangial proliferative glomerulonephritis. To identify the active component involved, we examined the expression of known macrophage deactivators IL-10, IL-13, and TGF-beta 1 in mesangial cells. Under the basal culture conditions, strong expression of TGF-beta 1 mRNA was observed, whereas ...
Recent studies have demonstrated much larger variability in the total number of nephrons in norma... more Recent studies have demonstrated much larger variability in the total number of nephrons in normal populations than previously suspected. In addition, it has been suggested that individuals with a low nephron number may have an increased lifetime risk of hypertension or renal insufficiency, emphasizing the importance of evaluating the nephron number in each individual. In view of the fact that all previous reports of the nephron number were based on analyses of autopsy kidneys, the identification of surrogate markers detectable in living subjects is needed in order to enhance understanding of the clinical significance of this parameter. In this review, we summarize the clinicopathological factors and findings indicating a reduction in the nephron number, focusing particularly on those found at the time of a preserved renal function.
Mesenchymal stem cells (MSCs) have been applied to the treatment of various diseases, and MSC adm... more Mesenchymal stem cells (MSCs) have been applied to the treatment of various diseases, and MSC administration in marginal donor grafts may help avoid the ischemia-reperfusion injury associated with solid organ transplants. Given the reports of side effects after intravenous MSC administration, local MSC administration to the target organ might be a better approach. We administered adipose tissue-derived MSCs (AT-MSCs) ex vivo to donor rat kidneys obtained after cardiac death (CD). Using male Lewis rats (8-10 weeks), and a marginal transplant model of 1hr CD plus 1hr sub-normothermic ET-Kyoto solution preservation were conducted. AT-MSCs obtained from double-reporter (luciferase-LacZ) transgenic Lewis rats were injected either systemically (1.0 × 10(6) cells/0.5 mL) to bilaterally nephrectomized recipient rats that had received a marginal kidney graft (n = 6), or locally via the renal artery (500 μL ET-Kyoto solution containing the same number of AT-MSCs) to marginal kidney grafts, wh...
Recent findings have demonstrated that stem cells can differentiate into mature tissue when suppl... more Recent findings have demonstrated that stem cells can differentiate into mature tissue when supplied with a niche containing factors identical to those in the normal developmental program. A niche for the development of an organ can be provided by xenotransplantation of a similar developing organ. However, this process has many technical, safety, and ethical concerns. Here, we established xenotransplantation models that control endogenous mesenchymal stem cell (MSC) differentiation into mature erythropoietin (EPO)-producing tissue in a niche provided by a developing xenometanephros. Transplantation of rat metanephroi into mouse omentum, and similarly pig metanephroi into cat omentum, led to the recruitment of host cells and EPO production. EPO-expressing cells were not differentiated from integrating vessels because they did not coexpress endothelial markers (Tie-2 and VE-cadherin). Instead, EPO-expressing cells were shown to be derived from circulating host cells, as shown by enhan...
Proceedings of the National Academy of Sciences, 2005
The use of stem cells has enabled the successful generation of simple organs. However, anatomical... more The use of stem cells has enabled the successful generation of simple organs. However, anatomically complicated organs such as the kidney have proven more refractory to stem-cell-based regenerative techniques. Given the limits of allogenic organ transplantation, an ultimate therapeutic solution is to establish self-organs from autologous stem cells and transplant them as syngrafts back into donor patients. To this end, we have striven to establish an in vitro organ factory to build up complex organ structures from autologous adult stem cells by using the kidney as a target organ. Cultivation of human mesenchymal stem cells in growing rodent embryos enables their differentiation within a spatially and temporally appropriate developmental milieu, facilitating the first step of nephrogenesis. We show that a combination of whole-embryo culture, followed by organ culture, encourages exogenous human mesenchymal stem cells to differentiate and contribute to functional complex structures of...
In this study, we examined the effects of macrophage-colony stimulating factor (M-CSF) on glomeru... more In this study, we examined the effects of macrophage-colony stimulating factor (M-CSF) on glomerular macrophages in lipopolysaccharide (LPS)-induced murine nephritis. Mice injected intraperitoneally with either M-CSF plus LPS, LPS alone, M-CSF alone or saline every day for 8 days were examined for the degree of urine albumin excretion and lymphocyte-function associated antigen-1-positive (LFA-1+) cells in peripheral blood as well as renal pathology. From our results, LPS or M-CSF combined with LPS emphasized the degree of proteinuria, glomerular deposition of immunoglobulins and mesangial proliferation, associated with accumulation of macrophages in the glomeruli. However, in immunohistological examination of kidneys from these nephritic mice, neither intercellular adhesion molecule-1 (ICAM-1), which may play an important role in the recruitment of macrophages into glomeruli, M-CSF receptor nor the number of LFA-1+ cells in peripheral blood was enhanced by M-CSF. On the other hand, ...
