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Mesenchymal stromal cells-based therapy, antibody therapy, cold organ preservation in transplantation, Roles of clusterin in kidney disease, Novel methods for monitoring kidney damage and function, anti-inflammatory natural products.
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Graduate Student Supervision
Master's Student Supervision (2010 - 2021)
Peritoneal Dialysis (PD), a treatment modality for end-stage renal disease (ESRD) uses high concentrations of glucose in its dialysis solutions. Since, 40% ESRD patients are diabetic, use of glucose-based PD solutions is non-rational because of the local and systemic adverse effects of glucose. There is a need for a biocompatible, non-glucose based osmotic agent that can replace glucose in peritoneal dialysis solutions. Recent studies on low molecular weight hyperbranched polyglycerol (HPG) as an osmotic agent show good ultrafiltration and reduction of peritoneal membrane injury but elimination through the kidneys. Our studies show that HPG is excreted via kidneys, and in ESRD patients, kidney function is impaired which could potentially result in poor excretion of this osmotic agent. N-acetylgalactosamine (GalNAc) carrying proteins and macromolecules has shown to be effectively up taken by the liver through asialoglycoprotein receptor (ASGPR). Thus, we hypothesize that HPG modified by GalNAc conjugation can be recognized by ASGPR in the liver, taken up and excreted through feces. GalNAc epoxide was synthesized by standard organic modifications and conjugated with HPG (3K) at two different densities (one or three GalNAc molecules per HPG) (denoted as HPG+1S or HPG+3S). The polymers were labeled with alexa647 and screened for effective internalization in a panel of hepatocyte cell lines with different levels of ASGPR expression using flow cytometry. Results of dose-dependent uptake of polymers and ASGPR staining of hepatocytes suggested receptor mediated internalization of HPG+3S in HepG2 and HuH7.5.1 cells. Kₘ values were assessed to determine the efficacious HPG conjugate followed by competitive inhibition with natural ligands of ASGPR in HepG2 cells. HEK293 cells ectopically expressing ASGPR1 and ASGPR1&2 genes were used to confirm HPG+3S uptake through ASGPR specifically. Mice tissue distribution studies of radiolabeled HPG and HPG+3S showed better uptake of HPG+3S by the liver than HPG. Percentage of HPG+3S excreted via feces was significantly more than HPG in mice with normal kidney function, which support the enhanced binding of HPG+3S to ASGPR in hepatocytes. In conclusion, both our in vitro and in vivo results substantiate our claim that GalNAc conjugated HPG can be rerouted to be excreted via feces.
Cellular autophagy is a prosurvival mechanism in the kidney against ischemia reperfusion injury (IRI), but the molecular pathways to activating autophagy in ischemic kidneys are not fully understood. Clusterin (CLU) is a chaperone-like protein and its expression is associated with kidney resistance to IRI. This study investigated the role of CLU in the prosurvival autophagy in the kidney. Renal IRI was induced in mice by clamping renal pedicles at 32°C for 45 min. Hypoxia in renal tubular epithelial cell (TEC) cultures was induced by exposure to 1% O₂ atmosphere. Autophagy was determined by either LC3-BII expression in Western blot or LC3-GFP aggregation in confocal microscopy. Cell apoptosis was determined by flow cytometric analysis. Unfolded protein response (UPR) was determined by PCR array. Here, we showed that autophagy was significantly activated by IRI in wild type (WT) but not CLU deficient kidneys. Similarly, the autophagy was activated by hypoxia in human proximal TECs (HKC-8) and WT mouse primary TECs but was impaired in CLU null TECs. Hypoxia activated autophagy was CLU dependent and was positively correlated with cell survival, and inhibition of autophagy significantly promoted cell death both in HKC-8 and mouse WT/CLU expressing TECs, but not in CLU null TECs. Further studies showed that CLU-dependent prosurvival autophagy was associated with UPR activation in hypoxic kidney cells. In conclusion, these data suggest that activation of prosurvival autophagy by hypoxia in kidney cells is required CLU expression, and may be a cytoprotective mechanism of CLU in the protection of the kidney from hypoxia/ischemia-mediated injury.
