Maria Oliveira-Souza

Genomic and nongenomic dose-dependent biphasic effect of aldosterone on Na+/H+ exchanger in proximal S3 segment: role of cytosolic calcium

The effects of aldosterone on the intracellular pH recovery rate (pHirr) via Na+/H+ exchanger and on the [Ca2+]i were investigated in isolated rat S3 segment. Aldosterone [10(-12), 10(-10), or 10(-8) M with 1-h, 15- or 2-min preincubation (pi)] caused a dose-dependent increase in the pHirr, but aldosterone (10(-6) M with 1-h, 15- or 2-min pi) decreased it (these effects were prevented by HOE694 but not by S3226). After 1 min of aldosterone pi, there was a transient and dose-dependent increase of the [Ca2+]i and after 6-min pi there was a new increase of [Ca2+]i that persisted after 1 h. Spironolactone, actinomycin D, or cycloheximide did not affect the effects of aldosterone (15- or 2-min pi) but inhibited the effects of aldosterone (1-h pi) on pHirr and on [Ca2+]i. RU 486 prevented the stimulatory effect of aldosterone (10(-12) M, 15- or 2-min pi) on both parameters and maintained the inhibitory effect of aldosterone (10(-6) M, 15- or 2-min pi) on the pHirr but reversed its stimulatory effect on the [Ca2+]i to an inhibitory effect. The data indicate a genomic (1 h, via MR) and a nongenomic action (15 or 2 min, probably via GR) on [Ca2+]i and on the basolateral NHE1 and are compatible with stimulation of the NHE1 by increases in [Ca2+]i in the lower range (at 10(-12) M aldosterone) and inhibition by increases at high levels (at 10(-6) M aldosterone) or decreases in [Ca2+]i (at 10(-6) M aldosterone plus RU 486).

Insulin but Not Phlorizin Treatment Induces a Transient Increase in GLUT2 Gene Expression in the Kidney of Diabetic Rats

Background/Aims: Increases in the renal glucose transporter gene expression are involved in renal tubule-glomerular diseases.Here we investigate the GLUT2 gene expression changes in the kidney of diabetic rats, by using insulin or phlorizin treatment. Methods:Rats were rendered diabetic and studied 20 days later: 4–12 h after one single injection of insulin or phlorizin, and 1–6 days after insulin or phlorizin injection twice a day, comparing with diabetic rats injected with placebo. GLUT2 was investigated by Northern and Western analysis. Results: In 20-day diabetic rats, acute treatment with insulin lowered the plasma glucose and increased the GLUT2 mRNA (∼100%, p < 0.001) without changes in the protein content, while phlorizin lowered the plasma glucose, but changed neither the GLUT2 mRNA nor the protein expression. Twenty-four hours of insulin treatment increased both GLUT2 mRNA (∼100%, p < 0.001) and protein (∼50%, p < 0.01), but no effects of phlorizin were observed. After 6 days, insulin and phlorizin similarly reduced glycemia, with opposite effects upon plasma insulin and urinary glucose, and both treatments decreased GLUT2 mRNA and protein (p < 0.05). Conclusion: In kidney of diabetic rats, an initial and transient upregulation of GLUT2 was induced specifically by insulin only. The 6-day normalization of GLUT2, however, was induced by both insulin and phlorizin treatment, which seems to be related to the plasma glucose lowering.

Regulation of Na+/H+ Exchanger Isoform 1 (NHE1) by Calmodulin-binding Sites: Role of Angiotensin II

We examined the effect of Angiotensin II (Ang II) on the interaction between the Ca²⁺/CaM complex and hNHE1. Considering that calmodulin binds to NHE1 at two sites (A and B), amino acids at both sites were modified and two mutants were constructed: SA^1K3R/4E and SB^1K3R/4E. Wild type and mutants were transfected into PS120 cells and their activity was examined by H⁺ flux (J_H+). The basal J_H+ of wild type was 4.71 ± 0.57 (mM/min), and it was similar in both mutants. However, the mutations partially impaired the binding of CaM to hNHE1. Ang II (10^-12 and 10^-9 M) increased the J_H+ in wild type and SB. Ang II (10^-6 M) increased this parameter only in SA. Ang II (10^-9 M) maintained the expression of calmodulin in wild type or mutants, and Ang II (10^-6 M) decreased it in wild type or SA, but not in SB. Dimethyl-Bapta-AM (10^-7 M), a calcium chelator, suppressed the effect of Ang II (10^-9 M) in wild type. With Ang II (10^-6 M), Bapta failed to affect wild type or SA, but it increased the J_H+ in SB. W13 or calmidazolium chloride (10^-5 M), two distinct calmodulin inhibitors, decreased the effect of Ang II (10^-9 M) in wild type or SB. With Ang II (10^-6 M), W13 or calmidazolium chloride decreased the J_H+ in wild type or SA and increased it in SB. Thus, with Ang II (10^-12 and 10^-9 M), site A seems to be responsible for the stimulation of hNHE1 and with Ang II (10^-6 M), site B is important to maintain its basal activity.

