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. Author manuscript; available in PMC: 2013 Mar 1.
Published in final edited form as: Hypertension. 2012 Feb 6;59(3):599–606. doi: 10.1161/HYPERTENSIONAHA.111.173195

Aldosterone Blunts Tubuloglomerular Feedback by Activating Macula Densa Mineralocorticoid Receptors

Yiling Fu 1, John E Hall 1, Deyin Lu 1, Lin Lin 1, R Davis Manning Jr 1, Liang Cheng 1, Celso E Gomez-Sanchez 3, Luis A Juncos 1,2, Ruisheng Liu 1,2
PMCID: PMC3299000  NIHMSID: NIHMS356470  PMID: 22311906

Abstract

Chronic aldosterone administration increases glomerular filtration rate (GFR) while inhibition of mineralocorticoid receptors (MR) markedly attenuates glomerular hyperfiltration and hypertension associated with primary aldosteronism or obesity. However, the mechanisms by which aldosterone alters GFR regulation are poorly understood. In the present study, we hypothesized that aldosterone suppresses tubuloglomerular feedback (TGF) via activation of macula densa (MD) MR. First, we observed the expression of MR in MD cells isolated by laser capture microdissection (LCM) and by immunofluorescence in rat kidneys. Second, to investigate the effects of aldosterone on TGF in vitro, we microdissected the juxtaglomerular apparatus (JGA) from rabbit kidneys and perfused the afferent arteriole (Af-Art) and distal tubule simultaneously. Under control conditions, TGF was 2.8 ± 0.2µm. In the presence of aldosterone (10−8 mol/L), TGF was reduced by 50%. The effect of aldosterone to attenuate TGF was blocked by the MR antagonist eplerenone (10−5 mol/L). Third, to investigate the effect of aldosterone on TGF in vivo, we performed micropuncture and TGF was determined by maximal changes in stop-flow pressure Psf (ΔPsf) when tubular perfusion rate was increased from 0 to 40 nl/min. Aldosterone (10−7 mol/L) decreased ΔPsf from 10.1 ± 1.4 to 7.7 ± 1.2 mmHg. In the presence of L-NG-monomethyl arginine citrate (L-NMMA, 10−3 mol/L), this effect was blocked. We conclude that MR are expressed in MD cells and can be activated by aldosterone, which increases nitric oxide (NO) production in the MD and blunts the TGF response.

Keywords: tubuloglomerular feedback, aldosterone, mineralocorticoid receptor, nitric oxide

Introduction

Aldosterone-mediated activation of mineralocorticoid receptors (MR) plays a key role in water and electrolyte homeostasis through genomic effects on the colon and renal epithelial cells, especially the principal cells and intercalated cells of the late distal tubules, collecting tubules and collecting ducts. However, aldosterone may also have important effects on other target tissues through genomic as well as non-genomic actions 1.

Aldosterone, at levels that mimic those found in pathophysiological conditions such as primary aldosteronism, alters regulation of renal hemodynamics. Chronic aldosterone infusion, at rates that raise plasma concentration to 5–6 times normal, increased glomerular filtration rate (GFR) and renal plasma flow by approximately 20% 2. Likewise, increased GFR has been observed in patients with primary aldosteronism prior to the development of renal injury and nephron loss 3, 4. Previous studies have also shown that MR antagonism almost completely prevented the glomerular hyperfiltration associated with development of obesity in dogs fed a high fat diet 5. The mechanisms by which hyperaldosteronism increases GFR are poorly understood but are unlikely to be caused solely by volume expansion since aldosterone infusion causes only small increases in extracellular fluid volume due to rapid “escape from sodium retention” 2. Also, increased arterial pressure cannot fully explain the rise in GFR since servo-control of renal perfusion pressure did not prevent glomerular hyperfiltration during chronic aldosterone infusion 2. Normal physiological levels of aldosterone may also influence GFR regulation since MR antagonism decreased GFR by approximately 20% in normal, lean dogs while causing only small effects on arterial pressure and sodium balance 5.