In chronic inflammation, macrophages and neutrophils, which are derived from bone marrow, play a ... more In chronic inflammation, macrophages and neutrophils, which are derived from bone marrow, play a pivotal role. Therefore, reconstitution of bone marrow with anti-inflammatory stem cells may modify inflammation. In this study, transplantation-based gene therapy was applied to glomerular inflammation for a long-lasting suppression of the glomerular damage seen in chronic nephritis. Bone marrow cells were harvested from male donor mice, which had received 5-fluorouracil 3 days previously, and transduced with an interleukin 1 (IL-1) receptor antagonist (IL-1Ra) or a mock gene using a retrovirus vector. After confirmation that transduced cells possessed the transgene at approximately 0.7 copies per cell and secreted recombinant IL-1Ra, these cells were infused into sublethally irradiated (6 Gy) female recipients once daily for 4 consecutive days. These female recipient mice had the male Y antigen in bone marrow, liver, and spleen, and 10% to 20% of their spleen cells possessed the transg...
Previously, we reported that human mesenchymal stem cells (hMSCs) that were cultivated in growing... more Previously, we reported that human mesenchymal stem cells (hMSCs) that were cultivated in growing embryos differentiated in an appropriate developmental milieu, thereby facilitating the development of a functional renal unit. However, this approach required transfection with an adenovirus that expressed glial cell line‐derived neurotrophic factor (GDNF) to enhance the development of hMSC‐derived renal tissue, and safety issues restrict the clinical use of such viral vectors. To circumvent this problem, we tested an artificial polymer as a means to diffuse GDNF. This GDNF‐polymer, which exists in liquid form at 4°C but becomes a hydrogel upon heating to 37°C, was used as a thermoreversible switch, allowing the injection of hMSCs at low viscosity using a mouth pipette, with subsequent slow diffusion of GDNF as it solidified. The polymer, which was dissolved in a solution of GDNF at 4°C and then maintained at 37°C, acted as a diffuser of GDNF for more than 48 h. LacZ‐transfected hMSCs ...
Tubulointerstitial injury is a final common pathway for progressive renal injury in various renal... more Tubulointerstitial injury is a final common pathway for progressive renal injury in various renal diseases. Several cytokines, infiltrating cells and adhesion molecules are thought to be involved in the development of interstitial injury and fibrosis. However, there is currently no effective therapy for interstitial fibrosis. In this study, we examined the therapeutic efficacy of genetically modified bone marrow-derived CD1 lb%D18+ vehicle cells to deliver anti-inflammatory cytokines to inflamed interstitium in an animal model of unilateral ureteral obstruction (UUO). Vehicle cells that express intracellular adhesion molecule (1CAM)1 ligands, CD11 b and CD18, were obtained from bone marrow cells of DBA/2 mice and adenovirally transduced with interleukin1 receptor antagonist (IL1RA) or glucocerebrosidase (GC) cDNA ex vivo. In renal cortex of UUO-treated kidneys, interstitial expression of ICAM1 and infiltra-
Previous studies have indicated that the enhanced expression of osteopontin (OPN) in proximal tub... more Previous studies have indicated that the enhanced expression of osteopontin (OPN) in proximal tubular cells plays an important role in the recruitment of macrophages into the interstitium.The present study was performed to elucidate the role of OPN expression in the proximal tubular cells in the long-term prognosis of IgA nephropathy (IgAN). We analyzed 36 patients with IgAN: 17 patients showed stable renal function for more than 15 years and 19 progressed to end-stage renal disease (ESRD) during the long-term follow up. The expression of OPN in the renal tissue was analyzed using immunohistochemistry. Based on the intensity and distribution of OPN expression in the proximal tubules, the patients were divided in two groups: 16 patients categorized into OPN group (OPN+ group) and 20 were given no OPN (OPNgroup). Compared with the OPNgroup, the OPN+ group had significantly compromised creatinine clearance (Cr clearance) (85 ± 23 vs 65 ± 17 mL/min, P < 0.05), large amounts of proteinuria (0.8 ± 0.5 vs 2.3 ± 1.9 g/day, P < 0.01) and severe tubulointerstitial injury when evaluated by the percentage of the affected area over whole cortical area (13 ± 9 vs 32 ± 23%, P < 0.01). It is of note that the incidence of ESRD was significantly higher in OPN+ group than OPNgroup (63.6 vs 10%, P < 0.05). To further explore whether OPN expression is an early indicator for the progression of IgAN, we analyzed the incidence of ESRD separately