Concerted regulation of renal plasma flow and glomerular filtration rate by renal dopamine and NOS I in rats on high salt intake.

Under high sodium intake renal dopamine (DA) increases while NOS I expression in macula densa cells (MD) decreases. To explore whether renal DA and NOS I, linked to natriuresis and to the stability of the tubuloglomerular feedback, respectively, act in concert to regulate renal plasma flow (RPF) and glomerular filtration rate (GFR). Male Wistar rats were studied under a normal sodium intake (NS, NaCl 0.24%) or a high sodium intake (HS, NaCl 1% in drinking water) during the 5 days of the study. For the last two days, the specific D1-like receptor antagonist SCH 23390 (1 mg kg bwt-1 day-1, sc) or a vehicle was administered. HS intake increased natriuresis, diuresis, and urinary DA while it decreased cortical NOS I expression (P < 0.05 vs. NS), Nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) activity in MD (P < 0.001 vs. NS) and cortical nitrates+nitrites (NOx) production (NS 2.04 ± 0.22 vs. HS 1.28 ± 0.10 nmol mg protein-1, P < 0.01). Treatment with SCH 23390 to rats on HS sharply decreased hydroelectrolyte excretion (P < 0.001 vs. HS) while NOS I expression, NADPH-d activity and NOx production increased (P < 0.05 vs. HS for NOS I and P < 0.001 vs. HS for NADPH-d and NOx). SCH 23390 increased RPF and GFR in HS rats (P < 0.01 HS+SCH vs. HS). It did not cause variations in NS rats. Results indicate that when NS intake is shifted to a prolonged high sodium intake, renal DA through the D1R, and NOS I in MD cells act in concert to regulate RPF and GFR to stabilize the delivery of NaCl to the distal nephron.

The Role of Endothelin System in Renal Structure and Function during the Postnatal Development of the Rat Kidney.

Renal development in rodents, unlike in humans, continues during early postnatal period. We aimed to evaluate whether the pharmacological inhibition of Endothelin system during this period affects renal development, both at structural and functional level in male and female rats. Newborn rats were treated orally from postnatal day 1 to 20 with vehicle or bosentan (Actelion, 20 mg/kg/day), a dual endothelin receptor antagonist (ERA). The animals were divided in 4 groups: control males, control females, ERA males and ERA females. At day 21, we evaluated renal function, determined the glomerular number by a maceration method and by morphometric analysis and evaluated possible structural renal alterations by three methods: 〈alpha〉-Smooth muscle actin (α-SMA) immunohistochemistry, Masson's trichrome and Sirius red staining. The pharmacological inhibition of Endothelin system with a dual ERA during the early postnatal period of the rat did not leads to renal damage in the kidneys of male and female rats. However, ERA administration decreased the number of glomeruli, the juxtamedullary filtration surface area and the glomerular filtration rate and increased the proteinuria. These effects could predispose to hypertension or renal diseases in the adulthood. On the other hand, these effects were more pronounced in male rats, suggesting that there are sex differences that could be greater later in life. These results provide evidence that Endothelin has an important role in rat renal postnatal development. However these results do not imply that the same could happen in humans, since human renal development is complete at birth.

Renal response to L-arginine in diabetic rats. A possible link between nitric oxide system and aquaporin-2.

The aim of this study was to evaluate whether L-Arginine (L-Arg) supplementation modifies nitric oxide (NO) system and consequently aquaporin-2 (AQP2) expression in the renal outer medulla of streptozotocin-diabetic rats at an early time point after induction of diabetes. Male Wistar rats were divided in four groups: Control, Diabetic, Diabetic treated with L-Arginine and Control treated with L-Arginine. Nitric oxide synthase (NOS) activity was estimated by [14C] L-citrulline production in homogenates of the renal outer medulla and by NADPH-diaphorase staining in renal outer medullary tubules. Western blot was used to detect the expression of AQP2 and NOS types I and III; real time PCR was used to quantify AQP2 mRNA. The expression of both NOS isoforms, NOS I and NOS III, was decreased in the renal outer medulla of diabetic rats and L-Arg failed to prevent these decreases. However, L-Arg improved NO production, NADPH-diaphorase activity in collecting ducts and other tubular structures, and NOS activity in renal homogenates from diabetic rats. AQP2 protein and mRNA were decreased in the renal outer medulla of diabetic rats and L-Arg administration prevented these decreases. These results suggest that the decreased NOS activity in collecting ducts of the renal outer medulla may cause, at least in part, the decreased expression of AQP2 in this model of diabetes and constitute additional evidence supporting a role for NO in contributing to renal water reabsorption through the modulation of AQP2 expression in this pathological condition. However, we cannot discard that another pathway different from NOS also exists that links L-Arg to AQP2 expression.

