Elida Adalgisa Neri

Fructose Acutely Stimulates NHE3 Activity in Kidney Proximal Tubule

Background/Aims: Fructose causes a sodium-sensitive hypertension and acutely reduces the urinary Na+ excretion, suggesting that it may regulate the activity of renal tubular sodium transporters. NHE3 is highly expressed in proximal tubule (PT), along with proteins that mediate fructose transport and metabolism. The present work was outlined to investigate whether fructose modulates proximal NHE3 activity and to elucidate the molecular mechanisms underlying this modulation. Methods/Results: Using in vivo stationary microperfusion, we observed that fructose stimulates NHE3 mediated JHCO3- reabsorption. The MAPK pathway is not involved in this activation, as demonstrated by using of MEK/MAPK inhibitors, whereas experiments using a PKA inhibitor suggest that PKA inhibition plays a role in this response. These results were confirmed in vitro by measuring the cell pH recovery rate after NH4Cl pulse in LLC-PK1, a pig PT cell line, which showed reduced cAMP levels and NHE3 phosphorylation at serine-552 (PKA consensus site) after fructose treatment. Conclusions: NHE3 activity is stimulated by fructose, which increases proximal tubule Na+ reabsorption. The molecular mechanisms involved in this process are mediated, at least in part, by downregulation of the PKA signaling pathway. Future studies are needed to address whether fructose-stimulated NHE3 activity may contribute to renal injury and hypertension.

Transcriptional regulation of the Na⁺/H⁺ exchanger NHE3 by chronic exposure to angiotensin II in renal epithelial cells.

Angiotensin II (Ang II) exerts an acute bimodal effect on proximal tubule NHE3: while low doses stimulate the exchanger, high doses inhibit it. In the present study, we have investigated the chronic effects of Ang II on NHE3 expression and transcriptional regulation. Treatment of a tubular epithelial cell line, OKP, with Ang II 10(-11)M significantly increased NHE protein expression and mRNA levels, without evidence of bimodal effect. No change in mRNA half-life was detected, but transient transfection studies showed a significant increase in NHE3 promoter activity. Binding sites for Sp1/Egr-1 and AP2 transcription factors of the NHE3 proximal promoter were mutated and we observed that the Sp1/Egr-1 binding site integrity is necessary for Ang II stimulatory effects. Inhibition of cytochrome P450, PI3K, PKA and MAPK pathways prevented the Ang II stimulatory effect on the NHE3 promoter activity. Taking all the results together, our data reveal that chronic Ang II treatment exerts a stimulatory effect on NHE3 expression and promoter activity. The Ang II up-regulation of the NHE3 promoter activity appears to involve the Sp1/Egr-1 binding site and the interplay of several intracellular signaling pathways.

Integrated molecular, biochemical, and physiological assessment unravels key extraction method mediated influences on rat neonatal cardiomyocytes

Neonatal cardiomyocytes are instrumental for disease modeling, but the effects of different cell extraction methods on basic cell biological processes remain poorly understood. We assessed the influence of two popular methods to extract rat neonatal cardiomyocytes, Pre‐plating (PP), and Percoll (PC) on cell structure, metabolism, and function. Cardiomyocytes obtained from PP showed higher gene expression for troponins, titin, and potassium and sodium channels compared to PC. Also, PP cells displayed higher levels of troponin I protein. Cells obtained from PC displayed higher lactate dehydrogenase activity and lactate production than PP cells, indicating higher anaerobic metabolism after 8 days of culture. In contrast, reactive oxygen species levels were higher in PP cells as indicated by ethidium and hydroxyethidium production. Consistent with these data, protein nitration was higher in PP cells, as well as nitrite accumulation in cell medium. Moreover, PP cells showed higher global intracellular calcium under basal and 1 mM isoprenaline conditions. In a calcium‐transient assessment under electrical stimulation (0.5 Hz), PP cells displayed higher calcium amplitude than cardiomyocytes obtained from PC and using a traction force microscope technique we observed that PP cardiomyocytes showed the highest relaxation. Collectively, we demonstrated that extraction methods influence parameters related to cell structure, metabolism, and function. Overall, PP derived cells are more active and mature than PC cells, displaying higher contractile function and generating more reactive oxygen species. On the other hand, PC derived cells display higher anaerobic metabolism, despite comparable high yields from both protocols.