Martin A. D'Ambrosio

N-acetyl-seryl-aspartyl-lysyl-proline prevents cardiac remodeling and dysfunction induced by galectin-3, a mammalian adhesion/growth-regulatory lectin.

Galectin-3 (Gal-3) is secreted by activated macrophages. In hypertension, Gal-3 is a marker for hypertrophic hearts prone to develop heart failure. Gal-3 infused in pericardial sac leads to cardiac inflammation, remodeling, and dysfunction. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), a naturally occurring tetrapeptide, prevents and reverses inflammation and collagen deposition in the heart in hypertension and heart failure postmyocardial infarction. In the present study, we hypothesize that Ac-SDKP prevents Gal-3-induced cardiac inflammation, remodeling, and dysfunction, and these effects are mediated by the transforming growth factor (TGF)-beta/Smad3 signaling pathway. Adult male rats were divided into four groups and received the following intrapericardial infusion for 4 wk: 1) vehicle (saline, n = 8); 2) Ac-SDKP (800 microg x kg(-1) x day(-1), n = 8); 3) Gal-3 (12 microg/day, n = 7); and 4) Ac-SDKP + Gal-3 (n = 7). Left ventricular ejection fraction, cardiac output, and transmitral velocity were measured by echocardiography; inflammatory cell infiltration, cardiomyocyte hypertrophy, and collagen deposition in the heart by histological and immunohistochemical staining; and TGF-beta expression and Smad3 phosphorylation by Western blot. We found that, in the left ventricle, Gal-3 1) enhanced macrophage and mast cell infiltration, increased cardiac interstitial and perivascular fibrosis, and causes cardiac hypertrophy; 2) increased TGF-beta expression and Smad3 phosphorylation; and 3) decreased negative change in pressure over time response to isoproterenol challenge, ratio of early left ventricular filling phase to atrial contraction phase, and left ventricular ejection fraction. Ac-SDKP partially or completely prevented these effects. We conclude that Ac-SDKP prevents Gal-3-induced cardiac inflammation, fibrosis, hypertrophy, and dysfunction, possibly via inhibition of the TGF-beta/Smad3 signaling pathway.

Enhanced myogenic response in the afferent arteriole of spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHRs) have normal glomerular capillary pressure even though renal perfusion pressure is higher, suggesting that preglomerular vessels exhibit abnormally high resistance. This may be due to increased superoxide (O(2)(-)) production, which contributes to the vasoconstriction in hypertension. We tested the hypothesis that the myogenic response of the afferent arteriole (Af-Art) is exaggerated in SHRs because of increased levels of reactive oxygen species (ROS). Single Af-Arts were microdissected from kidneys of SHRs and Wistar-Kyoto (WKY) rats and microperfused in vitro. When perfusion pressure in the Af-Art was increased stepwise from 60 to 140 mmHg, the luminal diameter decreased by 8.4 + or - 2.9% in WKY Af-Arts but fell by 29.3 + or - 5.6% in SHR Af-Arts. To test whether ROS production is enhanced during myogenic response in SHRs, we measured chloromethyl-dichlorodihydrofluorescein diacetate acetyl ester (CM-H(2)DCFDA) florescence before and after increasing intraluminal pressure from 60 to 140 mmHg. Pressure-induced increases in ROS were fourfold greater in SHR Af-Arts compared with WKY Af-Arts (SHR, 48.0 + or - 2.2%; and WKY, 12.2 + or - 0.3%). To test whether O(2)(-) contributes to the myogenic response in SHRs, either the membrane-permeant O(2)(-) scavenger Tempol or the nox2-based NADPH oxidase (NOX2) inhibitor gp91ds-tat were added to the Af-Art lumen and bath and the myogenic response was tested before and after treatment. Both Tempol (10(-4) M) and gp91ds-tat (10(-5) M) significantly attenuated the pressure-induced constriction in SHR Af-Arts but not in WKY Af-Arts. We conclude that 1) pressure-induced constriction is exaggerated in SHR Af-Arts, 2) NOX2-derived O(2)(-) may contribute to the enhanced myogenic response, and 3) O(2)(-) exerts little influence on the myogenic response under normotensive conditions.