One explanation for the effects of MR activation on GFR is altered tubuloglomerular feedback (TGF). If aldosterone resets TGF, this would tend to increase GFR and permit increased distal NaCl delivery. Such an effect could have important adaptive value since stimulation of sodium reabsorption in the collecting tubules by aldosterone excess, as occurs in primary aldosteronism, would necessitate increased NaCl to these tubular segments in order to maintain NaCl balance if intake remained unchanged. Conversely, inhibition of NaCl reabsorption in distal nephron segments by MR antagonism would require decreased distal delivery of NaCl, possibly through reductions in GFR, in order to achieve salt balance if intake was unaltered. TGF resetting could be an important mechanism for mediating reduced GFR and distal NaCl delivery during MR antagonism.

Despite the potential adaptive value of TGF resetting and glomerular hyperfiltration in offsetting the renal sodium retaining actions of aldosterone and MR activation, this effect has been proposed to contribute to renal injury in patients with primary aldosteronism4 . Although MR activation by aldosterone is recognized to be an important cause of glomerular hyperfiltration, the mechanisms involved are poorly understood. In preliminary studies we found significant MR expression on macula densa (MD) cells suggesting a potential role in TGF. To our knowledge, aldosterone’s direct effects on TGF have not been previously reported. Therefore, in the present study we tested the hypothesis that aldosterone suppresses TGF through MR activation in MD cells. Multiple approaches were used to test this hypothesis, including investigation of the effects of aldosterone on TGF using microdissected and perfused juxtaglomerular apparatus (JGA) in vitro and micropuncture studies in vivo. Our studies reveal that aldosterone reduces TGF by stimulation of nitric oxide (NO) synthesis, and that this effect is mediated through activation of MR.

Methods

All procedures and experiments were approved by the Institutional Animal Care and Use Committee at the University of Mississippi Medical Center. All chemicals were purchased from Sigma (St. Louis, MO) except as indicated.

Microperfusion

Isolation and microperfusion of the rabbit Af-Art and attached MD: We used methods similar to those we described previously 6. (please see http://hyper.ahajournals.org.) After the 30-minute equilibration period, the MD perfusate was switched from 10 to 80mmol/L NaCl at a rate of 40 nL/min, and luminal diameter of the Af-Art perfused at 60 mmHg was measured for 5 minutes. We used the average change in diameter of the Af-Art as our control TGF response. Then the MD perfusate was switched back to 10mmol/L NaCl. To study the effect of aldosterone on regulation of the TGF response in vitro, aldosterone (10−8 mol/L) was added to the tubular perfusate for 15 min and a second TGF response was measured. To determine if the aldosterone-induced TGF alteration could be inhibited by MR blockade, a selective MR antagonist, eplerenone, 10−5 mol/L, was added to the tubular perfusate 30 min before aldosterone administration in separate experiments and the above protocol was repeated.

Micropuncture

Animal preparation: Male, Sprague-Dawley (SD) rats, 250g to 350g, were used. Methods for animal preparation were similar to those previously published7 (please see http://hyper.ahajournals.org.). TGF was determined by maximum changes of Psf when increasing tubular perfusion rate from 0 to 40 nl/min. First, the nephron was perfused with artificial tubular fluid (ATF) containing vehicle for 3–5 min to get the initial measurement of Psf. Next, it was perfused for 10 min with vehicle or aldosterone (10−7 mol/L or 10−8 mol/L). In other experiments, to determine the role of NO in aldosterone-induced TGF alteration, L-NG-monomethyl arginine citrate (L-NMMA, Cayman Chemical, Ann Arbor, MI), 10−3 mol/L, was added into the perfusate and the protocol above was repeated.

Laser capture microdissection and RT-PCR

LCM and RT-PCR were used to isolate MD cells in SD rats, and to measure MR mRNA with methods we have described previously 8 (please see http://hyper.ahajournals.org.).

Immunofluorescence

The MR antibodies were developed and characterized as previously reported9. An antibody for the Na+-K+-2Cl transporter (NKCC2) was a gift from Dr. Pablo Ortiz in Henry Ford Hospital in Detroit. Rat kidney slices were double-stained with MR and NKCC2 primary antibodies and subsequently fluorescent secondary antibodies, and observed under the Nikon microscope. (please see http://hyper.ahajournals.org.).

