Sónia Simão

Age-related changes in renal expression of oxidant and antioxidant enzymes and oxidative stress markers in male SHR and WKY rats

Oxidative stress has been hypothesized to play a role in aging and age-related disorders, such as hypertension. This study compared levels of oxidative stress and renal expression of oxidant and antioxidant enzymes in male normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) at different ages (3 and 12 months). In the renal cortex of 3-month old SHR increases in hydrogen peroxide (H(2)O(2)) were accompanied by augmented expression of NADPH oxidase subunit Nox4 and decreased expression of antioxidant enzymes SOD1 and SOD3. A further increase in renal H(2)O(2) production and urinary TBARS was observed in 12-month old WKY and SHR as compared with 3-month old rats. Similarly, expressions of NADPH oxidase subunit p22(phox), SOD2 and SOD3 were markedly elevated with age in both strains. When compared with age-matched WKY, catalase expression was increased in 3-month old SHR, but unchanged in 12-month old SHR. Body weight increased with aging in both rat strains, but this increase was more pronounced in WKY. In conclusion, renal oxidative stress in 12-month old SHR is an exaggeration of the process already observed in the 3-month old SHR, whereas the occurrence of obesity in 12-month old normotensive rats may partially be responsible for the age-related increase in oxidative stress.

H2O2 Stimulation of the Cl−/HCO3− Exchanger by Angiotensin II and Angiotensin II Type 1 Receptor Distribution in Membrane Microdomains

The present study tested the hypothesis that angiotensin II (Ang II)–induced oxidative stress and Ang II–stimulated Cl−/HCO3− exchanger are increased and related to the differential membrane Ang II type 1 (AT1) receptor and reduced nicotinamide-adenine dinucleotide phosphate oxidase expression in immortalized renal proximal tubular epithelial (PTE) cells from the spontaneously hypertensive rat (SHR) relative to its normotensive control (Wistar Kyoto rat [WKY]). The exposure of cells to Ang II increased Cl−/HCO3− exchanger activity with EC50s of 0.10 and 12.2 nmol/L in SHR and WKY PTE cells, respectively. SHR PTE cells were found to overexpress nicotinamide-adenine dinucleotide phosphate oxidase 2 and 4 and were endowed with an enhanced ability to generate H2O2. The reduced nicotinamide-adenine dinucleotide phosphate oxidase inhibitor apocynin reduced the production of H2O2 in SHR PTE cells and abolished their hypersensitivity to Ang II. The expression of the glycosylated form of the AT1 receptor in both lipid and nonlipid rafts were higher in SHR cells than in WKY PTE cells. Pretreatment with apocynin reduced the abundance of AT1 receptors in both microdomains, mainly the glycosylated form of the AT1 receptor in lipid rafts, in SHR cells but not in WKY PTE cells. In conclusion, differences between WKY and SHR PTE cells in their sensitivity to Ang II correlate with the higher H2O2 generation that provokes an enhanced expression of glycosylated and nonglycosylated AT1 receptor forms in lipid rafts.

Oxidative stress and α1‐adrenoceptor‐mediated stimulation of the Cl−/HCO3− exchanger in immortalized SHR proximal tubular epithelial cells

This study evaluated the signalling coupled to the α1‐adrenoceptor‐induced stimulation of the Cl−/HCO3− exchanger in hypertension.

Age-related changes in the renal dopaminergic system and expression of renal amino acid transporters in WKY and SHR rats

This study examined age-related changes in renal dopaminergic activity and expression of amino acid transporters potentially involved in renal tubular uptake of l-DOPA in Wistar Kyoto (WKY) and spontaneously hypertensive rats. Aging (from 13 to 91 weeks) was accompanied by increases in systolic blood pressure (SBP) in both WKY and SHR. The sum of urinary dopamine and DOPAC and the urinary dopamine/l-DOPA ratio were increased in aged SHR but not in aged WKY. The urinary dopamine/renal delivery of l-DOPA ratio was increased in both rat strains with aging. LAT2 abundance was increased in aged WKY and SHR. The expression of 4F2hc was markedly elevated in aged SHR but not in aged WKY. ASCT2 was upregulated in both aged WKY and SHR. Plasma aldosterone levels and urinary noradrenaline levels were increased in aged WKY and SHR though levels of both entities were more elevated in aged SHR. Activation of the renal dopaminergic system is more pronounced in aged SHR than in aged WKY and is associated with an upregulation of renal cortical ASCT2 in WKY and of LAT2/4F2hc and ASCT2 in SHR. This activation may be the consequence of a counter-regulatory mechanism for stimuli leading to sodium reabsorption.