in a subgroup of patients either with normal (Cr clearance >0.80 mL/min) or deteriorated renal function (Cr clearance <0.80 mL/min) at biopsy. Interestingly, in a subgroup of patients with deteriorated renal function, the incidence of ESRD was again significantly higher in the patients with OPN expression than those without expression (62.5 vs 0%, P < 0.05), while it was not significantly different in a subgroup of patients with normal renal function. Likewise, when we analyzed 12 patients with advanced glomerular sclerosis (globally sclerosed glomeruli >0.30%), ESRD was more frequently observed in the patients with OPN expression than those without expression (100 vs 20%, P < 0.05). Among these 12 patients with advanced glomerular sclerosis, the OPN+ group had significantly large amounts of proteinuria (1.0 ± 0.5 vs 2.5 ± 2.0 g/day, P < 0.05), mesangial cell proliferation and intracapillary cell infiltration score (1.02 ± 0.73 vs 2.01 ± 1.01, P < 0.05), compared with the OPNgroup. However, there was no statistically significant difference in Cr clearance, globally sclerosed glomeruli % and tubulointerstitial injury %. These data suggest that OPN expression in proximal tubular cells is not an early predictor for the progression of IgAN, but is closely associated with the subsequent progression, if their nephron mass is reduced by the precedent glomerular injury. The role of osteopontin expression in proximal tubular cells in the long-term prognosis of IgA nephropathy
Since IgA nephropathy (IgAN) is one of the inflammatory diseases, local delivery of anti-inflamma... more Since IgA nephropathy (IgAN) is one of the inflammatory diseases, local delivery of anti-inflammatory cytokine into glomeruli has been suggested to prevent the development of IgAN. However, most attempts made to establish a safe and effective system of gene delivery into inflamed glomeruli still remain incomplete. It was previously reported that macrophages are recruited into glomeruli in association with upregulated expression of adhesion molecules and play a pivotal role in the development of IgAN. Therefore, we focused on this characteristic of macrophage and established a novel technique which may allow a site-specific gene delivery into inflamed glomeruli using a bone marrow-derived cell as a vehicle which possesses the ligand of adhesion molecules. Bone marrow cells from DBA/2 mice were incubated for 7 days in L-929 cell conditioned medium. Flow cytometric analysis revealed that 99.1 ± 0.9% of the subcloned cells were positive with CD11b and CD18, both of which are ligands of the intercellular adhesion molecule-1 (ICAM-1). These vehicle cells were labeled with a fluorescent lipophilic probe and intravenously returned to the DBA/2 mice. The mice then received 1 week of intraperitoneal injection of either lipopolysaccharide (LPS) to enhance ICAM-1 expression in the glomerulus, or saline as a control. In the LPS-treated mice, labeled vehicle cells were detected within the glomerulus and the kinetics of the vehicle cell recruitment into the glomerulus corresponded to the level of ICAM-1 expression. However, in the saline-treated control group, only a negligible number of vehicle cells could be detected in the glomerulus. A secondary administration of LPS 4 weeks after injection of the vehicle cells was also able to promote accumulation in the glomerulus. On the assumption that the LPS-induced ICAM-1 expression may regulate the site and period for the delivery of vehicle cells into the glomerulus, vehicle cells were transduced with human glucocerebrosidase (GC) gene using an adenovirus vector and reintroduced to the mice. The basal expression of the GC gene in the isolated glomeruli of vehicle cell-treated mice elevated 1.7-fold compared with endogenous activity, while the GC activity was enhanced 3.2-fold by LPS treatment. Polymerase chain reaction on human GC specific sequence revealed that isolated glomeruli of vehicle cell-treated mice exclusively contained the vehicle cell-oriented GC. These findings suggest that our novel method allows a sitespecific gene delivery into inflamed glomeruli through interaction of adhesion molecules. We then examined the feasibility of this system as a therapy for experimental glomerulonephritis. The antiinflammatory cytokine, interleukin-1 receptor antagonist (IL-1ra) gene were delivered into an animal model of inflamed glomeruli evoked by anti-glomerular basement membrane antibody. Vehicle cells were indeed accumulated in the glomeruli upon the induction of nephritis and confirmed to secrete recombinant IL-1ra. Renal functions as well as morphology were preserved by this intervention up to 2 weeks after IL-1ra introduction. These data demonstrate the possible application of gene therapy for some