G.H. Luo

Inhibition of connective tissue growth factor by small interfering RNA prevents renal fibrosis in rats undergoing chronic allograft nephropathy.

AIM Connective tissue growth factor (CTGF) is a highly profibrogenic molecule implicated in renal fibrogenesis. Small interfering RNA (siRNA) is an effective tool to silence gene expression. This study determined whether caudal vein injection of siRNA targeting CTGF inhibited its expression in rat kidneys in vivo, and furthermore whether it protected the kidney from renal fibrosis in chronic allograft nephropathy (CAN). METHODS Male inbred Fischer (F344, RT1(lv1)) rat renal grafts were orthotopically transplanted into Lewis (LEW, RT1(1)) rats following the procedure of Kamada with our modification. At 6 weeks, recipients were divided into siRNA, normal saline (NS), and control siRNA groups, using daily siRNA-targeting CTGF (0.1 mg/kg), or NS, or a control siRNA via caudal vein injection for 14 days. At 4, 6, and 8 weeks, we observed the pathologic changes, expression of CTGF, E-cadherin, collagen I and IV, and anti-smooth muscle actin (alpha-SMA). RESULTS Serum creatinine level, Banff score, and the expression of CTGF were significantly lower among the siRNA than the NS or the control siRNA groups at 8 weeks (P < .05). The expressions of collagen I and IV, and alpha-SMA were also significantly downregulated and E-cadherin was lost in the siRNA versus the NS and control siRNA groups at 8 weeks. CONCLUSIONS This study showed that delivery of CTGF siRNA via the caudal vein significantly inhibited expression of CTGF in rat kidneys, effectively preventing fibrosis in CAN. The results suggest that siRNA-targeting of CTGF has the potential to be a novel strategy for amelioration of CAN.

Urinary connective tissue growth factor increases far earlier than histopathological damage and functional deterioration in early chronic renal allograft injury

Objective. To date, serum biochemistry examination and routine biopsy are the most commonly used methods to assess renal function after allogenic kidney transplantation. Connective tissue growth factor (CTGF) has been considered as a biomarker of chronic renal allograft injury characterized by tubular atrophy and interstitial fibrosis (TA/IF). This study explored the potential value of urinary CTGF as an early predictor of TA/IF using a rat model. Material and methods. A Fisher to Lewis allogenic rat kidney transplant model was established and the recipients were killed at weeks 4, 8 and 12 post-transplantation. TA/IF was graded based on Banff Schema 1997. The location and expression of CTGF mRNA were detected by oligonucleotide-primed in situ DNA synthesis and quantitative polymerase chain reaction. CTGF protein expression was examined with immunohistochemistry and immunoblotting. Urinary CTGF concentration was measured by enzyme-linked immunosorbent assay. The correlation between urinary CTGF concentration and serum creatinine (SCr) and Banff score was analysed statistically. Results. Typical morphological changes including TA/IF in allograft appeared at week 8 and became very severe at week 12 post-transplantation. CTGF expression in epithelium was up-regulated early and urinary CTGF was markedly elevated from week 4. SCr in recipients was stable before week 8 but increased tremendously at week 12. Urinary CTGF concentration was positively correlated with SCr and degree of interstitial fibrosis. Conclusion. Urinary CTGF increases earlier than the appearance of biochemical abnormalities and pathological changes. Measurement of urinary CTGF may offer a potential non-invasive strategy to predict the early onset of chronic renal allograft injury.