Cell culture and NO measurement

Experiments were undertaken, using MMDD1 cells, a MD-like cell line (kindly provided by Dr. J. Schnermann, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD) in a manner similar to our previously described studies 8, 10. The cell-permeable fluorescent NO indicator, 4,5-diaminofluorescein diacetate (DAF-2 DA), 10 µmol/L, was used to measure NO before incubation and 15min after incubation with aldosterone(10−8 mol/L) (please see http://hyper.ahajournals.org.).

Statistical analysis

Data were collected as repeated measures over time under different conditions. We tested only the effects of interest, using analysis of variance (ANOVA) for repeated measures and a post-hoc Fisher LSD test or a Student’s paired t-test when appropriate. The changes were considered to be significant if P< 0.05. Data are presented as mean ± SEM.

Results

MR expression in the MD

Figure 1 shows immunofluorescent results and positive staining for MR in the MD with intensity similar to that in thick ascending limbs (TAL) but weaker than in distal tubules or medullary collecting ducts. We also used a NKCC2 antibody to mark MD and TAL. Note that MR is expressed in MD as well as in TAL and distal tubules, whereas NKCC2 is expressed in TAL and MD but not in distal tubules.

Figure 1. MR on MD cells identified by immunofluorescence.

Figure 1

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NKCC2 antibody was used to identify MD and thick ascending limb (green). Specific antibodies were used to determine MR expression (red) on the macula densa cells. A. A merged image of MR staining (red), NKCC2 (green) and bright field (blue). Positive staining of MR was observed in distal tubules, MD cells and thick ascending limbs, but not in proximal tubules and glomeruli; B. High magnification of MD cells; C. Light microscopy of the MD cells. D. A merged image of MR staining in renal medulla. More intense staining of MR in collecting ducts (blue arrows) compared with thick ascending limbs (green arrows).

To confirm MR expression in MD, we also used LCM to microdissect MD cells from frozen rat kidney slides and performed RT-PCR on the captured cells to measure mRNA expression. As shown in Figure 2A, the anatomic location and morphology of MD cells were identified with the LCM microscope before dissection. The cells were then thawed and combined with the membrane (Figure 2B), and cells were removed by lifting the membrane (Figure 2C). Figure 2D indicates that MR mRNA was expressed in microdissected MD cells. The negative control did not contain cDNA. Thus, we confirmed MR expression in the MD with two independent methods.

Figure 2. MR mRNA in MD cells isolated with laser capture microdissection.

Figure 2

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A: MD cells in the frozen and stained kidney slide were identified by their anatomic location and morphology with the LCM microscope. B: During LCM, MD cells were melted and combined to the membrane of a cap. C. After microdissection, the membrane-cell composite was lifted with the cap and removed from the frozen slide. D: MR mRNA expressed in laser-captured MD cells. The negative control was without adding cDNA.

Aldosterone blunts in vitro TGF measured with microperfusion

To test the direct effect of aldosterone on TGF, we microdissected and perfused rabbit Af-Arts and glomeruli with the attached MD. As shown in Figure 3A, when sodium concentration in tubular perfusate was increased from 10 to 80mmol/L, the Af-Art diameter decreased from 18.8 ± 0.3 to 16.0 ± 0.4µm, and the TGF response, as shown in Figure 3B, was 2.8 ± 0.2µm. In the presence of aldosterone, the Af-Art diameter decreased from 19.9 ± 0.5 to 18.5 ± 0.5µm and the TGF response was thus reduced by 50% to only 1.4 ± 0.4µm (n = 10, p<0.05). These data indicated that aldosterone blunted TGF in vitro.

Figure 3. Aldosterone blunts TGF in vitro and MR antagonist attenuates this effect.

