Expression of growth arrest specific 1 (Gas1) in the distal tubules and collecting ducts in normal kidney and in the early stages of diabetic nephropathy.

The Growth Arrest-Specific protein 1 (Gas1) has been recently described in kidney as an endogenous inhibitor of cell proliferation in mesangial cells and with an important role in the maintenance of nephron progenitor cells. Furthermore, the expression of Gas1 was demonstrated in NCAM + progenitor parietal cells of Bowman's capsule. Thus, the aim of this study was to analyze the expression of Gas1 in the collecting ducts (CD) of healthy rats and to examine whether high glucose levels modify its expression during the early stages of diabetes in STZ-treated rats. Immunofluorescence reveals that principal cells AQP2 + express Gas1 in both healthy and diabetic conditions. Western blot from enriched fractions of medullary CD suggests that diabetes promotes the increase of Gas1. AQP2 + cells are also positive for the expression of CD24 and CD1133 in diabetic rats. In addition, diabetes modifies the cell morphology in the CD and favors the increase of principal cells (AQP2+/Gas1+), induces a significant decrease of intercalated cells (V-ATPase+/Gas1-) and the presence of intermediate cells (Gas1+/V-ATPase+) which express both principal and intercalated cell markers. The expression of Gas1 in the distal tubules was also determined by immunofluorescence, western blot and ELISA in diabetic rats. The results identify Gas1 as a specific marker of principal cells in healthy and diabetic rats and suggest that diabetes promotes the expression of Gas1. Gas1 may have an important role in the maintenance and differentiation to principal cells in the CD during early stages of diabetes.

Diabetes and exposure to peritoneal dialysis solutions alter tight junction proteins and glucose transporters of rat peritoneal mesothelial cells.

AIM: To evaluate alterations in tight junction (TJ) proteins and glucose transporters in rat peritoneal mesothelial cells (RPMC) from diabetic rats and after treatment with peritoneal dialysis solutions (PDS) in vitro. METHODS: Diabetes was induced in female Wistar rats by streptozotocin (STZ)-injection. Twenty-one days after STZ-injection, peritoneal thickness and mesothelial cell morphology were studied by light microscopy and microvilli length and density by atomic force microscopy. RPMC were obtained from healthy and diabetic rats. Mesothelial phenotype, evaluated by cytokeratin and pan-cadherin, epithelial to mesenchymal transition (EMT), evaluated by alpha-smooth muscle action (α-SMA) and vimentin, TJ proteins, claudins-1 and -2, and occludin, and glucose transporters, sodium and glucose co-transporters (SGLT) -1 and -2 and facilitative glucose transporters (GLUT) -1 and -2 were analyzed. Also, transepithelial electrical resistance (TER) was measured. Oxidative stress was estimated by measuring reactive oxygen species production, and protein carbonylation, receptor for advanced glycation end products (RAGE), nuclear factor erythroid related factor-2 (Nrf-2), and expression of antioxidant enzymes. KEY FINDINGS: Peritoneal damage was present 21days after STZ-injection. Diabetes induced changes in TJ and glucose transporters in RPMC together with decreased TER. RPMC from diabetic rats showed oxidative stress, which was enhanced by exposure to PDS. In addition, RPMC from diabetic rats showed early EMT. SIGNIFICANCE: To our knowledge, this is the first study that shows changes in TJ proteins and glucose transporters of RPMC from diabetic rats. All these alterations might explain the increased peritoneal permeability observed in diabetic patients undergoing peritoneal dialysis.

Retinoic acid improves morphology of cultured peritoneal mesothelial cells from patients undergoing dialysis.

Patients undergoing continuous ambulatory peritoneal dialysis are classified according to their peritoneal permeability as low transporter (low solute permeability) or High transporter (high solute permeability). Factors that determine the differences in permeability between them have not been fully disclosed. We investigated morphological features of cultured human peritoneal mesothelial cells from low or high transporter patients and its response to All trans retinoic Acid (ATRA, vitamin A active metabolite), as compared to non-uremic human peritoneal mesothelial cells. Control cells were isolated from human omentum. High or low transporter cells were obtained from dialysis effluents. Cells were cultured in media containing ATRA (0, 50, 100 or 200 nM). We studied length and distribution of microvilli and cilia (scanning electron microscopy), epithelial (cytokeratin, claudin-1, ZO-1 and occludin) and mesenchymal (vimentin and α-smooth muscle actin) transition markers by immunofluorescence and Western blot, and transforming growth factor ß1 expression by Western blot. Low and high transporter exhibited hypertrophic cells, reduction in claudin-1, occludin and ZO-1 expression, cytokeratin and vimentin disorganization and positive α-smooth muscle actin label. Vimentin, α-smooth muscle actin and transforming growth factor-ß1 were overexpressed in low transporter. Ciliated cells were diminished in low and high transporters. Microvilli number and length were severely reduced in high transporter. ATRA reduced hypertrophic cells number in low transporter. It also improved cytokeratin and vimentin organization, decreased vimentin and α-smooth muscle actin expression, and increased claudin 1, occludin and ZO-1 expression, in low and high transporter. In low transporter, ATRA reduced transforming growth factor-ß1 expression. ATRA augmented percentage of ciliated cells in low and high transporter. It also augmented cilia length in high transporter. Alterations in structure, epithelial mesenchymal markers and transforming growth factor-ß1 expression were differential between low and high transporter. Beneficial effects of ATRA were improved human peritoneal mesothelial cells morphology tending to normalize structures.