Effects of Mycophenolate Mofetil on Chronic Allograft Nephropathy by Affecting RHO/ROCK Signal Pathways

AIM We sought to investigate the effects of mycophenolate mofetil (MMF) on chronic allograft nephropathy (CAN) by affecting Rho and ROCK signal pathways. METHODS Male inbred F344 rat renal grafts orthotopically transplanted into Lewis rats were first treated with CsA (10 mg/kg(-1).d(-1) x 10 d) and then divided into 3 groups (each n = 9): (1) orally vehicle, (2) cyclosporine (CsA, 6 mg/kg(-1).d(-1)) and (3) MMF (20 mg/kg(-1).d(-1)). In addition we performed autografts of F344 (n = 10) at 4, 8, and 12 weeks, serum creatinine (SCr) was measured and pathologic changes assessed. Expression of RhoA and ROCK-1 was determined by real-time reverse transcriptase polymerase chain reaction. Expressions of alpha-smooth muscle actin (alpha-SMA) and connective tissue growth factor (CTGF) were observed by immunohistochemistry. RESULTS SCr and Banff score began to increase at 4 weeks in all 3 allografted groups with obvious deterioration in both the vehicle and CsA groups at 8 and 12 weeks. The differences between vehicle/CsA and autografts were significant (P = .000). SCr and Banff score among the MMF group increased mildly and moderately at 8 and 12 weeks, respectively, but were significantly lower than those in the vehicle/CsA cohort (P < .05). Expressions of RhoA and ROCK-1 mRNAs and proteins were observed in mesangial and tubular cells, increasing gradually along with the progression of chronic allograft nephropathy (CAN). There was a negative correlation between RhoA/ROCK-1 mRNA and Banff score (r = -.637, p = .000; r = -.676, P = .000) or SCr (r = -.705, P = .000; r = -.756, P = .000). MMF downregulated gene and protein expressions of RhoA and ROCK-1. CsA had little effect on these expressions. Expressions of alpha-SMA and CTGF were observed in renal epithelial and tubular cells. CONCLUSION Herein we have demonstrated abnormal expression of RhoA and ROCK-1 signal pathways, which may play roles in CAN. MMF may attenuate CAN by downregulating the expression of RhoA/ROCK-1, alpha-SMA, and CTGF.

C4d deposition is correlated with the level of antivimentin antibody in rat kidneys undergoing chronic allograft nephropathy.

AIMS Antivimentin antibody is often produced as an autoantibody after transplantation. C4d deposition, a marker of humoral immunity during transplantation, is believed to reflect alloantibodies. This study investigated the relationship between C4d deposition and humoral immunity to vimentin among rat kidneys undergoing chronic allograft nephropathy (CAN). METHODS Fisher 344 rat renal grafts were orthotopically transplanted into Lewis rats following the procedure of Kamada with our modification. All recipients were administered cyclosporine (CsA) (10 mg/kg(-1).d(-1) x 10 d) before being divided into 3 groups of oral treatments: (1) vehicle, (2) CsA (6 mg/kg(-1).d(-1)), and (3) mycophenolate mofetil (MMF; 20 mg/kg(-1).d(-1)). At 4, 8 and 12 weeks after transplantation, the rats were killed, the renal allografts harvested, and the sera collected. Serum creatinine (SCr) was measured and pathologic changes assessed according to the Banff 97 criteria. The antivimentin antibody was quantified by enzyme-linked immunosorbent assay. The deposition of C4d detected by immunofluorescence was analyzed by integrated optical density (IOD). RESULTS Antivimentin antibody was observed in sera of all transplanted rats. The level of antivimentin antibody (IgGDeltaOD) increased gradually during the development of CAN from 4 weeks. Simultaneously, C4d deposition in peritubular capillaries also progressively strengthened. There was a strong positive correlation between the content of antivimentin antibody and C4d deposition (r = 0.892; P = .000). MMF simultaneously decreased antivimentin antibody formation and C4d deposition. In contrast, CsA had no significant effect. CONCLUSIONS We demonstrated the production of antivimentin antibodies and the deposition of C4d during the development of CAN. There was a positive correlation between them. Whether humoral immunity to vimentin contributes to C4d deposition is not clear and further studies are needed to elucidate this issue.