type of glomerulonephritis. At present there are several hurdles to address before making major investments for clinical use of this system for IgAN. Although the present system may achieve one of the longest control (up to 2 weeks) among glomerulus-targeted gene delivery systems, it is still not enough for clinical applications because IgAN usually progresses over a span of several years. We are currently investigating the novel system which establish ‘vehicle cell producing tissue’ by repetitive injection of retrovirally modified hematopoietic stem cells to non-ablated mice for long-lasting gene delivery. Another obstacle is the small amount of recombinant IL-1ra which is secreted from circulating vehicle cells may play a unpredictable role in non-targeted organs, especially if a multipotent cytokine like IL-1ra is used. In this regard, the on/off switching system by which vehicle cells are activated at the only recruited site will be necessary. We are currently trying to combine our gene delivery system with the Cre/loxP system which is activated by IL-1b promoter so that IL-1ra may be secreted in a situated environment of IL-1b production. Preliminary date using LacZ gene as a marker shows that certain genes can be switched on in the situation in which IL-1b is expressed in vitro. The feasibility of combining the Cre/loxP system and the vehicle cell are now under investigation. Finally, we believe that these trials precede a long avenue of research toward clinical use of gene therapy for IgAN. Treatment of IgA nephropathy using novel therapeutic strategy: Inflamed site specific gene delivery
Cross-communication between glomerular cells and infiltrating mononuclear cells plays an importan... more Cross-communication between glomerular cells and infiltrating mononuclear cells plays an important role in the generation of or recovery from glomerular diseases. We found that cultured mesangial cells secrete a factor that inhibits production of proinflammatory cytokines by activated macrophages. Treatment of J774.2 macrophages with conditioned media from rat mesangial cells blunted the transcriptional induction of IL-1 beta, IL-6, and TNF-alpha by LPS. None of the media conditioned by other fibroblastic, epithelial, or endothelial cell lines exhibited the inhibitory effect. Media conditioned by normal rat glomeruli contained a similar inhibitory activity, which was enhanced in an acute model of mesangial proliferative glomerulonephritis. To identify the active component involved, we examined the expression of known macrophage deactivators IL-10, IL-13, and TGF-beta 1 in mesangial cells. Under the basal culture conditions, strong expression of TGF-beta 1 mRNA was observed, whereas ...
Recent studies have demonstrated much larger variability in the total number of nephrons in norma... more Recent studies have demonstrated much larger variability in the total number of nephrons in normal populations than previously suspected. In addition, it has been suggested that individuals with a low nephron number may have an increased lifetime risk of hypertension or renal insufficiency, emphasizing the importance of evaluating the nephron number in each individual. In view of the fact that all previous reports of the nephron number were based on analyses of autopsy kidneys, the identification of surrogate markers detectable in living subjects is needed in order to enhance understanding of the clinical significance of this parameter. In this review, we summarize the clinicopathological factors and findings indicating a reduction in the nephron number, focusing particularly on those found at the time of a preserved renal function.
Mesenchymal stem cells (MSCs) have been applied to the treatment of various diseases, and MSC adm... more Mesenchymal stem cells (MSCs) have been applied to the treatment of various diseases, and MSC administration in marginal donor grafts may help avoid the ischemia-reperfusion injury associated with solid organ transplants. Given the reports of side effects after intravenous MSC administration, local MSC administration to the target organ might be a better approach. We administered adipose tissue-derived MSCs (AT-MSCs) ex vivo to donor rat kidneys obtained after cardiac death (CD). Using male Lewis rats (8-10 weeks), and a marginal transplant model of 1hr CD plus 1hr sub-normothermic ET-Kyoto solution preservation were conducted. AT-MSCs obtained from double-reporter (luciferase-LacZ) transgenic Lewis rats were injected either systemically (1.0 × 10(6) cells/0.5 mL) to bilaterally nephrectomized recipient rats that had received a marginal kidney graft (n = 6), or locally via the renal artery (500 μL ET-Kyoto solution containing the same number of AT-MSCs) to marginal kidney grafts, wh...
