Ac-SDKP decreases mortality and cardiac rupture after acute myocardial infarction.

The natural peptide N-Acetyl-Seryl-Aspartyl-Lysyl-Proline (Ac-SDKP) decreases inflammation in chronic diseases such as hypertension and heart failure. However, Ac-SDKP effects on acute inflammatory responses during myocardial infarction (MI) are unknown. During the first 72 hours post-MI, neutrophils, M1 macrophages (pro-inflammatory), and M2 macrophages (pro-resolution) and release of myeloperoxidase (MPO) and matrix metalloproteinases (MMP) are involved in cardiac rupture. We hypothesized that in the acute stage of MI, Ac-SDKP decreases the incidence of cardiac rupture and mortality by preventing immune cell infiltration as well as by decreasing MPO and MMP expression. MI was induced by ligating the left descending coronary artery in C57BL/6 mice. Vehicle or Ac-SDKP (1.6 mg/kg/d) was infused via osmotic minipump. Cardiac immune cell infiltration was assessed by flow cytometry, cardiac MPO and MMP levels were measured at 24-48 hrs post-MI. Cardiac rupture and mortality incidence were determined at 7 days post-MI. In infarcted mice, Ac-SDKP significantly decreased cardiac rupture incidence from 51.0% (26 of 51 animals) to 27.3% (12 of 44) and mortality from 56.9% (29 of 51) to 31.8% (14 of 44). Ac-SDKP reduced M1 macrophages in cardiac tissue after MI, without affecting M2 macrophages and neutrophils. Ac-SDKP decreased MMP-9 activation in infarcted hearts with no changes on MPO expression. Ac-SDKP prevents cardiac rupture and decreases mortality post-acute MI. These protective effects of Ac-SDKP are associated with decreased pro-inflammatory M1 macrophage infiltration and MMP-9 activation.

Cardiac-deleterious role of galectin-3 in chronic angiotensin II-induced hypertension.

Galectin-3 (Gal-3), a member of the ß-galactoside lectin family, has an important role in immune regulation. In hypertensive rats and heart failure patients, Gal-3 is considered a marker for an unfavorable prognosis. Nevertheless, the role and mechanism of Gal-3 action in hypertension-induced target organ damage are unknown. We hypothesized that, in angiotensin II (ANG II)-induced hypertension, genetic deletion of Gal-3 prevents left ventricular (LV) adverse remodeling and LV dysfunction by reducing the innate immune responses and myocardial fibrosis. To induce hypertension, male C57BL/6J and Gal-3 knockout (KO) mice were infused with ANG II (3 µg·min-1·kg-1 sc) for 8 wk. We assessed: 1) systolic blood pressure by plethysmography, 2) LV function and remodeling by echocardiography, 3) myocardial fibrosis by histology, 4) cardiac CD68+ macrophage infiltration by histology, 5) ICAM-1 and VCAM-1 expression by Western blotting, 6) plasma cytokines, including interleukin-6 (IL-6), by enzyme-linked immunosorbent assay, and 7) regulatory T (Treg) cells by flow cytometry as detected by their combined expression of CD4, CD25, and FOXP3. Systolic blood pressure and cardiac hypertrophy increased similarly in both mouse strains when infused with ANG II. However, hypertensive C57BL/6J mice suffered impaired ejection and shortening fractions. In these mice, the extent of myocardial fibrosis and macrophage infiltration was greater in histological sections, and cardiac ICAM-1, as well as plasma IL-6, expression was higher as assessed by Western blotting. However, all these parameters were blunted in Gal-3 KO mice. Hypertensive Gal-3 KO mice also had a higher number of splenic Treg lymphocytes. In conclusion, in ANG II-induced hypertension, genetic deletion of Gal-3 prevented LV dysfunction without affecting blood pressure or LV hypertrophy. This study indicates that the ANG II effects are, in part, mediated or triggered by Gal-3 together with the related intercellular signaling (ICAM-1 and IL-6), leading to cardiac inflammation and fibrosis.

