Vanda Pinto

Oxidative stress and the genomic regulation of aldosterone-stimulated NHE1 activity in SHR renal proximal tubular cells

This study evaluated the effects of aldosterone upon Na+/H+ exchange (NHE) activity in immortalized proximal tubular epithelial (PTE) cells from the spontaneously hypertensive rat (SHR) and the normotensive controls (Wistar Kyoto rat; WKY). Increases in NHE activity after exposure to aldosterone occurred in time- and concentration-dependent manner in SHR PTE cells, but not in WKY PTE cells. The aldosterone-induced increases in NHE activity were prevented by spironolactone, but not by the glucocorticoid receptor antagonist Ru 38486. The presence of the mineralocorticoid receptor transcript was confirmed by PCR and NHE1, NHE2, and NHE3 proteins were detected by immunoblot analysis. Cariporide and EIPA, but not S3226, inhibited the aldosterone-induced increase in NHE activity, indicating that NHE1 is the most likely involved NHE isoform. Pretreatment of SHR PTE cells with actinomycin D attenuated the aldosterone-induced increases in NHE activity. The SHR PTE cells had an increased rate of H2O2 production when compared with WKY PTE cells. Treatment of cells with apocynin, a NADPH oxidase inhibitor, markedly reduced the rate of H2O2 production. The aldosterone-induced increase in NHE activity SHR PTE cells was completely prevented by apocynin. In conclusion, the aldosterone-induced stimulation of NHE1 activity is a genomic event unique in SHR PTE cells, which involves the activation of the mineralocorticoid receptor, but ultimately requires the availability of H2O2 in excess.

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.

Renal amino acid transport systems and essential hypertension

Several clinical and animal studies suggest that “blood pressure goes with the kidney,” that is, a normotensive recipient of a kidney genetically programmed for hypertension will develop hypertension. Intrarenal dopamine plays an important role in the pathogenesis of hypertension by regulating epithelial sodium transport. The candidate transport systems for L‐DOPA, the source for dopamine, include the sodium‐dependent systems B0, B0,+, and y+L, and the sodium‐independent systems L (LAT1 and LAT2) and b0+. Renal LAT2 is overexpressed in the prehypertensive spontaneously hypertensive rat (SHR), which might contribute to enhanced L‐DOPA uptake in the proximal tubule and increased dopamine production, as an attempt to overcome the defect in D1 receptor function. On the other hand, it has been recently reported that impaired arginine transport contributes to low renal nitric oxide bioavailability observed in the SHR renal medulla. Here we review the importance of renal amino acid transporters in the kidney and highlight pathophysiological changes in the expression and regulation of these transporters in essential hypertension. The study of the regulation of renal amino acid transporters may help to define the underlying mechanisms predisposing individuals to an increased risk for development of hypertension.—Pinto, V., Pinho, M. J., Soares‐da‐Silva, P., Renal amino acid transport systems and essential hypertension. FASEB J. 27, 2927–2938 (2013). www.fasebj.org

Salt-inducible kinase 1 regulates E-cadherin expression and intercellular junction stability

The protein kinase liver kinase B1 (LKB1) regulates cell polarity and intercellular junction stability. Also, LKB1 controls the activity of salt‐inducible kinase 1 (SIK1). The role and relevance of SIK1 and its downstream effectors in linking the LKB1 signals within these processes are partially understood. We hypothesize that SIK1 may link LKB1 signals to the maintenance of epithelial junction stability by regulating E‐cadherin expression. Results from our studies using a mouse lung alveolar epithelial (MLE‐12) cell line or human renal proximal tubule (HK2) cell line transiently or stably lacking the expression of SIK1 (using SIK1 siRNAs or shRNAs), or with its expression abrogated (sik1+/+ vs. sik1−/− mice), indicate that suppression of SIK1 (∼40%) increases the expression of the transcriptional repressors Snail2 (∼12‐fold), Zeb1 (∼100%), Zeb2 (∼50%), and TWIST (∼20‐fold) by activating cAMP‐response element binding protein. The lack of SIK1 and activation of transcriptional repressors decreases the availability of E‐cadherin (mRNA and protein expression by ∼100 and 80%, respectively) and the stability of intercellular junctions in epithelia (decreases in transepithelial resistance). Furthermore, LKB1‐mediated increases in E‐cadherin expression are impaired in cells where SIK1 has been disabled. We conclude that SIK1 is a key regulator of E‐cadherin expression, and thereby contributes to the stability of intercellular junctions.—Eneling, K., Brion, L., Pinto, V., Pinho, M. J., Sznajder, J. I., Mochizuki, N., Emoto, K., Soares‐da‐Silva, P., Bertorello, A. M. Salt‐inducible kinase 1 regulates E‐cadherin expression and intercellular junction stability. FASEB J. 26, 3230–3239 (2012). www.fasebj.org

Increases in intracellular sodium activate transcription and gene expression via the salt-inducible kinase 1 network in an atrial myocyte cell line