R2: Identification of renal potential progenitor/stem cells that participate in the renal regeneration processes of kidney allograft fibrosis

Aim:  Many strategies are explored to ameliorate kidney allograft tubular atrophy and interstitial fibrosis (TA/IF), but little progress has been achieved. The latest evidence suggested that CD133+ cell in kidney represent a potential multipotent adult resident stem cell population that may contribute to the renal injury repair. Here we investigate whether the CD133+ cells exist in transplanted renal and exert a growth and self‐repair procedure in TA/IF.

Urinary connective tissue growth factor is a biomarker in a rat model of chronic nephropathy.

AIM This study sought to determine whether urinary connective tissue growth factor (CTGF) was a molecular marker for chronic allograft nephropathy (CAN). METHODS F344 rat renal grafts orthotopically transplanted into Lewis rats following the procedure of Kamada were harvested at 4,8,12, or 16 weeks. Morphological changes were studied using hematoxylin eosin (HE) and Masson trichrome stains. Serum creatinine (SCr) was measured. CAN grades were evaluated according to the Banff97 schema. Expressions of CTGF in the kidney and urine were determined using real-time polymerase chain reaction (PCR) Western blots, and competitive indirect enzyme-linked immunosorbent assay (ELISA). Spearman correlation analysis was used to compare urinary CTGF expression and CAN development. RESULTS SCr levels and Banff scores increased in a time-dependent manner. The expression of CTGF in the graft was markedly elevated compared with the control group. Urine CTGF increased by week 4, and maintained high levels up to week 16. The urinary levels correlated positively with the histological presence of CAN. Thus, urine CTGF concentrations reflected the course of CAN, especially at an early stage. CONCLUSION CTGF plays a significant role in the pathological changes of CAN after kidney transplantation. Urinary CTGF has the potential to be a biomarker for CAN.

Epithelial to Mesenchymal Transformation in Tubular Epithelial Cells Undergoing Anoxia

AIM Epithelial-mesenchymal transformation (EMT) has been proved to be a critical event in fibrogenesis of renal allografts. This study sought to determine whether anoxia could induce EMT from tubular epithelial cells (TEC). METHODS Rat TEC-line (NRK-52E) was cultured in Dulbelcos modified Eagles medium (DMEM) without glucose under 100% N2 for 4 hours. After 6, 12, 24, 48, and 72 hours, the expressions of connective tissue growth factor (CTGF) mRNA and protein were measured by real-time RT-PCR and Western blot, respectively. Morphologic changes and cytoskeleton remodeling were observed in NRK-52E cells under laser confocal microscopy. Immunohistochemistry and flow cytometry were used to detect expression changes of E-cadherin, alpha-smooth muscle actin (SMA), types I and IV collagen, all of which are involved in TEC, EMT. RESULTS After stimulation by anoxia, NRK-52E cells became round and enlarged with a remodeled cytoskeleton. The expressions of CTGF mRNA and protein were upregulated after 6 hours, reaching their peak at 48 hours. The expressions of types I and IV collagen, and alpha-SMA were all upregulated except for E-cadherin. CONCLUSIONS Anoxia upregulated the expression of CTGF and other EMT-associated genes in NRK-52E cells.

Rapamycin and cyclosporine have different effects on expression of Ang-1 and Ang-2 and Tie2 in rat renal allograft with chronic allograft nephropathy.