The regulation of NHE1 and NHE3 activity by angiotensin II is mediated by the activation of the angiotensin II type I receptor/phospholipase C/calcium/calmodulin pathway in distal nephron cells

Angiotensin II (Ang II), acting via the AT1 receptor, induces an increase in intracellular calcium [Ca(2+)]i that then interacts with calmodulin (CaM). The Ca(2+)/CaM complex directly or indirectly activates sodium hydrogen exchanger 1 (NHE1) and phosphorylates calmodulin kinase II (CaMKII), which then regulates sodium hydrogen exchanger 3 (NHE3) activity. In this study, we investigated the cellular signaling pathways responsible for Ang II-mediated regulation of NHE1 and NHE3 in Madin-Darby canine kidney (MDCK) cells. The NHE1- and NHE3-dependent pHi recovery rates were evaluated by fluorescence microscopy using the fluorescent probe BCECF/AM, messenger RNA was evaluated with the reverse transcription polymerase chain reaction (RT-PCR), and protein expression was evaluated by immunoblot. We demonstrated that treatment with Ang II (1pM or 1 nM) for 30 min induced, via the AT1 but not the AT2 receptor, an equal increase in NHE1 and NHE3 activity that was reduced by the specific inhibitors HOE 694 and S3226, respectively. Ang II (1 nM) did not change the total expression of NHE1, NHE3 or calmodulin, but it induced CaMKII, cRaf-1, Erk1/2 and p90(RSK) phosphorylation. The stimulatory effects of Ang II (1 nM) on NHE1 or NHE3 activity or protein abundance was reduced by ophiobolin-A (CaM inhibitor), KN93 (CaMKII inhibitor) or PD98059 (Mek inhibitor). These results indicate that after 30 min, Ang II treatment may activate G protein-dependent pathways, including the AT1/PLC/Ca(2+)/CaM pathway, which induces CaMKII phosphorylation to stimulate NHE3 and induces cRaf-1/Mek/Erk1/2/p90(RSK) activity to stimulate NHE1.

High Glucose Concentration Stimulates NHE-1 Activity in Distal Nephron Cells: the Role of the Mek/Erk1/2/p90RSK and p38MAPK Signaling Pathways

Aims: In models of diabetes, distal nephron cells contribute to glucose uptake and oxidation. How these cells contribute to the use of glucose for the regulation of H+ extrusion remains unknown. We used Madin-Darby Canine Kidney (MDCK) cells to investigate the effect of acute or chronic high glucose concentration on the abundance and activity of the Na+/H+ exchanger (NHE-1). Methods: Using RT-PCR, we also evaluated the mRNA expression for sodium glucose co-transporters SGLT1 and SGLT2. Protein abundance was analyzed using immunoblotting, and intracellular pH (pHi) recovery was evaluated using microscopy in conjunction with the fluorescent probe BCECF/AM. The Na+-dependent pHi recovery rate was monitored with HOE-694 (50 µM) and/or S3226 (10 µM), specific NHE-1 and NHE-3 inhibitors. Results: MDCK cells did not express the mRNA for SGLT1 or SGLT2 but did express the GLUT2, NHE-1 and NHE-3 proteins. Under control conditions, we observed a greater contribution of NHE-1 to pHi recovery relative to the other H+ transporters. Acute high glucose treatment increased the HOE-694-sensitive pHi recovery rate and p-Erk1/2 and p90RSK abundance. These parameters were reduced by PD-98059, a Mek inhibitor (1 µM). Chronic high glucose treatment also increased the HOE-694-sensitive pHi recovery rate and p-p38MAPK abundance. Both parameters were reduced by SB-203580, a p38MAPK inhibitor (10 µM). Conclusion: These results suggested that extracellular high glucose stimulated NHE-1 acutely and chronically through Mek/Erk1/2/p90RSK and p38MAPK pathways, respectively.

