Hongli Lin

Mesenchymal Stem Cells Attenuate Peritoneal Injury through Secretion of TSG-6

Background Mesothelial cell injury plays an important role in peritoneal fibrosis. Present clinical therapies aimed at alleviating peritoneal fibrosis have been largely inadequate. Mesenchymal stem cells (MSCs) are efficient for repairing injuries and reducing fibrosis. This study was designed to investigate the effects of MSCs on injured mesothelial cells and peritoneal fibrosis. Methodology/Principal Findings Rat bone marrow-derived MSCs (5 ×106) were injected into Sprague-Dawley (SD) rats via tail vein 24 h after peritoneal scraping. Distinct reductions in adhesion formation; infiltration of neutrophils, macrophage cells; number of fibroblasts; and level of transforming growth factor (TGF)-β1 were found in MSCs-treated rats. The proliferation and repair of peritoneal mesothelial cells in MSCs-treated rats were stimulated. Mechanically injured mesothelial cells co-cultured with MSCs in transwells showed distinct increases in migration and proliferation. In vivo imaging showed that MSCs injected intravenously mainly accumulated in the lungs which persisted for at least seven days. No apparent MSCs were observed in the injured peritoneum even when MSCs were injected intraperitoneally. The injection of serum-starved MSCs-conditioned medium (CM) intravenously reduced adhesions similar to MSCs. Antibody based protein array of MSCs-CM showed that the releasing of TNFα-stimulating gene (TSG)-6 increased most dramatically. Promotion of mesothelial cell repair and reduction of peritoneal adhesion were produced by the administration of recombinant mouse (rm) TSG-6, and were weakened by TSG-6-RNA interfering. Conclusions/Significance Collectively, these results indicate that MSCs may attenuate peritoneal injury by repairing mesothelial cells, reducing inflammation and fibrosis. Rather than the engraftment, the secretion of TSG-6 by MSCs makes a major contribution to the therapeutic benefits of MSCs.

Blocking core fucosylation of TGF-β1 receptors downregulates their functions and attenuates the epithelial-mesenchymal transition of renal tubular cells.

Posttranslational modification of proteins could regulate their multiple biological functions. Transforming growth factor-β receptor I and II (ALK5 and TGF-βRII), which are glycoproteins, play important roles in the renal tubular epithelial-mesenchymal transition (EMT). In the present study, we examined the role of core fucosylation of TGF-βRII and ALK5, which is regulated by α-1,6 fucosyltransferase (Fut8), in the process of EMT of cultured human renal proximal tubular epithelial (HK-2) cells. The typical cell model of EMT induced by TGF-β1 was constructed to address the role of core fucosylation in EMT. Core fucosylation was found to be essential for both TGF-βRII and ALK5 to fulfill their functions, and blocking it with Fut8 small interfering RNA greatly reduced the phosphorylation of Smad2/3 protein, caused the inactivation of TGF-β/Smad2/3 signaling, and resulted in remission of EMT. More importantly, even with high levels of expressions of TGF-β1, TGF-βRII, and ALK5, blocking core fucosylation also could attenuate the EMT of HK-2 cells. Thus blocking core fucosylation of TGF-βRII and ALK5 may attenuate EMT independently of the expression of these proteins. This study may provide new insight into the role of glycosylation in renal interstitial fibrosis. Furthermore, core fucosylation may be a novel potential therapeutic target for treatment of renal tubular EMT.

Tissue inhibitor of metalloproteinase-1 exacerbated renal interstitial fibrosis through enhancing inflammation

BACKGROUND Tissue inhibitor of metalloproteinase-1 (TIMP-1) is associated with renal fibrosis. Furthermore, it is a multi-functional protein, and whether it has other roles in renal fibrosis is unknown. As several inflammatory mediators are substrates of matrix metalloproteinases (MMPs), TIMP-1 might affect renal fibrosis via inflamatory pathways. METHODS Plasmids containing the sense and antisense human TIMP-1 sequence were stably transfected into the human kidney proximal tubular epithelial cell line (HKC), MMP-2 and MMP-9 siRNA were transiently transfected into HKC and the transfected cells were stimulated with phorbol 12-myristate 13-acetate (PMA). In vivo, we established unilateral ureteral obstruction (UUO) models by using homozygote human TIMP-1 transgenic mice. The expression of intercellular adhesion molecule-1 (ICAM-1) in transfected cells and F4/80-positive cells in the renal interstitium were examined by indirect immunofluorescence. Protein levels in the cells and UUO models were examined by western blot, and the activities of the gelatinases and TIMP-1 were examined by gelatin zymography and reverse zymography, respectively. RESULTS After stimulation with PMA, the activities of the gelatinases were decreased, ICAM-1 was upregulated, and soluble ICAM-1 in the supernatant was decreased, in HKC transfected with sense TIMP-1, and ICAM-1 was increased in HKC transfected with MMP-9 siRNA. At 14 days after UUO, it was found that compared with wild-type mice, in transgenic mice, with upregulation of TIMP-1, activities of gelatinases were downregulated, ICAM-1, transforming growth factor-beta1 (TGF-beta1), collagens I and III were upregulated, and the extent of renal fibrosis and infiltration of macrophages was more severe. CONCLUSION Overexpression of TIMP-1 could promote renal interstitial fibrosis through the inflammatory pathway, which might be partly induced by upregulating ICAM-1.