Loss of oxidative stress tolerance in hypertension is linked to reduced catalase activity and increased c-Jun NH2-terminal kinase activation

Hypertension is accompanied by increased levels of reactive oxygen species, which may contribute to progressive renal injury and dysfunction. Here we tested the hypothesis that sensitivity to exogenous hydrogen peroxide (H(2)O(2)) is enhanced in immortalized renal proximal tubular epithelial cells from spontaneously hypertensive rats (SHR) compared to normotensive control Wistar Kyoto rats (WKY). We found that SHR cells were more sensitive to H(2)O(2)-induced cell death than WKY cells. Lower survival in SHR cells correlated with increased DNA fragmentation, chromatin condensation, and caspase-3 activity, indicating apoptosis. H(2)O(2) degradation was slower in SHR than in WKY cells, suggesting that reduced antioxidant enzyme activity might be the basis for their increased sensitivity. In fact, catalase activity was downregulated in SHR cells, whereas glutathione peroxidase activity was similar in both cell types. We next examined whether MAPK signaling pathways contributed to H(2)O(2)-mediated apoptosis. Inhibition of c-Jun NH(2)-terminal kinase (JNK) with SP600125 partially rescued H(2)O(2)-induced apoptosis in WKY but not in SHR cells. In addition, p54 JNK2 isoform was robustly phosphorylated by H(2)O(2), this effect being more pronounced in SHR cells. Together, these results suggest that the survival disadvantage of SHR cells upon exposure to H(2)O(2) stems from impaired antioxidant mechanisms and activated JNK proapoptotic signaling pathways.

Renal aging in WKY rats: changes in Na+,K+ -ATPase function and oxidative stress.

It has been suggested that alterations in Na(+),K(+)-ATPase mediate the development of several aging-related pathologies, such as hypertension and diabetes. Thus, we evaluated Na(+),K(+)-ATPase function and H(2)O(2) production in the renal cortex and medulla of Wistar Kyoto (WKY) rats at 13, 52 and 91 weeks of age. Creatinine clearance, proteinuria, urinary excretion of Na(+) and K(+) and fractional excretion of Na(+) were also determined. The results show that at 91 weeks old WKY rats had increased creatinine clearance and did not have proteinuria. Despite aging having had no effect on urinary Na(+) excretion, urinary K(+) excretion was increased and fractional Na(+) excretion was decreased with age. In renal proximal tubules and isolated renal cortical cells, 91 week old rats had decreased Na(+),K(+)-ATPase activity when compared to 13 and 52 week old rats. In renal medulla, 91 week old rats had increased Na(+),K(+)-ATPase activity, paralleled by an increase in protein expression of α(1)-subunit of Na(+),K(+)-ATPase. In addition, renal H(2)O(2) production increased with age and at 91 weeks of age renal medulla H(2)O(2) production was significantly higher than renal cortex production. The present work demonstrates that although at 91 weeks of age WKY rats were able to maintain Na(+) homeostasis, aging was accompanied by alterations in renal Na(+),K(+)-ATPase function. The observed increase in oxidative stress may account, in part, for the observed changes. Possibly, altered Na(+),K(+)-ATPase renal function may precede the development of age-related pathologies and loss of renal function.

Short-term regulation of the Cl-/HCO3(-) exchanger in immortalized SHR proximal tubular epithelial cells.

The present study evaluated the activity of Cl(-)/HCO(3)(-) exchanger and the abundance of Slc26a6 in immortalized renal proximal tubular epithelial (PTE) cells from the Wistar-Kyoto rat (WKY) and spontaneously hypertensive rat (SHR) and identified the signaling pathways that regulate the activity of the transporter. The affinity for HCO(3)(-) was identical in WKY and SHR PTE cells, but V(max) values (in pH units/min) in SHR PTE cells (0.4016) were significantly higher than in WKY PTE cells (0.2304). The expression of Slc26a6 in SHR PTE cells was sevenfold that in WKY PTE cells. Dibutyryl-cAMP (db-cAMP) or forskolin, which increased endogenous cAMP, phorbol-12,13-dibutyrate (PDBu) and anisomycin, significantly (P<0.05) increased the Cl(-)/HCO(3)(-) exchanger activity in WKY and SHR PTE cells to a similar extent. The stimulatory effects of db-cAMP and forskolin were prevented by the PKA inhibitor H89, but not by chelerythrine. The stimulatory effects of PDBu were prevented by both chelerythrine and SB 203580, but not by H89 or the MEK inhibitor PD 98059. The stimulatory effect of anisomycin was prevented by SB 203580, but not by chelerythrine. Increases in phospho-p38 MAPK by anisomycin were identical in WKY and SHR PTE cells, this being sensitive to SB 203580 but not to chelerythrine. It is concluded that SHR PTE cells, which overexpress the Slc26a6 protein, are endowed with an enhanced activity of the Cl(-)/HCO(3)(-) exchanger. The Cl(-)/HCO(3)(-) exchanger is an effector protein for PKA, PKC and p38 MAPK in both WKY and SHR PTE cells.