N-Acetyl-Seryl-Aspartyl-Lysyl-Proline Attenuates Renal Injury and Dysfunction in Hypertensive Rats With Reduced Renal Mass. Council for High Blood Pressure Research

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a naturally occurring peptide of which the plasma concentration is increased 4- to 5-fold by angiotensin-converting enzyme inhibitors. We reported previously that, in models of both hypertension and postmyocardial infarction, Ac-SDKP reduces cardiac inflammation and fibrosis. However, it is unknown whether Ac-SDKP can prevent or reverse renal injury and dysfunction in hypertension. In the present study, we tested the hypothesis that, in rats with 5/6 nephrectomy (5/6Nx)-induced hypertension, Ac-SDKP reduces renal damage, albuminuria, and dysfunction by decreasing inflammatory cell infiltration and renal fibrosis and by increasing nephrin protein. Ac-SDKP (800 &mgr;g/kg per day, SC via osmotic minipump) or vehicle was either started 7 days before 5/6Nx (prevention) and continued for 3 weeks or started 3 weeks after 5/6Nx (reversal) and continued for another 3 weeks. Rats with 5/6Nx developed high blood pressure, left ventricular hypertrophy, albuminuria, decreased glomerular filtration rate, and increased macrophage infiltration (inflammation) and renal collagen content (fibrosis). Ac-SDKP did not affect blood pressure or left ventricular hypertrophy in either group; however, it significantly reduced albuminuria, renal inflammation, and fibrosis and improved glomerular filtration rate in both prevention and reversal groups. Moreover, slit diaphragm nephrin protein expression in the glomerular filtration barrier was significantly decreased in hypertensive rats. This effect was partially prevented or reversed by Ac-SDKP. We concluded that Ac-SDKP greatly attenuates albuminuria and renal fibrosis and improves renal function in rats with 5/6Nx. These effects may be related to decreased inflammation (macrophages) and increased nephrin protein.

Prevention of aortic fibrosis by N-acetyl-seryl-aspartyl-lysyl-proline in angiotensin II-induced hypertension

Fibrosis is an important component of large conduit artery disease in hypertension. The endogenous tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) has anti-inflammatory and antifibrotic effects in the heart and kidney. However, it is not known whether Ac-SDKP has an anti-inflammatory and antifibrotic effect on conduit arteries such as the aorta. We hypothesize that in ANG II-induced hypertension Ac-SDKP prevents aortic fibrosis and that this effect is associated with decreased protein kinase C (PKC) activation, leading to reduced oxidative stress and inflammation and a decrease in the profibrotic cytokine transforming growth factor-beta1 (TGF-beta1) and phosphorylation of its second messenger Smad2. To test this hypothesis we used rats with ANG II-induced hypertension and treated them with either vehicle or Ac-SDKP. In this hypertensive model we found an increased collagen deposition and collagen type I and III mRNA expression in the aorta. These changes were associated with increased PKC activation, oxidative stress, intercellular adhesion molecule (ICAM)-1 mRNA expression, and macrophage infiltration. TGF-beta1 expression and Smad2 phosphorylation also increased. Ac-SDKP prevented these effects without decreasing blood pressure or aortic hypertrophy. Ac-SDKP also enhanced expression of inhibitory Smad7. These data indicate that in ANG II-induced hypertension Ac-SDKP has an aortic antifibrotic effect. This effect may be due in part to inhibition of PKC activation, which in turn could reduce oxidative stress, ICAM-1 expression, and macrophage infiltration. Part of the effect of Ac-SDKP could also be due to reduced expression of the profibrotic cytokine TGF-beta1 and inhibition of Smad2 phosphorylation.

Deletion of interleukin-6 prevents cardiac inflammation, fibrosis and dysfunction without affecting blood pressure in angiotensin II-high salt-induced hypertension.

Objective: Inflammation has been proposed as a key component in the development of hypertension and cardiac remodeling associated with different cardiovascular diseases. However, the role of the proinflammatory cytokine interleukin-6 in the chronic stage of hypertension is not well defined. Here, we tested the hypothesis that deletion of interleukin-6 protects against the development of hypertension, cardiac inflammation, fibrosis, remodeling and dysfunction induced by high salt diet and angiotensin II (Ang II). Methods: Male C57BL/6J and interleukin-6-knock out (KO) mice were implanted with telemetry devices for blood pressure (BP) measurements, fed a 4% NaCl diet, and infused with either vehicle or Ang II (90 ng/min per mouse subcutaneously) for 8 weeks. We studied BP and cardiac function by echocardiography at baseline, 4 and 8 weeks. Results: Myocyte cross-sectional area (MCSA), macrophage infiltration, and myocardial fibrosis were also assessed. BP increased similarly in both strains when treated with Ang II and high salt (Ang II-high salt); however, C57BL/6J mice developed a more severe decrease in left ventricle ejection fraction, fibrosis, and macrophage infiltration compared with interleukin-6-KO mice. No differences between strains were observed in MCSA, capillary density and MCSA to capillary density ratio. Conclusion: In conclusion, absence of interleukin -6 did not alter the development of Ang II-high salt-induced hypertension and cardiac hypertrophy, but it prevented the development of cardiac dysfunction, myocardial inflammation, and fibrosis. This indicates that interleukin-6 plays an important role in hypertensive heart damage but not in the development of hypertension.