Inhibition of TGF-β1-receptor posttranslational core fucosylation attenuates rat renal interstitial fibrosis

The profibrotic cytokine transforming growth factor-β1 (TGF-β1) causes renal fibrosis by binding to receptors at the cell surface; however, it is not clear which of the TGF-β superfamily receptors correlates with renal fibrosis. To resolve this, we quantified TGF-β superfamily receptor expression in the kidneys of rats with unilateral ureteral obstruction using a real-time PCR gene array. Expression of activin receptor-like kinase (ALK)-5, ALK7, and TGF-β receptor II (TGF-βRII) mRNA increased significantly, while ALK6 mRNA expression was significantly decreased in the obstructed rat kidney. Core fucosylation is essential for the proper function of both TGF-βRII and ALK5 in cultured human renal proximal tubular epithelial cells in vitro. Therefore, we targeted posttranslational core fucosylation, regulated by α-1,6 fucosyltransferase (FUT8), by adenoviral-mediated knockdown of FUT8 mRNA in vivo and measured TGF-βRII and ALK5 expression and the progression of renal fibrosis. Despite long-term obstruction injury, inhibition of TGF-βRII and ALK5 of core fucosylation ameliorated the progression of renal fibrosis, an effect independent of TGF-βRII and ALK5 expression. Thus, the regulation of TGF-β1-receptor core fucosylation may provide a novel potential therapeutic strategy for treating renal fibrosis.

Efficacy and Safety of Abelmoschus manihot for Primary Glomerular Disease: A Prospective, Multicenter Randomized Controlled Clinical Trial

BACKGROUND Abelmoschus manihot, a single medicament of traditional Chinese medicine, has been widely used to treat kidney disease. This is the first randomized controlled clinical trial to assess its efficacy and safety in patients with primary glomerular disease. STUDY DESIGN Prospective, open-label, multicenter, randomized, controlled, clinical trial. SETTING & PARTICIPANTS From May 2010 to October 2011, a total of 417 patients with biopsy-proven primary glomerular disease from 26 hospitals participated in the study. INTERVENTIONS A manihot in the form of a huangkui capsule, 2.5 g, 3 times per day; losartan potassium, 50mg/d; or combined treatment, a huangkui capsule at 2.5 g 3 times per day, was combined with losartan potassium, 50mg/d. The duration of intervention was 24 weeks. OUTCOMES & MEASUREMENTS The primary outcome was change in 24-hour proteinuria from baseline after treatment. Change in estimated glomerular filtration rate (eGFR) from baseline after treatment was a secondary outcome. The 24-hour proteinuria was measured every 4 weeks and eGFR was measured at 0, 4, 12, and 24 weeks. RESULTS Mean baseline urine protein excretion was 1,045, 1,084, and 1,073 mg/d in the A manihot, losartan, and combined groups, respectively, and mean eGFR was 108, 106, and 106 mL/min/1.73 m2, respectively. After 24 weeks of treatment, mean changes in proteinuria were protein excretion of -508, -376, and -545 mg/d, respectively (P=0.003 for A manihot vs losartan and P<0.001 for the combined treatment vs losartan). Mean eGFR did not change significantly. The incidence of adverse reactions was not different among the 3 groups (P>0.05), and there were no severe adverse events in any group. LIMITATIONS Results cannot be generalized to those with nephrotic syndrome or reduced eGFR. CONCLUSIONS A manihot is a promising therapy for patients with primary kidney disease (chronic kidney disease stages 1-2) with moderate proteinuria.

Induction of epithelial-to-mesenchymal transition in proximal tubular epithelial cells on microfluidic devices.