Figure 3

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In microdissected and perfused rabbit JGA, TGF was determined by the changes of the diameter of Af-Art when switching the tubular perfusion solution from 10 to 80 mmol/L NaCl. A: Af-Art constricted when tubular NaCl was increased; in the presence of aldosterone, Af-Art constriction was blunted. B: the control TGF was 2.8 ± 0.2 µm; in the presence of aldosterone (10−8 mol/L), TGF was only 1.4 ± 0.4 µm (n = 10, #p<0.01 v.s.10mmol/L, *p<0.01 vs. 10mmol/L+aldosterone, &p<0.05 vs. control). C. The MR antagonist eplerenone (10−5 mol/L) was added to the tubular perfusate 30min before TGF measurements. Af-Art constriction was not altered when tubular NaCl was increased in the presence of eplerenone before and after adding aldosterone into perfusate. D: In the presence of eplerenone, TGF response was 2.8 ± 0.7µm; when aldosterone was added into tubular perfusate, TGF response was 2.4 ± 0.5µm. Aldosterone’s effect on TGF was attenuated by MR inhibition. (n=5, #p<0.01 vs. 10mmol/L +eplerenone, *p<0.01 vs. 10mmol/L +eplerenone+aldosterone)

To determine the role of MR in the acute effect of aldosterone on TGF, eplerenone (10−5 mol/L), a selective MR antagonist, was used. As shown in Figure 3C, eplerenone was added in the tubular perfusate for 30 min and present during the rest of the experiment. When sodium concentration in tubular perfusate was increased from 10 to 80mmol/L, the Af-Art diameter decreased from 18.9 ± 0.4 to 16.1 ± 0.7µm. TGF, as shown in Figure 3D, was 2.8 ± 0.7µm. Then, the tubular perfusate was switched back to 10 mmol/L NaCl and aldosterone was added for 15 min. When we increased tubular NaCl to 80 mmol/L in the presence of aldosterone, the Af-Art diameter decreased from 18.9 ± 0.7 to 16.5 ± 0.3µm. TGF was thus 2.4 ± 0.5µm (n = 5). These data indicate that aldosterone’s effect on TGF was attenuated by MR antagonism, suggesting that the inhibitory effect of aldosterone on TGF was primarily mediated via MR activation.

Aldosterone blunts in vivo TGF measured by micropuncture

We performed micropuncture to test if aldosterone affects the TGF response in vivo. The results of time control experiments are shown in Figure 4A (n = 4 rats, 7 tubules). When the tubular perfusion rate of vehicle was increased from 0 to 40 nl/min, Psf decreased from 38.9 ± 1.3 to 30.1 ± 1.1 mmHg, and the change in Psf (ΔPsf), used as the TGF response, was 8.8 ± 0.9 mmHg. Then we stopped tubular perfusion and waited for the Psf to return to baseline, which was 38.2 ± 1.3mmHg. When we increased tubular perfusion rate to 40nl/min again, Psf decreased to 30.0 ± 1.5mmHg. ΔPsf was 8.1 ± 1.0mmHg. There was no significant difference between the two TGF responses, indicating that the TGF response is reversible and was a suitable control for the following experiments.

Figure 4. Aldosterone blunts TGF in vivo.

Figure 4

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In micropuncture study in SD rats, maximum change of Psf (ΔPsf) when increasing tubular flow from 0 to 40nl/min was used to determine TGF. In the presence of aldosterone (10−7 mol/L) in tubular perfusate, ΔPsf decreased from 10.1 ± 1.4 to 7.7 ± 1.2 mmHg (Figure 5C, n = 4 rats, 9 tubules, p<0.05). There was no difference of ΔPsf when TGF responses were repeated with only vehicle (Figure 5A). Aldosterone at 10−8 mol/L had no effect on TGF response (Figure 5B). Figure 2D shows a representative micropuncture experiment of TGF measurement induced by switching tubular perfusion rate and measured by change of Psf. Blood pressure was stable during measurement. Arrows indicate where Psf was measured.