Connecting tubule glomerular feedback antagonizes tubuloglomerular feedback in vivo

In vitro experiments showed that the connecting tubule (CNT) sends a signal that dilates the afferent arteriole (Af-Art) when Na(+) reabsorption in the CNT lumen increases. We call this process CNT glomerular feedback (CTGF) to differentiate it from tubuloglomerular feedback (TGF), which is a cross talk between the macula densa (MD) and the Af-Art. In TGF, the MD signals the Af-Art to constrict when NaCl transport by the MD is enhanced by increased luminal NaCl. CTGF is mediated by CNT Na(+) transport via epithelial Na(+) channels (ENaC). However, we do not know whether CTGF occurs in vivo or whether it opposes the increase in Af-Art resistance caused by TGF. We hypothesized that CTGF occurs in vivo and opposes TGF. To test our hypothesis, we conducted in vivo micropuncture of individual rat nephrons, measuring stop-flow pressure (P(SF)) as an index of glomerular filtration pressure. To test whether activation of CTGF opposes TGF, we used benzamil to block CNT Na(+) transport and thus CTGF. CTGF inhibition with the ENaC blocker benzamil (1 μM) potentiated the decrease in P(SF) at 40 and 80 nl/min. Next, we tested whether we could augment CTGF by inhibiting NaCl reabsorption in the distal convoluted tubule with hydrochlorothiazide (HCTZ, 1 mM) to enhance NaCl delivery to the CNT. In the presence of HCTZ, benzamil potentiated the decrease in P(SF) at 20, 40, and 80 nl/min. We concluded that in vivo CTGF occurs and opposes the vasoconstrictor effect of TGF.

Possible Mediators of Connecting Tubule Glomerular Feedback

In the renal cortex, the connecting tubule (CNT) returns to the glomerular hilum and contacts the afferent arteriole (Af-Art). Increasing Na delivery to the CNT dilates the Af-Art by activating epithelial Na channels, a process that we call connecting tubule glomerular feedback (CTGF). However, the mediator(s) of CTGF are unknown. We tested the hypothesis that Na reabsorption by the CNT induces release of arachidonic acid metabolites that diffuse to and dilate the Af-Art. Microdissected rabbit Af-Arts and adherent CNTs were simultaneously microperfused. CTGF was measured as the increase in diameter of norepinephrine-preconstricted Af-Arts in response to switching NaCl concentration in the lumen of the CNT from 10 to 80 mmol/L. Under control conditions, CTGF was repeatable and completely reversed norepinephrine-induced vasoconstriction. In the presence of 5,8,11,14-eicosatetraynoic acid, an inhibitor of arachidonic acid metabolism, CTGF was completely blocked (−0.7±0.3 versus 7.3±0.5 &mgr;m), suggesting that arachidonic acid metabolites mediate CTGF. Because both cyclooxygenase-derived prostaglandins and epoxygenase-derived epoxyeicosatrienoic acids are known vasodilatory arachidonic acid metabolites, we tested whether indomethacin or MS-PPOH (a cyclooxygenase and an epoxygenase inhibitor) could block CTGF. Both indomethacin and MS-PPOH partially blocked CTGF (2.3±0.8 versus 6.5±0.5 &mgr;m, and 2.9±0.8 versus 6.6±1.1 &mgr;m, respectively). When combined, they completely blocked CTGF (−0.4±0.3 versus 6.6±1.1 &mgr;m). We confirmed these findings by using the epoxyeicosatrienoic acid antagonist 14,15-EEZE. The combination of indomethacin plus 14,15-EEZE completely abolished CTGF (−0.3±0.2 versus 8.0±1.0 &mgr;m). We conclude that increasing Na concentrations in the CNT stimulate release of prostaglandins and epoxyeicosatrienoic acids, which mediate CTGF.

Heme oxygenase metabolites inhibit tubuloglomerular feedback (TGF)

Tubuloglomerular feedback (TGF) is the mechanism by which the macula densa (MD) senses increases in luminal NaCl concentration and sends a signal to constrict the afferent arteriole (Af-Art). The kidney expresses constitutively heme oxygenase-2 (HO-2) and low levels of HO-1. HOs release carbon monoxide (CO), biliverdin, and free iron. We hypothesized that renal HOs inhibit TGF via release of CO and biliverdin. Rabbit Af-Arts and attached MD were simultaneously microperfused in vitro. The TGF response was determined by measuring Af-Art diameter before and after increasing NaCl in the MD perfusate. When HO activity was inhibited by adding stannous mesoporphyrin (SnMP) to the MD perfusate, the TGF response increased from 2.1+/-0.2 to 4.1+/-0.4 microm (P=0.003, control vs. SnMP, n=7). When a CO-releasing molecule, (CORM-3; 50 microM), was added to the MD perfusate, the TGF response decreased by 41%, from 3.6+/-0.3 to 2.1+/-0.2 microm (P<0.001, control vs. CORM-3, n=12). When CORM-3 at 100 microM was added to the perfusate, it completely blocked the TGF response, from 4.2+/-0.4 to -0.2+/-0.3 microm (P<0.001, control vs. CORM-3, n=6). When biliverdin was added to the perfusate, the TGF response decreased by 79%, from 3.4+/-0.3 to 0.7+/-0.4 microm (P=0.001, control vs. biliverdin, n=6). The effects of SnMP and CORM-3 were not blocked by inhibition of nitric oxide synthase. We concluded that renal HO inhibits TGF probably via release of CO and biliverdin. HO regulation of TGF is a novel mechanism that could lead to a better understanding of the control of renal microcirculation and function.