In proteinuric nephropathy, epithelial-to-mesenchymal transition (EMT) is an important mechanism that causes renal interstitial fibrosis. The precise role of EMT in the pathogenesis of fibrosis remains controversial, partly due to the absence of suitable in vitro or in vivo models. We developed two microfluidic and compartmental chips that reproduced the fluidic and three-dimensional microenvironment of proximal tubular epithelial cells in vivo. Using one microfluidic device, we stimulated epithelial cells with a flow of healthy human serum, heat-inactivated serum and complement C3a, which mimicked the flow of urine within the proximal tubule. We observed that epithelial cells exposed to serum proteins became apoptotic or developed a mesenchymal phenotype. Incubating cells with C3a induced similar features. However, cells exposed to heat-inactivated serum did not adopt the mesenchymal phenotype. Furthermore, we successfully recorded the cellular morphological changes and the process of transmigration into basement membrane extract during EMT in real-time using another three-dimensional microdevice. In conclusion, we have established a cell-culture system that mimics the native microenvironment of the proximal tubule to a certain extent. Our data indicates that EMT did occur in epithelial cells that were exposed to serum proteins, and C3a plays an essential role in this pathological process.

Novel mechanism for mesenchymal stem cells in attenuating peritoneal adhesion: accumulating in the lung and secreting tumor necrosis factor α-stimulating gene-6.

IntroductionWe previously found that mesenchymal stem cells (MSCs) injected intravenously could attenuate peritoneal adhesion by secreting tumor necrosis alpha-stimulating gene (TSG)-6, while MSCs injected intraperitoneally could not. However, the underlying mechanism remains unclear. This study was designed to investigate the means by which MSCs exert their effects.MethodsRat bone marrow-derived MSCs/red fluorescent protein (RFP) were injected either intraperitoneally or intravenously into Sprague-Dawley (SD) rats at different time points after peritoneal scraping. Peritoneal adhesions were evaluated macroscopically at day 14 after scraping. The distribution of MSCs injected intraperitoneally or intravenously was traced by two-photon fluorescence confocal imaging and immunofluorescence microscopy. The co-localization of MSCs and macrophages in the lung and the spleen, and the expression of TSG-6 in MSCs trapped in the lung or the spleen were evaluated by immunofluorescence microscopy. The concentration of TSG-6 in serum was evaluated by ELISA. After intravenous injection of TSG-6- small interfering (si) RNA-MSCs, the expression of TSG-6 in MSCs and the concentration of TSG-6 in serum were reevaluated, and peritoneal adhesions were evaluated macroscopically and histologically.ResultsMSCs injected intraperitoneally failed to reduce peritoneal adhesion, and MSCs injected intravenously markedly improved peritoneal adhesion. Two-photon fluorescence confocal imaging showed that MSCs injected intravenously accumulated mainly in the lung, where they remained for seven days, and immunofluorescence microscopy showed few MSCs phagocytosed by macrophages. In contrast, large numbers of MSCs accumulated in the spleen with obvious phagocytosis by macrophages even at 4 hours after intraperitoneal injection. Immunofluorescence microscopy showed that MSCs that accumulated in the lung after intravenous injection could express TSG-6 within 12 hours, but TSG-6-siRNA-MSCs or MSCs accumulated in the spleen after intraperitoneal injection did not. ELISA showed that the concentration of TSG-6 in serum was increased at 4 hours after intravenous injection of MSCs, while there was no increase after injection of TSG-6-siRNA-MSCs or after intraperitoneal injection of MSCs. Moreover, intravenous injection of TSG-6-siRNA-MSCs failed to attenuate peritoneal adhesion.ConclusionsOur findings suggest that intravenously injected MSCs accumulated in the lung and attenuated peritoneal adhesion by secreting TSG-6, but intraperitoneally injected MSCs were phagocytosed by macrophages in the spleen and failed to attenuate peritoneal adhesion.

Phase 2 studies of oral hypoxia-inducible factor prolyl hydroxylase inhibitor FG-4592 for treatment of anemia in China.