We tested whether aldosterone had any effect on TGF in vivo by adding aldosterone to the tubular perfusate and measuring TGF. When tubular perfusate was increased from 0 to 40 nl/min with vehicle, Psf decreased from 39.7 ± 2.1 to 31.0 ± 2.9mmHg. The ΔPsf was 9.1±1.0 mmHg. Then we stopped tubular perfusion and waited for the Psf to return to baseline. When we increased tubular perfusate to 40 nl/min in the presence of 10−8 mol/L aldosterone, Psf decreased from 38.4 ± 2.1 to 29.7 ± 2.1mmHg. The ΔPsf was 9.0 ± 0.9 mmHg. There was no significant difference in TGF with and without aldosterone (Figure 4B, n = 4 rats, 7 tubules). Then we increased the concentration of aldosterone to 10−7 mol/L in the above experiments. As shown in Figure 4C, when perfused with vehicle only (control), Psf was reduced from 38.8 ± 1.3 to 28.7±1.9mmHg and ΔPsf was 10.1 ± 1.4 mmHg. In the presence of aldosterone, the Psf was reduced from 39.3 ± 1.6 to 31.5 ± 1.6mmHg and ΔPsf was 7.7 ± 1.2 mmHg (n = 4 rats, 9 tubules, p<0.05). Figure 4D shows a representative experiment demonstrating the changes of stop flow pressure responding to vehicle or aldosterone perfusion. Arrows indicate where Psf was measured. These data suggest that aldosterone blunted the TGF response in vivo.

To determine if NO is involved in aldosterone-induced TGF inhibition, we used L-NMMA, a non-selective NOS inhibitor in the lumen. First, L-NMMA (10−3 mol/L) alone was tested in control experiments. When tubular perfusate was increased from 0 to 40 nl/min with L-NMMA, Psf decreased from 36.8 ± 2.4 to 24.7 ± 2.7mmHg, and ΔPsf was 12.1 ± 1.4 mmHg. In a repeat of control conditions, after Psf returned to baseline, tubular perfusion rate was increased from 0 to 40nl/min and Psf decreased from 36.4 ± 2.3 to 22.9 ± 3.1mmHg. ΔPsf was13.6 ± 1.6 mmHg (Figure 5A, n = 5 rats, 8 tubules, p<0.05). In the next group of experiments, aldosterone, 10−7 mol/L, was added to the tubular perfusate in the presence of L-NMMA. When perfused with L-NMMA alone, Psf decreased from 37.5 ± 1.9 to 24.3 ± 1.7mmHg, and ΔPsf was 13.2 ± 1.4mmHg. In the presence of aldosterone, Psf decreased from 37.5 ± 1.7 to 21.8 ± 1.8mmHg, and ΔPsf was 15.7 ± 1.0mmHg. The delta changes between the 1st and 2nd TGF responses in the absence and presence of aldosterone were not significantly different. As shown in Figure 5B, aldosterone’s effect on TGF was totally blocked when NO was inhibited (n = 4 rats, 7 tubules). We also compared the difference between TGF response to aldosterone alone (Fig 4C) and aldosterone plus L-NMMA (Fig 5B). The difference was significant (p < 0.01). These data suggest that NO was involved in mediating the acute effect of aldosterone on TGF since blockade of NOS totally prevented the attenuation of TGF by aldosterone.

Figure 5. Aldosterone-induced inhibition of TGF is mediated by nitric oxide.

Figure 5

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L-NMMA, 10−3 mol/L, was perfused into tubules to inhibit NO. A. By L-NMMA alone, ΔPsf was decreased from 12.1 ± 1.4 to 13.6±1.6 mmHg (n=5 rats, 8 tubules, #p<0.05). B. In the presence of L-NMMA, the acute effect of aldosterone on TGF was blocked, with ΔPsf decreased from 13.2±1.4 to 15.7±1.0mmHg (n=4 rats, 7 tubules, *p<0.05). C. NO production induced by aldosterone was measured in MMDD1 cells with DAF-2 DA. In basal condition, the rate of increase in NO generation was 40.4 ± 4.3 units/min (n = 16). In the presence of aldosterone (10−8 mol/L), the rate of NO production increased significantly to 644.1 ± 118.5 units/min (n = 16, * p<0.01 vs basal). In time control experiments, the rate of increase in NO generation was not significantly changed (n = 11).