OBJECTIVE Previous studies have indicated that angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) and tyrosine kinase receptor Tie2 regulate the maintenance and integrity of blood vessels and have potential anti-inflammatory properties. This study of cardiac allografts investigated whether there is a difference between rapamycin and cyclosporine A (CsA) in the ability to affect expression of Ang1, Ang2, and Tie2 in rat renal allografts with chronic allograft nephropathy (CAN). METHODS A male inbred F344 to Lewis rat renal CAN model was established via a modified Kamada procedure. The recipients were first treated with CsA, 10 mg/kg/d, for 10 days and then allocated randomly to three oral treatment groups: control; CsA, 6 mg/kg/d; and rapamycin, 0.8 mg/kg/d. At 4, 8, and 12 weeks posttransplantation, the rats were killed to harvest the renal allografts. The serum creatinine concentration (SCr) was measured, and the pathologic changes were assessed according to Banff 97 criteria. The expression of messengerRNA and proteins of Ang1, Ang2, and Tie2 was determined using real-time fluorescence quantitative polymerase chain reaction and immunohistochemistry. RESULTS The elevation of SCr and the pathologic changes of CAN were observed in the control and CsA groups at 8 and 12 weeks; the differences between the 2 groups were not significant (P > .05). The levels of SCr and Banff score in the rampamycin group were lower than those in other 2 groups (P < .01). The expression of Ang1 and Ang2 was localized to epithelial cells and endothelium of vascular bundles of glomeruli, and Tie2 was specifically expressed in the endothelium of vessels in all 3 groups. At 4 weeks, the differences in mRNA expression of Ang1, Ang2, and Tie2 between 3 groups were not significant (P > .05). In a comparison of the control and CsA groups, mRNA expression of Ang1 was increased (P < .05), and mRNA expression of Ang2 and Tie2 was decreased (P < .05) in the rapamycin group at 8 and 12 weeks. The differences between the control and CsA groups were not significant at 8 or 12 weeks (P > .05). CONCLUSIONS Our results show that compared with CsA, rapamycin modulates the expression of Ang1, Ang2, and Tie2 in rat renal allografts with CAN, which suggests that rapamycin may improve the long-term survival of renal allografts through its vasculoprotective properties.

Expressions of Angiopoietin-1, Angiopoietin-2, and Tie2 and their roles in rat renal allografts with chronic allograft nephropathy.

OBJECTIVE Angiopoietin-1 (Ang1) and -2(Ang2) are 2 ligands for the endothelium-specific tyrosine kinase Tie2. Previous studies have shown that reciprocal regulation of Ang1, Ang2, and Tie2 plays an important role in chronic cardiac allograft vasculopathy. This study investigated the expressions of Ang1, Ang2, and Tie2 in rat renal allografts undergoing chronic allograft nephropathy (CAN). MATERIALS AND METHODS Renal transplantations following the procedure of Kamada with our modification were orthotopically performed using Fisher (F344, RT1(1v1)) rats as both donors and recipients in the autograft group. Fisher and Lewis (LEW, RT1(1)) rats were used as donors and recipients in the allograft group, respectively, which was treated with cyclosporine (CsA; 10 mg/kg/d x 10 d). At 4w, 8w, and 12 weeks posttransplantation, serum creatinine (SCr) was measured and pathologic changes assessed according to the Banff 97 criteria. The mRNA (Deltact) and protein expressions of Ang1, Ang2, and Tie2 were localized by real-time fluorescence quantitative polymerase chain reaction (PCR) and by immunohistochemistry. RESULTS The elevation in SCr and the pathologic changes in CAN were observed in all allografts at 8 and 12 weeks. The expressions of Ang1 and Ang2 were localized to epithelial cells and endothelium of the vascular bundles of the glomeruli; Tie2 was specifically expressed in endothelium of vessels both in auto- and allografts at all time points posttransplantation. At 4 weeks, the differences in mRNA expression of Ang1, Ang2, and Tie2 between the 2 groups were not significant (P > .05). Compared with autografts, the mRNA expression of Ang1 decreased significantly (P = .008 and .003 for 8 and 12 weeks, respectively), and the mRNA expressions of Ang2 and Tie2 significantly increased (P = .001/.006 and .005/.001 for 8 and 12 weeks, respectively). The changes in expression of all 3 genes showed significant correlation with the Banff score in the allografts. CONCLUSION This study suggested that the abnormal expression and reciprocal regulation of Ang1, Ang2, and Tie2 may play important roles in the development of CAN in rat renal allografts.