Increased responsiveness to JNK1/2 mediates the enhanced H2O2-induced stimulation of Cl−/HCO3− exchanger activity in immortalized renal proximal tubular epithelial cells from the SHR

We have previously demonstrated that exogenous H2O2 stimulates Cl(-)/HCO3(-) exchanger activity in immortalized renal proximal tubular epithelial (PTE) cells from both the Wistar-Kyoto (WKY) rat and the spontaneously hypertensive rat (SHR), this effect being more pronounced in SHR cells. The aim of the present study was to examine the mechanism of H2O2-induced stimulation of Cl(-)/HCO3(-) exchanger activity in WKY and SHR cells. It is now reported that the SHR PTE cells were endowed with an enhanced capacity to produce H2O2, comparatively with WKY cells and this was accompanied by a decreased expression of SOD2, SOD3, and catalase in SHR PTE cells. The stimulatory effect of H2O2 on the exchanger activity was blocked by SP600125 (JNK inhibitor), but not by U0126 (MEK1/2 inhibitor) or SB203580 (p38 inhibitor) in both cell lines. Basal JNK1 and JNK2 protein expression was higher in SHR PTE cells than in WKY PTE cells. H2O2 had no effect on p-JNK1/2 in WKY PTE cells over time. By contrast, H2O2 treatment resulted in a rapid and sustained increase in JNK1/2 phosphorylation in SHR PTE cells, which was completely abolished by apocynin. Treatment of SHR PTE cells with apocynin significantly decreased the H2O2-induced stimulation of Cl(-)/HCO3(-) exchanger activity. It is concluded that H2O2-induced stimulation of Cl(-)/HCO3(-) exchanger activity is regulated by JNK1/2, particularly by JNK2, in SHR PTE cells. The imbalance between oxidant and antioxidant mechanisms in SHR PTE cells enhances the response of JNK1/2 to H2O2, which contributes to their increased sensitivity to H2O2.

Identification of SLC26A transporters involved in the Cl /HCO3 exchange in proximal tubular cells from WKY and SHR

AIMS slc26a proteins are responsible for a large number of functions either in normal physiology or in human disease. We have previously reported that proximal tubular epithelial (PTE) cells immortalized from spontaneously hypertensive rats (SHRs) were endowed with increased Cl(-)/HCO3(-) exchanger activity and slc26a6 protein expression compared with PTE cells immortalized from normotensive Wistar Kyoto (WKY) rats. The aim of the present study was to identify slc26a members responsible for the Cl(-)/HCO3(-) exchange in WKY and SHR PTE cells. MAIN METHODS Cl(-)/HCO3(-) exchanger activity was assessed as the initial rate of pHi recovery after removal of HCO3(-) or after removal of Cl(-). The presence of slc26a genes was evaluated by means of reverse transcriptase-PCR (RT-PCR) in WKY and SHR PTE cell lines and in the kidney of WKY and SHR. Transcript abundance was measured by quantitative real-time polymerase chain reaction (PCR). KEY FINDINGS We detected slc26a4, slc26a6, slc26a7 and slc26a9 transcripts in the rat kidney of WKY and SHR. In WKY and SHR PTE cell lines we detected slc26a4, slc26a6 and slc26a9 transcripts, which were, respectively, 12-, 4- and 15-fold upregulated in SHR cells. Gene silencing with small interfering RNAs (siRNAs) targeting slc26a4, slc26a6 and slc26a9 reduced Cl(-)/HCO3(-) exchanger activity in both cell lines. SIGNIFICANCE These results indicate that Cl(-)/HCO3(-) exchanger activity is mediated by, at least in part, slc26a4, slc26a6 and slc26a9 in cultured WKY and SHR cells. The overexpression of these slc26a members in SHR cells may correspond to an adaptive process to cope with the sustained increase in proximal tubular sodium reabsorption.

H2O2 stimulates Cl−/HCO 3− exchanger activity through oxidation of thiol groups in immortalized SHR renal proximal tubular epithelial cells

Cl−/HCO  3− exchanger and Na+/H+ exchanger 3 are the main transporters responsible for NaCl reabsorption in kidney proximal tubules (PT). It is well accepted that membrane exchangers can be regulated by reactive oxygen species (ROS). In the kidney, ROS are known to contribute to decreases in Na+ excretion and consequently increase blood pressure. The present study investigated mechanisms by which H2O2‐induced stimulation of Cl−/HCO  3− exchanger activity is enhanced in proximal tubular epithelial (PTE) cells immortalized from spontaneously hypertensive rats (SHR) as compared to normotensive Wistar Kyoto (WKY). H2O2 decreased Km values for Cl−/HCO  3− exchanger activity in SHR PTE cells, but had no effect on the kinetic parameters in WKY cells. DTDP stimulated in a concentration‐dependent manner Cl−/HCO  3− exchanger activity in both cell lines, but SHR PTE cells were 2.4‐fold more responsive to this oxidant. In contrast, thimerosal had no effect on exchanger activity in both cell lines. The effects of H2O2 and DTDP upon the exchanger activity were blocked by DTT in WKY and SHR PTE cells. Similar to H2O2, DTDP decreased Km values for Cl−/HCO  3− exchanger activity in SHR PTE cells. Basal content of free thiol groups was higher in WKY PTE cells than in SHR. Upon H2O2 treatment the free thiol groups decreased in both cell lines; however, this decrease was more pronounced in WKY cells. In conclusion, in SHR PTE cells H2O2 stimulates Cl−/HCO  3− exchanger activity via modification of thiol groups of intracellular and/or transmembrane protein. Furthermore, the thiol oxidation‐dependent pathway also increases the HCO  3− affinity in SHR PTE cells. J. Cell. Biochem. 112: 3660–3665, 2011.