Renovascular remodeling and renal injury after extended angiotensin II infusion

Chronic angiotensin II (ANG II) infusion for 1 or 2 wk leads to progressive hypertension and induces inward hypertrophic remodeling in preglomerular vessels, which is associated with increased renal vascular resistance (RVR) and decreased glomerular perfusion. Considering the ability of preglomerular vessels to exhibit adaptive responses, the present study was performed to evaluate glomerular perfusion and renal function after 6 wk of ANG II infusion. To address this study, male Wistar rats were submitted to sham surgery (control) or osmotic minipump insertion (ANG II 200 ng·kg(-1)·min(-1), 42 days). A group of animals was treated or cotreated with losartan (10 mg·kg(-1)·day(-1)), an AT1 receptor antagonist, between days 28 and 42 Chronic ANG II infusion increased systolic blood pressure to 185 ± 4 compared with 108 ± 2 mmHg in control rats. Concomitantly, ANG II-induced hypertension increased intrarenal ANG II level and consequently, preglomerular and glomerular injury. Under this condition, ANG II enhanced the total renal plasma flow (RPF), glomerular filtration rate (GFR), urine flow and induced pressure natriuresis. These changes were accompanied by lower RVR and enlargement of the lumen of interlobular arteries and afferent arterioles, consistent with impairment of renal autoregulatory capability and outward preglomerular remodeling. The glomerular injury culminated with podocyte effacement, albuminuria, tubulointerstitial macrophage infiltration and intrarenal extracellular matrix accumulation. Losartan attenuated most of the effects of ANG II. Our findings provide new information regarding the contribution of ANG II infusion over 2 wk to renal hemodynamics and function via the AT1 receptor.

The effect of angiotensin II on intracellular pH is mediated by AT1 receptor translocation

The effect of ANG II on intracellular pH (pH(i)) recovery rate and AT(1) receptor translocation was investigated in transfected MDCK cells. The pH(i) recovery rate was evaluated by fluorescence microscopy using the fluorescent probe BCECF-AM. The human angiotensin II receptor isoform 1 (hAT(1)) translocation was analyzed by immunofluorescence and confocal microscope. Our data show that transfected cells in control situation have a pH(i) recovery rate of 0.219 +/- 0.017 pH U/min (n = 11). This value was similar to nontransfected cells [0.211 +/- 0.009 pH U/min (n = 12)]. Both values were significantly increased with ANG II (10(-9) M) but not with ANG II (10(-6) M). Losartan (10(-7) M) and dimethyl-BAPTA-AM (10(-7) M) decreased significantly the stimulatory effect of ANG II (10(-9) M) and induced an increase in Na(+)/H(+) exchanger 1 (NHE-1) activity with ANG II (10(-6) M). Immunofluorescence studies indicated that in control situation, the hAT(1) receptor was predominantly expressed in cytosol. However, it was translocated to plasma membrane with ANG II (10(-9) M) and internalized with ANG II (10(-6) M). Losartan (10(-7) M) induced hAT(1) translocation to plasma membrane in all studied groups. Dimethyl-BAPTA-AM (10(-7) M) did not change the effect of ANG II (10(-9) M) on the hAT(1) receptor distribution but induced its accumulation at plasma membrane in cells treated with ANG II (10(-6) M). With ionomycin (10(-6) M), the receptor was accumulated in cytosol. The results indicate that, in MDCK cells, the effect of ANG II on NHE-1 activity is associated with ligand binding to AT(1) receptor and intracellular signaling events related to AT(1) translocation.

Beta-2-microglobulin (B2M) expression in the urinary sediment correlates with clinical markers of kidney disease in patients with type 1 diabetes

PURPOSE After observing variation in the expression of the housekeeping gene B2M in cells of the urinary sediment during a study of candidate genes potentially involved in diabetic kidney disease (DKD), we hypothesized that B2M mRNA expression in the urinary sediment could reflect the presence of DKD. METHODS qPCR was used to quantify B2M mRNA expression in cells of the urinary sediment of 51 type 1 diabetes (T1D) patients (61% women, 33.5 [27.0-39.7] years old, with diabetes duration of 21.0 [15.0-28.0] years and HbA1c of 8.2% [7.3-8.9]; median [interquartile interval]) sorted according to the diabetic nephropathy (DN) stages; 8 focal segmental glomerulosclerosis (FSGS) patients and 10 healthy controls. B2M mRNA expression was also evaluated in human embryonic kidney epithelium-like (HEK-293) cells exposed to 25mM glucose and to albumin in order to mimic, respectively, a diabetic and a proteinuric milieu. RESULTS No differences were found in B2M mRNA expression among healthy controls, FSGS and T1D patients. Nonetheless B2M mRNA expression was higher in the group composed by T1D patients with incipient or overt DN combined with FSGS patients versus T1D patients without DN combined with healthy controls (P=0.0007). B2M mRNA expression was higher in T1D patients with incipient or overt DN versus without DN (P=0.03). B2M mRNA expression positively correlated with albuminuria in the overall T1D population (r=0.43; P=0.01) and negatively correlated with estimated glomerular filtration rate in male T1D patients (r=- 0.57; P=0.01). Increased B2M expression was observed in HEK-293 cells exposed to 25mM glucose and to albumin. CONCLUSIONS Β2M mRNA expression in cells of the urinary sediment is higher in T1D patients with DKD and in patients with FSGS in comparison to healthy subjects, maybe reflecting a tubulointerstitial injury promoted by albumin. Given the proinflammatory nature of B2M, we suggest that this protein contributes to diabetic (and possibly, to non-diabetic) tubulopathy.

