Research Projects
Immuno-Pathogenetic Mechanisms in Kidney Disease of Systemic Lupus Erythematous

Objective and Significance:

Lupus nephritis is characterized by the production of anti-DNA antibodies. Prominent features of LN include infiltration of inflammatory cells into the glomerulus and tubulo-interstitium, induction of pro-fibrotic growth factors and pro-inflammatory cytokines, and modulation of matrix components. To date, much attention has focused on the pathogenesis of glomerular injury in LN whilst the immuno-pathogenetic mechanisms of tubulo-interstitial alterations remain obscure.

Tubular epithelial cells constitute the predominant cell type within the tubulo-interstitium. Although previously considered to play a primary role in the regulation and homeostasis of water, electrolytes and nutrients, it has now become apparent that proximal tubular epithelial cells {PTEC) play a pivotal role in the patho-physiology of renal disease. Studies have demonstrated that under certain stress/stimulation, F°TEC may become activated in which the phenotype of PTEC transform to a more fibroblastic morphology. How anti-DNA antibodies modulate PTEC morphology, viability, and cellular functions have not been elucidated and thus constitute the theme of this project.

Objectives:

1. To elucidate the role of anti-DNA antibodies in modulating cell viability [lactate dehydrogenase (LDH) release, apoptosis, and necrosis] and proliferation.
2. To assess the synthesis of growth factors [transforming growth factor-betal (TGF-P l}, platelet derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF)], and cytokines (interleukin-lbeta (IL-lp), IL-6, IL-12, tumor necrosis factor-alpha (TNF-a) and interferon-y (IFN-y)] by PTEC in the presence/absence of anti-DNA antibodies at the transcriptional and translational level.
3. To examine whether anti-DNA antibodies induce epithelial-mesenchymal transformation.

Significance:

Understanding the mechanisms of lupus nephritis is prerequisite to providing novel strategies in alleviating pathogenesis of disease.

Research Plan and Methodology:

Culture of PTEC: PTEC will be obtained from nephrectomized renal specimens by differential sieving and maintained in DMEM/Ham's 12 medium supplemented with 10% FCS. Confluent PTEC will be characterized by their morphology and specific epithelial markers (alkaline phosphatase, vimentin and cytokeratin).

Isolation of IgG anti-DNA antibodies from the sera of LN patients: This will be achieved by protein A-Sepharose chromatography foll.owed by DNA-cellulose affinity chromatography.

LDH release: Confluent PTEC will be cultured under control and experimental conditions in 96-well tissue culture plates for selective time periods up to 48h. Supematants will be collected, centrifuged for l 0min at 2000 g, and assessed for LDH release using a commercially available cytotoxicity kit according to the manufacturer's instructions.

Determination of cell apoptosis and necrosis: Cells will be cultured under control and experimental conditions and monitored for apoptosis and necrosis by staining with annexin V conjugated with FITC, and propidium iodide respectively.

Determination of cell proliferation: PTEC will be seeded into 96-well plates at a density of 10,000 cells/cm2 and cultured in DMEM/Ham's Fl2 supplemented with 10% FCS for 48 h. Thereafter, the cells will be washed with PBS and incubated under control or experimental conditions. At selective time periods (0.5-5 days), cell proliferation will be assessed by the addition ofMTT.

Gene expression of growth factors and cytokines: This will investigated by Reverse Transcription and

Polymerase Chain Reaction (RT-PCR) using specific primers for TGF-Pl, PDGF, bFGF, EGF, IL-1 p, IL-6, IL-12, TNF-a, IFN-y.

Determination of growth factor and cytokine synthesis: The aforementioned growth factors and cytokines will be measured using commercial ELISAs.

Expression of epithelial and mesenchymal markers: PTEC cell-associated samples will be obtained after stimulation under control and experimental conditions and monitored for epithelial-mesenchymal transition using Western blot analysis and immunohistochemistry using antibodies against cytokeratin and E-cadherin (epithelial markers), and smooth muscle actin and OB-cadherin (mesenchymal markers).

Professor DTM Chan, Department of Medicine

Biography
dtmchan@hku.hk

For more information or to express interest for this project, please email the supervisor or the specified contact point in the project description.  Interested candidates are advised to enclose with your email:

1. your CV,
2. a brief description of your research interest and experience, and
3. two reference letters (not required for HKUMed UG students seeking MRes[Med]/URIS projects).

Information on the research programme, funding support and admission documentations could be referenced online at the Research Postgraduate Admissions website. General admission enquiries should be directed to rpgmed@hku.hk.

HKUMed MBBS students interested in the Master of Research in Medicine (MRes[Med]) programme may visit the programme website for more information.  

HKUMed UG students interested in the Undergraduate Research Internship Scheme (URIS) may visit the scheme’s website for more information.