Cardiac hypertrophy (CH) generally occurs as the result of the sustained mechanical stress caused by elevated systemic arterial blood pressure (BP). However, in animal models, elevated salt intake is associated with CH even in the absence of significant increases in BP. We hypothesize that CH is not exclusively the consequence of mechanical stress but also of other factors associated with elevated BP such as abnormal cell sodium homeostasis. We examined the effect of small increases in intracellular sodium concentration ([Na(+)](i)) on transcription factors and genes associated with CH in a cardiac cell line. Increases in [Na(+)](i) led to a time-dependent increase in the expression levels of mRNA for natriuretic peptide and myosin heavy chain genes and also increased myocyte enhancer factor (MEF)2/nuclear factor of activated T cell (NFAT) transcriptional activity. Increases in [Na(+)](i) are associated with activation of salt-inducible kinase 1 (snflk-1, SIK1), a kinase known to be critical for cardiac development. Moreover, increases in [Na(+)](i) resulted in increased SIK1 expression. Sodium did not increase MEF2/NFAT activity or gene expression in cells expressing a SIK1 that lacked kinase activity. The mechanism by which SIK1 activated MEF2 involved phosphorylation of HDAC5. Increases in [Na(+)](i) activate SIK1 and MEF2 via a parallel increase in intracellular calcium through the reverse mode of Na(+)/Ca(2+)-exchanger and activation of CaMK1. These data obtained in a cardiac cell line suggest that increases in intracellular sodium could influence myocardial growth by controlling transcriptional activation and gene expression throughout the activation of the SIK1 network.

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.

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.

DBA2J db/db mice are susceptible to early albuminuria and glomerulosclerosis that correlates with systemic insulin resistance

Diabetic nephropathy (DN) is the leading cause of kidney failure in the world. To understand important mechanisms underlying this condition, and to develop new therapies, good animal models are required. In mouse models of type 1 diabetes, the DBA/2J strain has been shown to be more susceptible to develop kidney disease than other common strains. We hypothesized this would also be the case in type 2 diabetes. We studied db/db and wild-type (wt) DBA/2J mice and compared these with the db/db BLKS/J mouse, which is currently the most widely used type 2 DN model. Mice were analyzed from age 6 to 12 wk for systemic insulin resistance, albuminuria, and glomerular histopathological and ultrastructural changes. Body weight and nonfasted blood glucose were increased by 8 wk in both genders, while systemic insulin resistance commenced by 6 wk in female and 8 wk in male db/db DBA/2J mice. The urinary albumin-to-creatinine ratio (ACR) was closely linked to systemic insulin resistance in both sexes and was increased ~50-fold by 12 wk of age in the db/db DBA/2J cohort. Glomerulosclerosis, foot process effacement, and glomerular basement membrane thickening were observed at 12 wk of age in db/db DBA/2J mice. Compared with db/db BLKS/J mice, db/db DBA/2J mice had significantly increased levels of urinary ACR, but similar glomerular histopathological and ultrastructural changes. The db/db DBA/2J mouse is a robust model of early-stage albuminuric DN, and its levels of albuminuria correlate closely with systemic insulin resistance. This mouse model will be helpful in defining early mechanisms of DN and ultimately the development of novel therapies.

Long-term food restriction attenuates age-related changes in the expression of renal aldosterone-sensitive sodium transporters in Wistar-Kyoto rats: A comparison with SHR

In the present study we hypothesized that age-associated changes in the renal aldosterone/mineralocorticoid receptor (MR) system may differ between normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). In WKY, body mass index significantly increased with age. Fat mass may operate as a confounding factor; therefore, WKY (WKY-FR) was pair-fed with SHR. Pair-feeding resulted in a 14% body weight reduction at the age of 52 weeks in WKY-FR. Renal oxidative stress was increased in aged WKY and SHR. Aged WKY and SHR had increased MR functionality, which correlated positively with increased plasma aldosterone levels, nuclear MR content and abundance of aldosterone effectors in the renal medulla. In contrast, decreases in nuclear MR content were observed in the renal cortex of both strains with aging. When compared to aged SHR, aged WKY-FR had decreased plasma aldosterone levels and decreased activation of the aldosterone/MR system in the renal medulla. Increases in renal oxidative stress and plasma aldosterone in aged WKY, to levels observed in SHR, were not sufficient to result in sustained increases in blood pressure. In conclusion, activation of the aldosterone/MR system is intensified by aging in SHR, whereas increases in body fat mass in WKY associate with hyperaldosteronism and oxidative stress.

Regulation of Renal LAT2 and 4F2hc Expression by Aldosterone

In the spontaneous hypertensive rat, overexpression of the renal Na + -independent L-amino acid transporter LAT2 is organ specific, precedes the onset of hypertension, correlates negatively with plasma aldosterone, and parallels the enhanced ability to take up L-DOPA and form renal dopamine. The present study evaluated the role of aldosterone on transcript and protein abundance of Na + -independent and Na + -dependent amino acid transporters. Na + -independent het- erodimeric amino acid transporters LAT1/4F2hc, LAT2/4F2hc and a Na + -dependent transporter ASCT2 transcript and protein abundance was determined in the renal cortex of normotensive Wistar rats chronically treated with aldosterone (1.5 mg), spironolactone (200 mg) or aldosterone plus spironolactone. Aldosterone significantly increased renal cortical LAT2 mRNA levels (45 % increase), with no changes in LAT1, 4F2hc and ASCT2 transcript levels. The effect of aldos- terone upon LAT2 mRNA levels was completely prevented by spironolactone. At the protein level, aldosterone treatment did not significantly affect LAT1 and LAT2 expression, but markedly reduced (51 % decrease) the abundance of 4F2hc and the urinary excretion of dopamine and DOPAC. The effect of aldosterone upon 4F2hc protein abundance was not re- versed by spironolactone. Increases in renal LAT2 transcript during chronic treatment with aldosterone occur through a spironolactone-sensitive genomic mechanism. This effect parallels with a decrease in LAT2 functionality, resulting from decreases in 4F2hc protein abundance, which appears to be either a non-genomic effect or an indirect effect of aldoster- one. The decrease in LAT2 functionality by aldosterone correlates well with the reduction in urinary dopamine.