MP098
FACTORS RELEASED BY HUMAN MESENCHYMAL STEM CELLS SUPPRESS GLUCOSE-INDUCED INFLAMMATORY RESPONSES OF STABLE RENAL PROXIMAL TUBULAR EPITHELIAL CELL MONOLAYERS Free

INTRODUCTION AND AIMS: Although diabetic kidney disease (DKD) is often viewed as being driven by glomerular injury, direct pro-inflammatory and pro-fibrotic effects of prolonged hyperglycaemia on renal proximal tubular epithelial cells (RPTEC) also play an important pathogenic role. Bone marrow derived mesenchymal stem cells (MSC) have shown promise as therapeutic modulators of the inflammatory components of DKD pathogenesis. The aims of this study were to develop an in vitro system for modelling inflammatory response of stable human (h)RPTEC monolayers to prolonged high glucose concentration and to investigate the potential for hMSC-associated factor to modulate this response.

METHODS: Human RPTEC/TERT1 cells were seeded at pre-optimized density to generate stable confluent monolayers during 12-day culture. Media containing “Normal” D-glucose (5 mM), “High” (25mM) D-Glucose or D-Mannitol (osmotic control) was added for a further 5 days (with exchanges on days 2 and 4) following which supernatants and cells were collected for analysis by ELISA (for IL-6, IL-8, MCP-1 and NGAL) and flow cytometry (for apoptotic and necrotic cell death by Annexin V/PI staining) respectively. Similar experiments were then performed in which High glucose- and Mannitol-exposed RPTECT/TERT1 monolayers were cultured for the final 24 hours in the presence or absence of 10X concentrated 50:50 v/v “full” and “extracellular vesicle (EV) depleted” hMSC conditioned medium (CM). Results were statistically analysed with Graphpad Prism 6.0®.

RESULTS: Five-day exposure to high glucose caused significant increases in RPTEC/TERT1 cell secretion of IL-6 (~1.5 fold), IL-8 (~1.7 fold), MCP-1 (~2 fold) and NGAL (~2 fold) compared to normal glucose and mannitol without an increase in apoptotic or necrotic cell death. Addition of human serum albumin (100 ug/ml) was associated with further increases in these analytes, again without increased cell death. Addition of both “full” and “EV depleted” hMSC-CM resulted in ≥ 50% decrease in RPTEC/TERT1 cell secretion of levels of IL-6, IL-8 and MCP-1 but not NGAL under high glucose conditions (Figure 1). In the case of MCP-1, suppression by EV depleted hMSC CM was less than suppression by full hMSC CM.

CONCLUSIONS: This study demonstrates that: (a) High glucose (± albumin) induces secretion of pro-inflammatory mediators by hRPTEC stable monolayers. (b) Factors released by hMSC suppress high glucose-induced hRPTEC secretion of inflammatory cytokines but not NGAL. (c) hMSC-EVs may partially mediate the suppression of high glucose-induced MCP-1 secretion.