Metabolic syndrome (diabetes, hypertension, obesity and hypercholesteremia) increases the risk of high-mortality chronic diseases including chronic kidney disease, which accounts for 50% of end-stage renal disease (ESRD) in the developed world. Over 1/3 of the world’s adult population have metabolic syndrome. Oxidative stress plays a central role in metabolic syndrome pathophysiology. Grape is one of the broadly studied natural anti-oxidants. Literature demonstrates grape antioxidant’s significant protective effects on metabolic syndrome, however, not yet on metabolic syndrome-related kidney disease. This study evaluates the effect of whole grape on kidney disease associated with metabolic syndrome. Material and methods: Preclinical model of metabolic syndrome-related kidney disease, Obese ZSF-1 rats, ingested whole grape powder (5% of daily diet) for 6 months. Blood and urine samples were analyzed monthly to assess renal function parameters including 24-hour urine volumes, proteinuria, and urine protein to creatinine ratio (PCR). Rats’ kidney tissue histopathology and PCR array studies were conducted. In vitro kidney cell death was examined in cultured podocytes using flow cytometry. Results: Here, collective data from 6-month preclinical study showed chronic kidney disease consistent with an early stage diabetic nephropathy picture in both experimental and control groups. Renal function in rats of the experimental group was significantly enhanced compared with those of the control group, indicated by less 24-hour urine volumes (34.79 ± 15.77 mL vs. 55.8 ± 20.27 mL, p = 0.0147) and less proteinuria (8.56 ± 5.71 g vs. 24.01 ± 37.51 g, p = 0.0412) in the experimental group. Urine PCR was significantly lower in the experimental group versus control (3.42 ± 1.289 vs. 9.722 ± 9.156, p = 0.0084). Histopathology and PCR array analysis showed less oxidative stress picture in experimental group versus control. In vitro antioxidant assays showed significantly reduced H2O2-induced cell death in podocytes treated with grape extract versus control. Conclusion: This pilot study indicates that daily intake of whole grape powder has a protective effect on the kidney in obese ZSF-1 rats, suggesting the potential of grape antioxidants as a prevention strategy for reducing kidney disease progression in metabolic syndrome patients. Further investigations are required to support this preliminary study.
Antifreeze proteins from natural sources have been discovered to have cryoprotective function against freezing temperature, and have been tested for the application for cryopreservation of biological materials. However, none has been shown to match the effectiveness of current chemical cryoprotectants, such as dimethyl sulfoxide. One potential limitation with the application of antifreeze proteins is that they may only stay in the extracellular space around cells whereas chemical cryoprotectants can be penetrative. In this thesis project, we have designed, purified and explored the function of antifreeze proteins that were engineered with an intracellular delivery signal peptide, known as iPTD. We showed that iPTD-engineered antifreeze proteins had effective cell surface coverage within 30 minutes of incubation as shown by flow cytometry; however no intracellular protein delivery was observed under multiphoton microscopy. The plasma membrane was protected by iPTD-engineered antifreeze proteins during cryopreservation as seen in Calcein dye release assay, but cell recovery or proliferation was not observed after thawing. Given these properties of iPTD-engineered antifreeze proteins, we used them as red blood cell cryopreservation additives. By adding these modified antifreeze proteins, we were able to reduce the amount of glycerol (used for RBC cryopreservation) necessary to control freeze-induced hemolysis. Furthermore, the quality of thawed red blood cells is higher as protein addition resulted in high retention of intracellular ATP.
Background: Clusterin (CLU) is a chaperone-like protein. Our previous studies have demonstrated that CLU protects kidney from ischemia-reperfusion injury (IRI) and enhances renal repair after IRI; however, the molecular pathways for its functions in the kidney are not fully understood. This study was designed to investigate CLU-mediating pathways in kidney cells by using bioinformatics analysis.Materials and Methods: An in vitro model of kidney tissue using CLU null renal tubular epithelial cells (TECs) was established for this research project. An immortalized CLU null TEC cell line was generated from a CLU knockout (KO) mouse, and was stably expressing pHEX6300 plasmid containg human CLU cDNA (TEC-CLUhCLU), so that this cell line constitutively expresses human CLU protein, whereas control cell line (TEC-CLU-/-) was generated from the same parental CLU null TEC cell line by expressing empty pHEX6300. Both TEC-CLUhCLU and TEC-CLU-/- cell lines were exposed to either normoxia or hypoxia (1% O2). Transcriptome profiling with a significant 2-fold change (FC) (FC ≥ 2, p ≤ 0.05) was performed using SurePrint G3 Mouse Gene Expression 8×60K microarray, and the signaling pathways was ranked by using Ingenuity pathway analysis (IPA).Results: Here, we showed that compared to CLU null TEC-CLU-/- controls ectopic expression of human CLU in CLU null kidney cells (TEC-CLUhCLU) promoted cell growth but inhibited migration in normoxia, and enhanced cell survival in hypoxia. CLU affected expression of 3864 transcripts (1893 up-regulated) in normoxia and 3670 transcripts (1925 up-regulated) in hypoxia. CLU functions including cell proliferation, survival and adhesion in normoxia were associated mostly with AKT2 dependent PI3K/AKT, PTEN, VEGF and ERK/MAPK signaling and as well with GSK3B-mediated cell cycle progression. In addition to unfolded protein response (UPR) and/or endoplasmic reticulum (ER) stress, CLU-enhanced cell survival in hypoxia was also associated with Foxo3/PIK3CD/MAPK1-dependent PI3K/AKT, HIF-α, PTEN, VEGF and ERK/MAPK signaling. Conclusion: Our data showed that CLU functions in kidney cells were mediated in a cascade manner mainly by PI3K/AKT, PTEN, VEGF and ERK/MAPK signaling, and specifically by activation of UPR/ER stress in hypoxia, providing new insights into the protective role of CLU in the kidney.