Aldosterone sensitizes connecting tubule glomerular feedback via the aldosterone receptor GPR30

Increasing Na delivery to epithelial Na channels (ENaC) in the connecting tubule (CNT) dilates the afferent arteriole (Af-Art), a process we call connecting tubule glomerular feedback (CTGF). We hypothesize that aldosterone sensitizes CTGF via a nongenomic mechanism that stimulates CNT ENaC via the aldosterone receptor GPR30. Rabbit Af-Arts and their adherent CNTs were microdissected and simultaneously perfused. Two consecutive CTGF curves were elicited by increasing luminal NaCl in the CNT. During the control period, the concentration of NaCl that elicited a half-maximal response (EC50) was 37.0 ± 2.0 mmol/l; addition of aldosterone 10(-8) mol/l to the CNT lumen caused a left-shift (decrease) in EC50 to 19.3 ± 1.3 mmol/l (P = 0.001 vs. control; n = 6). Neither the transcription inhibitor actinomycin D nor the translation inhibitor cycloheximide prevented the effect of aldosterone (control EC50 = 34.7 ± 1.9 mmol/l; aldosterone+actinomycin D EC50 = 22.6 ± 1.6 mmol/l; P < 0.001 and control EC50 = 32.4 ± 4.3 mmol/l; aldosterone+cycloheximide EC50 = 17.4 ± 3.3 mmol/l; P < 0.001). The aldosterone antagonist eplerenone prevented the sensitization of CTGF by aldosterone (control EC50 = 33.2 ± 1.7 mmol/l; aldosterone+eplerenone EC50 = 33.5 ± 1.3 mmol/l; n = 7). The GPR30 receptor blocker G-36 blocked the sensitization of CTGF by aldosterone (aldosterone EC50 = 16.5 ± 1.9 mmol/l; aldosterone+G-36 EC50 = 29.0 ± 2.1 mmol/l; n = 7; P < 0.001). Finally, we found that the sensitization of CTGF by aldosterone was mediated, at least in part, by the sodium/hydrogen exchanger (NHE). We conclude that aldosterone in the CNT lumen sensitizes CTGF via a nongenomic effect involving GPR30 receptors and NHE. Sensitized CTGF induced by aldosterone may contribute to renal damage by increasing Af-Art dilation and glomerular capillary pressure (glomerular barotrauma).

Mechanism of impaired afferent arteriole myogenic response in Dahl salt-sensitive rats: role of 20-HETE

The afferent arteriole (Af-Art) controls glomerular capillary pressure, an important determinant of glomerular injury. Af-Art myogenic response is mediated by ATP, and ATP signaling is in turn mediated by 20-HETE. Dahl salt-sensitive rats (Dahl SS) have decreased renal 20-HETE production. We hypothesized that Dahl SS have an impaired myogenic response and constrictor response to ATP, due to decreased 20-HETE. Af-Arts from Dahl SS or Dahl salt-resistant rats (Dahl SR) were microdissected and perfused. When myogenic response was induced by increasing Af-Art perfusion pressure from 60 to 140 mmHg, luminal Af-Art diameter decreased in Dahl SR but not in Dahl SS (-3.1 ± 0.8 vs. 0.5 ± 0.8 μm, P < 0.01). The 20-HETE antagonist 20-HEDE (10(-6) M) blocked the myogenic response in Dahl SR but had no effect in Dahl SS. Addition of a subconstrictor concentration of 20-HETE (but not a subconstrictor concentration of norepinephrine) restored the myogenic response in Dahl SS. We then perfused Af-Arts at 60 mmHg and tested the effects of the ATP analog α,β-methylene-ATP (10(-6) M). Maximum ATP-induced constriction was attenuated in Dahl SS compared with Dahl SR (1.5 ± 0.5 vs. 7.4 ± 0.8 μm, P < 0.001). 20-HEDE attenuated ATP-induced Af-Art constriction in Dahl SR but not in Dahl SS, and consequently, ATP-induced constriction was no longer different between strains. In conclusion, Dahl SS have an impaired myogenic response and ATP-induced Af-Art constriction due to a decrease in Af-Art 20-HETE. The impaired myogenic responses may contribute to the nephrosclerosis that develops in Dahl SS.