Signaling pathways in the biphasic effect of ANG II on Na+/H+ exchanger in T84 cells.

The effect of ANG II on pHi, [Ca2+]i and cell volume was investigated in T84 cells, a cell line originated from colon epithelium, using the probes BCECF-AM, Fluo 4-AM and acridine orange, respectively. The recovery rate of pHi via the Na+/H+ exchanger was examined in the first 2 min following the acidification of pHi with a NH4Cl pulse. In the control situation, the pHi recovery rate was 0.118 ± 0.001 (n = 52) pH units/min and ANG II (10−12M or 10−9M) increased this value (by 106% or 32%, respectively) but ANG II (10−7M) decreased it to 47%. The control [Ca2+]i was 99 ± 4 (n = 45) nM and ANG II increased this value in a dose-dependent manner. The ANG II effects on cell volume were minor and late and should not interfere in the measurements of pHi recovery and [Ca2+]i. To document the signaling pathways in the hormonal effects we used: Staurosporine (a PKC inhibitor), W13 (a calcium-dependent calmodulin antagonist), H89 (a PKA inhibitor) or Econazole (an inhibitor of cytochrome P450 epoxygenase). Our results indicate that the biphasic effect of ANG II on Na+/H+ exchanger is a cAMP-independent mechanism and is the result of: 1) stimulation of the exchanger by PKC signaling pathway activation (at 10−12 – 10−7M ANG II) and by increases of [Ca2+]i in the lower range (at 10−12M ANG II) and 2) inhibition of the exchanger at high [Ca2+]i levels (at 10−9 – 10−7M ANG II) through cytochrome P450 epoxygenase-dependent metabolites of the arachidonic acid signaling pathway.

Thioredoxin interacting protein expression in the urinary sediment associates with renal function decline in type 1 diabetes

ABSTRACT Aims: Thioredoxin interacting protein (TXNIP), an inhibitor of antioxidant thioredoxin (Trx), is upregulated by hyperglycemia and implicated in pathogenesis of diabetes complications. We evaluated mRNA expressions of genes encoding TXNIP and Trx (TXN) in urinary sediment and peripheral blood mononuclear cells (PBMC) of type 1 diabetes (T1D) patients with different degrees of chronic complications. Methods: qPCR was employed to quantify target genes in urinary sediment (n = 55) and PBMC (n = 161) from patients sorted by presence or absence of diabetic nephropathy (DN), retinopathy, peripheral and cardiovascular neuropathy; 26 healthy controls and 13 patients presenting non-diabetic nephropathy (focal and segmental glomerulosclerosis, FSGS) were also included. Results: Regarding the urinary sediment, TXNIP (but not TXN) expression was higher in T1D (p = 0.0023) and FSGS (p = 0.0027) patients versus controls. Expressions of TXNIP and TXN were higher, respectively, in T1D patients with versus without DN (p = 0.032) and in those with estimated glomerular filtration rate (eGFR) < 60 versus ≥60 mL/min/1.73 m2 (p = 0.008). eGFR negatively correlated with TXNIP (p = 0.04, r = −0.28) and TXN (p = 0.04, r = −0.30) expressions. T1D patients who lost ≥5 mL/min/1.73 m2 yearly of eGFR presented higher basal TXNIP expression than those who lost <5 mL/min/1.73 m2 yearly after median follow-up of 24 months. TXNIP (p < 0.0001) and TXN (p = 0.002) expressions in PBMC of T1D patients were significantly higher than in controls but no differences were observed between patients with or without chronic complications. Conclusions: TXNIP and TXN are upregulated in urinary sediment of T1D patients with diabetic kidney disease (DKD), but only TXNIP expression is associated with magnitude of eGFR decline.