One-Lung Ventilation: A Pediatric Simulation Case for Anesthesiology Residents.

INTRODUCTION: This activity is designed for midlevel and senior anesthesia trainees to experience the complexities of one-lung ventilation in pediatrics in a high-fidelity simulated environment. With the use of video-assisted thoracoscopic surgery (VATS) becoming increasingly common in pediatrics, we identified this area as an opportunity for the development of a dedicated educational simulation activity. METHODS: Our simulated patient is a 3-year-old girl with empyema presenting for decortication via VATS who subsequently develops hypoxemia. The main challenges for the trainee include airway selection and insertion, lung isolation with fiber optic confirmation, and management of hypoxemia in the setting of one-lung ventilation. A pediatric medical simulator suitable for practicing resuscitation is required, and a tracheobronchial tree model is highly desirable. Basic knowledge of thoracic and pediatric anesthesia is required, but specific experience with pediatric lung isolation is not. RESULTS: Learners who experienced the content of this simulation expressed a strong sentiment of value. All pilot trainees were surveyed and indicated they either agree or strongly agree (4 or 5, respectively, on a 5-point Likert scale) that "This simulation enhanced my understanding of how to select lung isolation devices for pediatric patients" and "This simulation enhanced my understanding of how to manage hypoxia in context in one-lung ventilation." Comments were overall positive, including "I am better prepared to manage pediatric one lung ventilation cases." DISCUSSION: At the University of Iowa, this activity is part of a core curriculum of simulation training that resident physicians in anesthesiology experience during their training. It functions as a tool for education, evaluation, and self-identification of weaknesses in the learner's knowledge base as it relates to the perioperative management of pediatric one-lung ventilation, as well as for reinforcing material learned in the classroom and operating room. Numerous anesthesiology residents and faculty have pilot-tested this simulation, and necessary modifications have been made based on their feedback.

Renal protective effect of N-acetyl-seryl-aspartyl-lysyl-proline in dahl salt-sensitive rats.

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a natural tetrapeptide with anti-inflammatory and antifibrotic properties. Its effect on salt-sensitive (SS) hypertension is unknown. We hypothesized that in Dahl SS rats on high-salt (HS) diet, Ac-SDKP prevents loss of nephrin expression and renal immune cell infiltration, leading to a decrease in albuminuria, renal inflammation, fibrosis, and glomerulosclerosis. To test this, Dahl SS rats and consomic SS13BN controls were fed either a low-salt (0.23% NaCl) or HS (4% NaCl) diet and treated for 6 weeks with vehicle or Ac-SDKP at either low or high dose (800 or 1600 µg/kg per day, respectively). HS increased systolic blood pressure in SS rats (HS+vehicle, 186±5 versus low salt+vehicle, 141±3 mm Hg; P<0.005) but not in SS13BN rats. Ac-SDKP did not affect blood pressure. Compared with low salt, HS-induced albuminuria, renal inflammation, fibrosis, and glomerulosclerosis in both strains, but the damages were higher in SS than in SS13BN. Interestingly, in SS13BN rats, Ac-SDKP prevented albuminuria induced by HS (HS+vehicle, 44±8 versus HS+low Ac-SDKP, 24±3 or HS+high Ac-SDKP, 8±1 mg/24 h; P<0.05), whereas in SS rats, only high Ac-SDKP dose significantly attenuated albuminuria (HS+vehicle, 94±10 versus HS+high Ac-SDKP, 57±7 mg/24 h; P<0.05). In both strains, Ac-SDKP prevented HS-induced inflammation, interstitial fibrosis, and glomerulosclerosis. In summary, in SS rats on HS diet, at low and high doses, Ac-SDKP prevented renal damage without affecting the blood pressure. Only the high dose of Ac-SDKP attenuated HS-induced albuminuria. Conversely, in SS13BN rats, both doses of Ac-SDKP prevented HS-induced renal damage and albuminuria.

N-Acetyl-Seryl-Aspartyl-Lysyl-Proline: mechanisms of renal protection in mouse model of systemic lupus erythematosus.