Peritoneal Dialysis (PD) is an effective method of renal replacement therapy for patients with end-stage renal disease. PD solution is instilled into the peritoneal cavity and water, solutes, and waste products are removed across the peritoneal membrane, which serves as a natural filter between the peritoneal cavity and the bloodstream. The current conventional PD solution uses hypertonic glucose as an osmotic agent to remove water – a process termed ultrafiltration (UF). Although effective, chronic daily exposure to glucose causes systemic metabolic complications for PD patients; it also directly damages the peritoneal membrane and eventually causes the filter to fail.Hyperbranched Polyglycerol (HPG) is a non-toxic, non-immunogenic synthetic polymer that contains no starch or glucose. HPG has shown very limited organ accumulation after intravenous injection. HPG offers many theoretical advantages over glucose-based PD including the ability to synthesize HPG over a range of molecular weights. This current thesis tests HPG as a glucose-sparing osmotic agent in PD solution.We used a rodent model of PD to evaluate solute and waste removal, ultrafiltration, and peritoneal biocompatibility over 0-8 hours of peritoneal exposure. We compared HPG solutions of molecular weights 0.5, 1, and 3 kDa with conventional glucose-based solution (Dianeal™ 2.5%) and buffered glucose-based solution (Physioneal™ 2.27%).We demonstrated that HPG solutions can induce superior and sustained UF for 8 hours, in contrast to glucose-based solutions that lose UF capacity after 4 hours. Sodium and urea removal was superior for HPG solutions, in part because HPG polymer acts as a colloid - as opposed to crystalloid - osmotic agent. We used neutrophil infiltration and peritoneal mesothelial cell detachment as markers of biocompatibility. We found that HPG solutions, particularly lower molecular weight polymers, demonstrate superior biocompatibility profiles when compared to glucose-based PD solutions.Taken together, these experiments support the proof-of concept of HPG as a promising novel osmotic agent in PD. Future studies are required to investigate the chronic effects of HPG exposure on the peritoneal membrane, as well as the metabolic and pharmacokinetic profiles of HPG PD solutions.
Inactivation of T cells is a widely used strategy for immunosuppression. Halofuginone (HF) is an antiprotozoal agent for treating parasites in veterinary medicine, and has been demonstrated to inhibit collagen type 1 synthesis, T helper 17 cell differentiations and cytokine production in activated T cells. The present study was designed to examine its actions against T cell proliferation, and in a combined therapy with conventional immunosuppressant rapamycin. To examine the anti-proliferative ability of HF, T cell proliferation in cultured murine splenocytes, cell apoptosis, and cell cycle analysis as well as autophagy markers were investigated. The expression of cleaved Poly ADP ribose polymerase and DNA fragmentation were further to characterize apoptosis. Here, I showed that addition of HF in naïve splenocyte cultures significantly suppressed cell proliferation in response to antigen- or anti-CD3 antibody or in activated T cell cultures in response to Interleukin (IL)-2 in a dose-dependent manner with activation of apoptosis, and cell cycle arrest at S phase. Further studies showed that proline supplement in cell culture medium significantly prevented HF-mediated suppression of T cell proliferation by reducing cell apoptosis and proliferation arrest. In addition, HF synergistically enhanced the anti-proliferative ability of rapamycin, which was correlated with increased induction of apoptosis in T cell cultures. My data suggests that HF interferes proline incorporation in protein synthesis machinery resulting in apoptosis and autophagy-induced cell cycle arrest via amino acid starvation response in T cells. The data of this study suggest that HF may be a potential adjuvant that can enhance anti-proliferative activity of rapamycin in preventing T cell proliferation.