Abstract Background FG-4592 (roxadustat) is an oral hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor (HIF-PHI) promoting coordinated erythropoiesis through the transcription factor HIF. Two Phase 2 studies were conducted in China to explore the safety and efficacy of FG-4592 (USAN name: roxadustat, CDAN name: ), a HIF-PHI, in patients with anemia of chronic kidney disease (CKD), both patients who were dialysis-dependent (DD) and patients who were not dialysis-dependent (NDD). Methods In the NDD study, 91 participants were randomized to low (1.1–1.75 mg/kg) or high (1.50–2.25 mg/kg) FG-4592 starting doses or to placebo. In the DD study, 87 were enrolled to low (1.1–1.8 mg/kg), medium (1.5–2.3 mg/kg) and high (1.7–2.3 mg/kg) starting FG-4592 doses or to continuation of epoetin alfa. In both studies, only oral iron supplementation was allowed. Results In the NDD study, hemoglobin (Hb) increase ≥1 g/dL from baseline was achieved in 80.0% of subjects in the low-dose cohort and 87.1% in the high-dose cohort, versus 23.3% in the placebo arm (P < 0.0001, both). In the DD study, 59.1%, 88.9% (P = 0.008) and 100% (P = 0.0003) of the low-, medium- and high-dose subjects maintained their Hb levels after 5- and 6-weeks versus 50% of the epoetin alfa-treated subjects. In both studies, significant reductions in cholesterol were noted in FG-4592-treated subjects, with stability or increases in serum iron, total iron-binding capacity (TIBC) and transferrin (without intravenous iron administration). In the NDD study, hepcidin levels were significantly reduced across all FG-4592-treated arms as compared with no change in the placebo arm. In the DD study, hepcidin levels were also reduced in a statistically significant dose-dependent manner in the highest dose group as compared with the epoetin alfa-treated group. Adverse events were similar for FG-4592-treated and control subjects. Conclusions FG-4592 may prove an effective alternative for managing anemia of CKD. It is currently being investigated in a pivotal global Phase 3 program.

Tanshinone IIA ameliorates bleomycin-induced pulmonary fibrosis and inhibits transforming growth factor-beta-β–dependent epithelial to mesenchymal transition

BACKGROUND Epithelial to mesenchymal transition (EMT) of alveolar epithelial cells occurs in lung fibrotic diseases. Tanshinone IIA (Tan IIA) has been reported to exert anti-inflammatory effects in pulmonary fibrosis. Nonetheless, whether Tan IIA affects lung fibrosis-related EMT remains unknown and requires for further investigations. MATERIALS AND METHODS A single intratracheal instillation of saline containing bleomycin (BLM; 5 mg/kg body weight) was performed to induce pulmonary fibrosis in Sprague-Dawley rats. Rats receiving an instillation of equivoluminal normal saline served as controls. Then, these rats were given a daily intraperitoneal administration of Tan IIA (15 mg/kg body weight) for 28 d before sacrifice. In vitro, recombinant transforming growth factor-beta 1 (TGF-β1; 10 ng/mL) was used to treat human alveolar epithelial A549 cells for 48 h. Tan IIA (10 μM) or control DMSO was used to pretreat cells for 2 h before TGF-β1 stimulation. Rat lung tissue samples and A549 cells were then subjected to further assessments. RESULTS Tan IIA was noted to alleviate BLM-induced pulmonary collagen deposition and macrophage infiltration in rats. Epithelial-cadherin expression was decreased after BLM stimulation, whereas α-smooth muscle actin, fibronectin, and vimentin were increased. These expression alterations were partially reversed by Tan IIA. Moreover, Tan IIA suppressed BLM-induced increases in TGF-β1, phosphorylated Smad-2, and -3 in rats. Additionally, pretreatment of Tan IIA inhibited TGF-β1-triggered EMT, reduced collagen Ⅰ production, and blocked TGF-β signal transduction in A549 cells. CONCLUSIONS Our research suggests that Tan IIA mitigates BLM-induced pulmonary fibrosis and suppresses TGF-β-dependent EMT of lung alveolar epithelial cells.

Development of a Functional Glomerulus at the Organ Level on a Chip to Mimic Hypertensive Nephropathy

Glomerular hypertension is an important factor exacerbating glomerular diseases to end-stage renal diseases because, ultimately, it results in glomerular sclerosis (especially in hypertensive and diabetic nephropathy). The precise mechanism of glomerular sclerosis caused by glomerular hypertension is unclear, due partly to the absence of suitable in vitro or in vivo models capable of mimicking and regulating the complex mechanical forces and/or organ-level disease processes. We developed a “glomerulus-on-a-chip” (GC) microfluidic device. This device reconstitutes the glomerulus with organ-level glomerular functions to create a disease model-on-a chip that mimics hypertensive nephropathy in humans. It comprises two channels lined by closely opposed layers of glomerular endothelial cells and podocytes that experience fluid flow of physiological conditions to mimic the glomerular microenvironment in vivo. Our results revealed that glomerular mechanical forces have a crucial role in cellular cytoskeletal rearrangement as well as the damage to cells and their junctions that leads to increased glomerular leakage observed in hypertensive nephropathy. Results also showed that the GC could readily and flexibly meet the demands of a renal-disease model. The GC could provide drug screening and toxicology testing, and create potential new personalized and accurate therapeutic platforms for glomerular disease.