Aquaporin-2 promoter is synergistically regulated by nitric oxide and nuclear factor of activated T cells.

BACKGROUND/AIMS: We have previously shown that aquaporin-2 (AQP2) is down-regulated in the renal medulla of rats made hypertensive by chronic inhibition of nitric oxide synthase. It has been shown that AQP2 expression is regulated by the calcineurin/nuclear factor of activated T cells (NFATc). Nitric oxide (NO) regulates the activity of NFATc via c-Jun-N-terminal kinase 2 (JNK2). Therefore, we hypothesized that increases in NO enhance NFATc-mediated up-regulation of AQP2 promoter activity. METHODS: AQP2 mRNA and protein expression were detected in mouse renal papilla. AQP2 promoter luciferase reporter- and NFAT luciferase reporter-transfected MDCK cells were used to determine AQP2 promoter activity and NFATc activity, respectively. Cells were incubated with classic activators and inhibitors of NFATc and the NO pathway. RESULTS: Our results demonstrate that both Ca(2+) and NO have a synergistic effect resulting in an increase in AQP2 mRNA and protein in mouse papilla and activation of the AQP2 promoter in kidney-derived cells. In addition, NO enhances Ca(2+)-induced NFATc activation. The underlying mechanism involves increased NFATc nuclear import and decreased export via protein kinase G-mediated inhibition of JNK1/2. CONCLUSIONS: This is the first study defining novel regulatory roles for NO and NFATc in the control of AQP2, which is an important renal protein.

Expression and activity of SGLT2 in diabetes induced by streptozotocin: relationship with the lipid environment.

BACKGROUND/AIMS: Diabetes mellitus may impact on the regulation of renal Na+-glucose cotransporter type 2 (SGLT2), however, previous studies have yielded conflicting results on the effects of streptozotocin (STZ)-induced diabetes on SGLT-mediated glucose transport. METHODS: Diabetes was induced in male Wistar rats. The studies were performed at 3 (D3), 7 (D7) and 14 (D14) days after a single i.p. injection of STZ. SGLT2 activity was measured using alpha-14C-methyl glucose uptake in brush-border vesicles (BBV) from renal cortex, and SGLT2 expression was assessed by immunoblotting. Phospholipids were quantified by a modification of Fiske-Subarow's method after being separated by thin-layer chromatography. RESULTS: Glucose uptake was reduced in all groups of diabetic rats. SGLT2 expression decreased in D3 and D7. There was a decrease in sphingomyelin (SM) content and an increase in phosphatidylcholine (PC) content in BBV from D14 versus control, without differences in phosphatidylinositol (PI), phosphatidylserine (PS) and phosphatidylethanolamine (PE). CONCLUSION: The downregulation of SGLT2 activity during STZ-induced diabetes may be a protective mechanism to control the excess of circulating glucose and could be a consequence of a decrease in SGLT2 expression in D3 and D7, whereas altered activity of SGLT2 in D14 could be a consequence of changes in membrane lipid composition. However, we cannot discard the possibility that the decrease in SGLT2 activity could be due to a covalent modification of the active site of the protein.

Renal sodium-glucose cotransporter activity and aquaporin-2 expression in rat kidney during chronic nitric oxide synthase inhibition.

BACKGROUND/AIMS: The renal sodium glucose cotransporter (SGLT2) and the water channel aquaporin-2 (AQP2) play a critical role in tubular sodium and water reabsorption and in the regulation of extracellular fluid volume both in physiologic and pathophysiologic conditions. However, there is little information about SGLT2 and AQP2 expression and/or activity in hypertension and there are no reports during hypertension induced by chronic nitric oxide synthase (NOS) inhibition. METHODS: Hypertension was induced in rats by oral administration of N(G)-nitro-L-arginine methyl ester (L-NAME) (20 mg/kg/24 h) for 6 (H6) or 12 (H12) weeks. SGLT2 activity was measured using alpha-(14)C-methylglucose active uptake. The expression level of transporters was assessed by immunohistochemistry and/or immunoblotting. RESULTS: SGLT2 activity was reduced in both H6 and H12; this was due neither to a decrease in SGLT2 expression nor to a change in membrane phospholipid composition. In H6, AQP2 expression diminished only in the inner medulla (IM), while in H12 it diminished in both outer (OM) and IM. This reduced expression of AQP2 may partially account for the increased urinary volume and decreased urinary osmolality in H12, since we obtained a strong correlation between AQP2 expression and these urinary parameters in both OM and IM. CONCLUSION: We propose that in rats in which hypertension is induced by NOS inhibition, SGLT2 activity and AQP2 expression are modified to compensate for the elevated arterial pressure. However, we cannot discount the possibility that the observed changes are due to the decrease in NO production itself.