Recent findings have demonstrated that stem cells can differentiate into mature tissue when suppl... more Recent findings have demonstrated that stem cells can differentiate into mature tissue when supplied with a niche containing factors identical to those in the normal developmental program. A niche for the development of an organ can be provided by xenotransplantation of a similar developing organ. However, this process has many technical, safety, and ethical concerns. Here, we established xenotransplantation models that control endogenous mesenchymal stem cell (MSC) differentiation into mature erythropoietin (EPO)-producing tissue in a niche provided by a developing xenometanephros. Transplantation of rat metanephroi into mouse omentum, and similarly pig metanephroi into cat omentum, led to the recruitment of host cells and EPO production. EPO-expressing cells were not differentiated from integrating vessels because they did not coexpress endothelial markers (Tie-2 and VE-cadherin). Instead, EPO-expressing cells were shown to be derived from circulating host cells, as shown by enhan...
Proceedings of the National Academy of Sciences, 2005
The use of stem cells has enabled the successful generation of simple organs. However, anatomical... more The use of stem cells has enabled the successful generation of simple organs. However, anatomically complicated organs such as the kidney have proven more refractory to stem-cell-based regenerative techniques. Given the limits of allogenic organ transplantation, an ultimate therapeutic solution is to establish self-organs from autologous stem cells and transplant them as syngrafts back into donor patients. To this end, we have striven to establish an in vitro organ factory to build up complex organ structures from autologous adult stem cells by using the kidney as a target organ. Cultivation of human mesenchymal stem cells in growing rodent embryos enables their differentiation within a spatially and temporally appropriate developmental milieu, facilitating the first step of nephrogenesis. We show that a combination of whole-embryo culture, followed by organ culture, encourages exogenous human mesenchymal stem cells to differentiate and contribute to functional complex structures of...
In this study, we examined the effects of macrophage-colony stimulating factor (M-CSF) on glomeru... more In this study, we examined the effects of macrophage-colony stimulating factor (M-CSF) on glomerular macrophages in lipopolysaccharide (LPS)-induced murine nephritis. Mice injected intraperitoneally with either M-CSF plus LPS, LPS alone, M-CSF alone or saline every day for 8 days were examined for the degree of urine albumin excretion and lymphocyte-function associated antigen-1-positive (LFA-1+) cells in peripheral blood as well as renal pathology. From our results, LPS or M-CSF combined with LPS emphasized the degree of proteinuria, glomerular deposition of immunoglobulins and mesangial proliferation, associated with accumulation of macrophages in the glomeruli. However, in immunohistological examination of kidneys from these nephritic mice, neither intercellular adhesion molecule-1 (ICAM-1), which may play an important role in the recruitment of macrophages into glomeruli, M-CSF receptor nor the number of LFA-1+ cells in peripheral blood was enhanced by M-CSF. On the other hand, ...
In chronic inflammation, macrophages and neutrophils, which are derived from bone marrow, play a ... more In chronic inflammation, macrophages and neutrophils, which are derived from bone marrow, play a pivotal role. Therefore, reconstitution of bone marrow with anti-inflammatory stem cells may modify inflammation. In this study, transplantation-based gene therapy was applied to glomerular inflammation for a long-lasting suppression of the glomerular damage seen in chronic nephritis. Bone marrow cells were harvested from male donor mice, which had received 5-fluorouracil 3 days previously, and transduced with an interleukin 1 (IL-1) receptor antagonist (IL-1Ra) or a mock gene using a retrovirus vector. After confirmation that transduced cells possessed the transgene at approximately 0.7 copies per cell and secreted recombinant IL-1Ra, these cells were infused into sublethally irradiated (6 Gy) female recipients once daily for 4 consecutive days. These female recipient mice had the male Y antigen in bone marrow, liver, and spleen, and 10% to 20% of their spleen cells possessed the transg...