Aldosterone stimulates NO production in cultured MD cells

To test if aldosterone enhances NO generation by the MD cells, we loaded MMDD1 cells with DAF-2 DA to measure NO generation with and without aldosterone. In basal conditions, the rate of NO generation was 40.4 ± 4.3 units/min. After adding aldosterone for 15 min, the rate of NO production increased significantly to 644.1 ± 118.5 units/min (Figure 5C, n = 16, p<0.01 vs basal). In time control experiments only with vehicle, the rate of increase in NO generation was 45.9 ± 5.2 units/min at basal and 53.4 ± 6.1 units/min in 15 min later (n = 11).

Discussion

A novel finding of the present study is that mRNA and protein for MR are expressed in the MD cells. Second, we found that aldosterone blunted the TGF response both in anesthetized rats in vivo and in microperfused JGA in vitro. Third, the MR antagonist, eplerenone, abolished aldosterone-induced TGF inhibition, and fourth, NOS inhibition restored the blunted TGF. Fifth, aldosterone markedly increased NO generation by MMDD1 cells. Taken together these data suggest that aldosterone attenuates TGF by a MR-mediated event resulting from release of NO by the MD.

Previous studies using autoradiographic methods and immunostaining have found MR in distal tubules, connecting and cortical collecting tubules, and in medullary and papillary collecting ducts 11, 12. MR was also found in the thick ascending limb of the loop of Henle using RT-PCR 13 and recently in glomeruli by immunostaining 14. Using highly specific antibodies MR was found in connecting tubules, distal convoluted tubules and cortical collecting tubules in rats, which is consistent with previous studies9, 12. However, we were surprised to find that substantial levels of MR are also expressed in MD cells, an observation that has not, to our knowledge, been previously reported. This finding was confirmed using laser-capture microscopy (LCM). In the past, it has been difficult to acquire sufficient MD cells to perform the necessary biochemical analysis and to study their cellular and molecular signaling mechanisms. The development of LCM, however, provides a tool to isolate and capture MD cells in sufficient numbers to measure mRNAs with single-cell RT-PCR 8. In the present study, we used LCM to acquire MD cells and demonstrated mRNA for MR in the MD. These observations, when combined with our immunohistochemistry data, clearly indicate significant MR expression in MD cells.

Our results also indicate that aldosterone-mediated activation of MR in the MD has a functional role in altering TGF. Using in vitro microperfused JGA as well as in vivo micropuncture experiments we found that aldosterone attenuated TGF by almost 50%. This effect was completely reversed by blocking NO synthesis, indicating that NOS plays a primary role in TGF resetting by aldosterone. To our knowledge, there have been no previous reports of aldosterone’s direct effects on TGF. Only a few studies have been conducted to examine the effects of MR activation on renal hemodynamics. Arima et al showed that acute administration of aldosterone caused constriction in rabbit arterioles and NOS inhibition further augmented this vasoconstriction 15, 16. In contrast, Uhrenholt et al found in renal afferent arterioles a vasodilator effect of aldosterone that was abolished by blockade of NOS 17. Schmidt et al also found in human forearms that aldosterone caused vasodilation and increased blood flow but after administration of L-NMMA forearm blood flow significantly decreased during aldosterone infusion 18. Thus, although there is still controversy concerning the vascular effects of aldosterone, the signaling pathway consistently points to NO, which is consistent with our observation that aldosterone’s rapid effect on TGF is mediated by NO.

To further test whether aldosterone enhances NO generation in the MD, we measured NO generation in MMDD1 cells with a fluorescent dye. We found that aldosterone significantly enhanced NO generation by MMDD1 cells. We recently reported that aldosterone also stimulated superoxide generation in MMDD1 cells10. In that study we excluded the effect of NO by using L-arginine-free solutions. However, in the present study L-arginine was present in solutions used for the experiments with MMDD1. Therefore, enzymes that generate both NO and superoxide should have been intact in the present study. The detection of either NO or superoxide should therefore reflect the net effect of the interaction between the NO and superoxide. A significant increase of NO was detected in MMDD1 cells stimulated by aldosterone, indicating that production of NO exceeded that of superoxide.

Our results also indicate that aldosterone’s effects to reduce TGF responses occur rapidly, within 10 minutes. When aldosterone functions through a genomic pathway, it couples with MR and this product functions as a transcription factor. However, rapid non-genomic effects of aldosterone that do not require transcription or protein synthesis have also been reported in various tissues such as the heart, colon, renal tubule, and vascular smooth muscle 1. As early as 1958, the rapid action of aldosterone on urinary electrolyte excretion was reported to occur in 5 minutes 19. In rat cortical collecting tubules an aldosterone-induced ion influx occurred within 30 minutes 20. A series of studies demonstrated that aldosterone acts within minutes to alter cellular pH and plasma membrane potassium conductance in various cell preparations by rapidly increasing net entry of Ca2+, activating membrane Na+/H+ exchanger, and modulating K+ channel activity21–23. These effects were shown to be spironolactone-insensitive and blocked by protein kinase C inhibitors 24. The involvement of ERK 1/2 in the rapid non-genomic action was shown in MDCK 25 and in the renal medullary thick ascending limb of the loop of Henle 26.

The concentration of aldosterone required for rapid non-genomic effects has varied from subnanomolar up to 10nM 27, 28. In the present study, the rapid effect of aldosterone occurred within 10 minutes in microperfused JGA and in micropuncture studies suggesting that a non-genomic pathway may be involved. For the in vivo micropuncture studies, a higher dose of aldosterone was required to alter TGF. The reason for this is not clear but may be due to the micropuncture technique used. For example, effective concentrations of NOS inhibitors needed to inhibit TGF in renal micropuncture studies 7, 29 are usually higher than used in JGA microperfusion 30. We cannot measure the actual concentration at the MD under current experimental settings, but assume that the aldosterone concentration that actually reaches the MD cells is considerably less than the concentration in the proximal tubule when using micropuncture methods. Overall, our results suggest a novel mechanism by which aldosterone may influence GFR regulation through a non-genomic pathway.

In the present study we found that eplerenone completely blocked the effect of aldosterone on TGF, indicating that the effects are mediated through activation of MR. In agreement with our observations, the acute effects of aldosterone on small resistance mesenteric vessels 31 and renal afferent arterioles 17 are also mediated by activation of MR. However, it also has been reported that acute infusion of aldosterrone had no effect on GFR both in humans 32 and animals 33. These data do not necessarily contradict our findings in the present study. We found that aldosterone suppressed TGF and dilated Af-Art through the MR of the MD. Arima 15, 16 reported that aldosterone directly constricted the Af-Art. Therefore, the effect of acute injection of aldosterone may reflect the balance of effects on the MD which would tend to inhibit TGF and raise GFR and direct vasoconstrictor effects on Af-Art which would tend to decrease GFR. The net acute effect of aldosterone could be unchanged GFR as a result of these offsetting effects. In secondary hyperaldosteronism which is often associated with high levels of Ang II which enhances TGF, the increased levels of aldosterone may serve to buffer the effect of Ang II on TGF and GFR.

Perspectives

Our observations provide a potential mechanism by which high levels of aldosterone may cause glomerular hyperfiltration, a risk factor for renal injury and chronic kidney disease in patients with primary aldosteronism as well as in obese subjects who have increased renal MR activation 2, 34. Our results also indicate that the rapid effects of aldosterone on TGF are mediated through synthesis of NO at the MD and may provide a potential target for treating obesity hypertension and associated chronic kidney disease. Further studies are needed to elucidate the physiological and pathophysiological significance of this novel effect of aldosterone on TGF.

Supplementary Material

1

Acknowledgments

Sources of Funding

This work was supported by National Institutes of Health Grants R01-HL086767(to R. L.), PO1-HL 051971 (to J.E.H.) and HL27255(to C.E.G) and American Heart Association Postdoctoral Fellowship Award 11POST7750023 (to Y. F.).

Footnotes

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Disclosure

No conflicts of interest are declared by the author(s).

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