Previously, we reported that human mesenchymal stem cells (hMSCs) that were cultivated in growing... more Previously, we reported that human mesenchymal stem cells (hMSCs) that were cultivated in growing embryos differentiated in an appropriate developmental milieu, thereby facilitating the development of a functional renal unit. However, this approach required transfection with an adenovirus that expressed glial cell line‐derived neurotrophic factor (GDNF) to enhance the development of hMSC‐derived renal tissue, and safety issues restrict the clinical use of such viral vectors. To circumvent this problem, we tested an artificial polymer as a means to diffuse GDNF. This GDNF‐polymer, which exists in liquid form at 4°C but becomes a hydrogel upon heating to 37°C, was used as a thermoreversible switch, allowing the injection of hMSCs at low viscosity using a mouth pipette, with subsequent slow diffusion of GDNF as it solidified. The polymer, which was dissolved in a solution of GDNF at 4°C and then maintained at 37°C, acted as a diffuser of GDNF for more than 48 h. LacZ‐transfected hMSCs ...
Tubulointerstitial injury is a final common pathway for progressive renal injury in various renal... more Tubulointerstitial injury is a final common pathway for progressive renal injury in various renal diseases. Several cytokines, infiltrating cells and adhesion molecules are thought to be involved in the development of interstitial injury and fibrosis. However, there is currently no effective therapy for interstitial fibrosis. In this study, we examined the therapeutic efficacy of genetically modified bone marrow-derived CD1 lb%D18+ vehicle cells to deliver anti-inflammatory cytokines to inflamed interstitium in an animal model of unilateral ureteral obstruction (UUO). Vehicle cells that express intracellular adhesion molecule (1CAM)1 ligands, CD11 b and CD18, were obtained from bone marrow cells of DBA/2 mice and adenovirally transduced with interleukin1 receptor antagonist (IL1RA) or glucocerebrosidase (GC) cDNA ex vivo. In renal cortex of UUO-treated kidneys, interstitial expression of ICAM1 and infiltra-
Previous studies have indicated that the enhanced expression of osteopontin (OPN) in proximal tub... more Previous studies have indicated that the enhanced expression of osteopontin (OPN) in proximal tubular cells plays an important role in the recruitment of macrophages into the interstitium.The present study was performed to elucidate the role of OPN expression in the proximal tubular cells in the long-term prognosis of IgA nephropathy (IgAN). We analyzed 36 patients with IgAN: 17 patients showed stable renal function for more than 15 years and 19 progressed to end-stage renal disease (ESRD) during the long-term follow up. The expression of OPN in the renal tissue was analyzed using immunohistochemistry. Based on the intensity and distribution of OPN expression in the proximal tubules, the patients were divided in two groups: 16 patients categorized into OPN group (OPN+ group) and 20 were given no OPN (OPNgroup). Compared with the OPNgroup, the OPN+ group had significantly compromised creatinine clearance (Cr clearance) (85 ± 23 vs 65 ± 17 mL/min, P < 0.05), large amounts of proteinuria (0.8 ± 0.5 vs 2.3 ± 1.9 g/day, P < 0.01) and severe tubulointerstitial injury when evaluated by the percentage of the affected area over whole cortical area (13 ± 9 vs 32 ± 23%, P < 0.01). It is of note that the incidence of ESRD was significantly higher in OPN+ group than OPNgroup (63.6 vs 10%, P < 0.05). To further explore whether OPN expression is an early indicator for the progression of IgAN, we analyzed the incidence of ESRD separately in a subgroup of patients either with normal (Cr clearance >0.80 mL/min) or deteriorated renal function (Cr clearance <0.80 mL/min) at biopsy. Interestingly, in a subgroup of patients with deteriorated renal function, the incidence of ESRD was again significantly higher in the patients with OPN expression than those without expression (62.5 vs 0%, P < 0.05), while it was not significantly different in a subgroup of patients with normal renal function. Likewise, when we analyzed 12 patients with advanced glomerular sclerosis (globally sclerosed glomeruli >0.30%), ESRD was more frequently observed in the patients with OPN expression than those without expression (100 vs 20%, P < 0.05). Among these 12 patients with advanced glomerular sclerosis, the OPN+ group had significantly large amounts of proteinuria (1.0 ± 0.5 vs 2.5 ± 2.0 g/day, P < 0.05), mesangial cell proliferation and intracapillary cell infiltration score (1.02 ± 0.73 vs 2.01 ± 1.01, P < 0.05), compared with the OPNgroup. However, there was no statistically significant difference in Cr clearance, globally sclerosed glomeruli % and tubulointerstitial injury %. These data suggest that OPN expression in proximal tubular cells is not an early predictor for the progression of IgAN, but is closely associated with the subsequent progression, if their nephron mass is reduced by the precedent glomerular injury. The role of osteopontin expression in proximal tubular cells in the long-term prognosis of IgA nephropathy
Since IgA nephropathy (IgAN) is one of the inflammatory diseases, local delivery of anti-inflamma... more Since IgA nephropathy (IgAN) is one of the inflammatory diseases, local delivery of anti-inflammatory cytokine into glomeruli has been suggested to prevent the development of IgAN. However, most attempts made to establish a safe and effective system of gene delivery into inflamed glomeruli still remain incomplete. It was previously reported that macrophages are recruited into glomeruli in association with upregulated expression of adhesion molecules and play a pivotal role in the development of IgAN. Therefore, we focused on this characteristic of macrophage and established a novel technique which may allow a site-specific gene delivery into inflamed glomeruli using a bone marrow-derived cell as a vehicle which possesses the ligand of adhesion molecules. Bone marrow cells from DBA/2 mice were incubated for 7 days in L-929 cell conditioned medium. Flow cytometric analysis revealed that 99.1 ± 0.9% of the subcloned cells were positive with CD11b and CD18, both of which are ligands of the intercellular adhesion molecule-1 (ICAM-1). These vehicle cells were labeled with a fluorescent lipophilic probe and intravenously returned to the DBA/2 mice. The mice then received 1 week of intraperitoneal injection of either lipopolysaccharide (LPS) to enhance ICAM-1 expression in the glomerulus, or saline as a control. In the LPS-treated mice, labeled vehicle cells were detected within the glomerulus and the kinetics of the vehicle cell recruitment into the glomerulus corresponded to the level of ICAM-1 expression. However, in the saline-treated control group, only a negligible number of vehicle cells could be detected in the glomerulus. A secondary administration of LPS 4 weeks after injection of the vehicle cells was also able to promote accumulation in the glomerulus. On the assumption that the LPS-induced ICAM-1 expression may regulate the site and period for the delivery of vehicle cells into the glomerulus, vehicle cells were transduced with human glucocerebrosidase (GC) gene using an adenovirus vector and reintroduced to the mice. The basal expression of the GC gene in the isolated glomeruli of vehicle cell-treated mice elevated 1.7-fold compared with endogenous activity, while the GC activity was enhanced 3.2-fold by LPS treatment. Polymerase chain reaction on human GC specific sequence revealed that isolated glomeruli of vehicle cell-treated mice exclusively contained the vehicle cell-oriented GC. These findings suggest that our novel method allows a sitespecific gene delivery into inflamed glomeruli through interaction of adhesion molecules. We then examined the feasibility of this system as a therapy for experimental glomerulonephritis. The antiinflammatory cytokine, interleukin-1 receptor antagonist (IL-1ra) gene were delivered into an animal model of inflamed glomeruli evoked by anti-glomerular basement membrane antibody. Vehicle cells were indeed accumulated in the glomeruli upon the induction of nephritis and confirmed to secrete recombinant IL-1ra. Renal functions as well as morphology were preserved by this intervention up to 2 weeks after IL-1ra introduction. These data demonstrate the possible application of gene therapy for some type of glomerulonephritis. At present there are several hurdles to address before making major investments for clinical use of this system for IgAN. Although the present system may achieve one of the longest control (up to 2 weeks) among glomerulus-targeted gene delivery systems, it is still not enough for clinical applications because IgAN usually progresses over a span of several years. We are currently investigating the novel system which establish ‘vehicle cell producing tissue’ by repetitive injection of retrovirally modified hematopoietic stem cells to non-ablated mice for long-lasting gene delivery. Another obstacle is the small amount of recombinant IL-1ra which is secreted from circulating vehicle cells may play a unpredictable role in non-targeted organs, especially if a multipotent cytokine like IL-1ra is used. In this regard, the on/off switching system by which vehicle cells are activated at the only recruited site will be necessary. We are currently trying to combine our gene delivery system with the Cre/loxP system which is activated by IL-1b promoter so that IL-1ra may be secreted in a situated environment of IL-1b production. Preliminary date using LacZ gene as a marker shows that certain genes can be switched on in the situation in which IL-1b is expressed in vitro. The feasibility of combining the Cre/loxP system and the vehicle cell are now under investigation. Finally, we believe that these trials precede a long avenue of research toward clinical use of gene therapy for IgAN. Treatment of IgA nephropathy using novel therapeutic strategy: Inflamed site specific gene delivery
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