Systemic lupus erythematosus is an autoimmune disease characterized by the development of auto antibodies against a variety of self-antigens and deposition of immune complexes that lead to inflammation, fibrosis, and end-organ damage. Up to 60% of lupus patients develop nephritis and renal dysfunction leading to kidney failure. N-acetyl-seryl-aspartyl-lysyl-proline, i.e., Ac-SDKP, is a natural tetrapeptide that in hypertension prevents inflammation and fibrosis in heart, kidney, and vasculature. In experimental autoimmune myocarditis, Ac-SDKP prevents cardiac dysfunction by decreasing innate and adaptive immunity. It has also been reported that Ac-SDKP ameliorates lupus nephritis in mice. We hypothesize that Ac-SDKP prevents lupus nephritis in mice by decreasing complement C5-9, proinflammatory cytokines, and immune cell infiltration. Lupus mice treated with Ac-SDKP for 20 wk had significantly lower renal levels of macrophage and T cell infiltration and proinflammatory chemokine/cytokines. In addition, our data demonstrate for the first time that in lupus mouse Ac-SDKP prevented the increase in complement C5-9, RANTES, MCP-5, and ICAM-1 kidney expression and it prevented the decline of glomerular filtration rate. Ac-SDKP-treated lupus mice had a significant improvement in renal function and lower levels of glomerular damage. Ac-SDKP had no effect on the production of autoantibodies. The protective Ac-SDKP effect is most likely achieved by targeting the expression of proinflammatory chemokines/cytokines, ICAM-1, and immune cell infiltration in the kidney, either directly or via C5-9 proinflammatory arm of complement system.

Tubuloglomerular and connecting tubuloglomerular feedback during inhibition of various Na transporters in the nephron.

Afferent (Af-Art) and efferent arterioles resistance regulate glomerular capillary pressure. The nephron regulates Af-Art resistance via: 1) vasoconstrictor tubuloglomerular feedback (TGF), initiated in the macula densa via Na-K-2Cl cotransporters (NKCC2) and 2) vasodilator connecting tubuloglomerular feedback (CTGF), initiated in connecting tubules via epithelial Na channels (ENaC). Furosemide inhibits NKCC2 and TGF. Benzamil inhibits ENaC and CTGF. In vitro, CTGF dilates preconstricted Af-Arts. In vivo, benzamil decreases stop-flow pressure (PSF), suggesting that CTGF antagonizes TGF; however, even when TGF is blocked, CTGF does not increase PSF, suggesting there is another mechanism antagonizing CTGF. We hypothesize that in addition to NKCC2, activation of Na/H exchanger (NHE) antagonizes CTGF, and when both are blocked CTGF dilates Af-Arts and this effect is blocked by a CTGF inhibitor benzamil. Using micropuncture, we studied the effects of transport inhibitors on TGF responses by measuring PSF while increasing nephron perfusion from 0 to 40 nl/min. Control TGF response (-7.9 ± 0.2 mmHg) was blocked by furosemide (-0.4 ± 0.2 mmHg; P < 0.001). Benzamil restored TGF in the presence of furosemide (furosemide: -0.2 ± 0.1 vs. furosemide+benzamil: -4.3 ± 0.3 mmHg; P < 0.001). With furosemide and NHE inhibitor, dimethylamiloride (DMA), increase in tubular flow increased PSF (furosemide+DMA: 2.7 ± 0.5 mmHg, n = 6), and benzamil blocked this (furosemide+DMA+benzamil: -1.1 ± 0.2 mmHg; P < 0.01, n = 6). We conclude that NHE in the nephron decreases PSF (Af-Art constriction) when NKCC2 and ENaC are inhibited, suggesting that in the absence of NKCC2, NHE causes a TGF response and that CTGF dilates the Af-Art when TGF is blocked with NKCC2 and NHE inhibitors.

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.

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,ß-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.

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).

Prostaglandin E2 mediates connecting tubule glomerular feedback.

Connecting tubule glomerular feedback (CTGF) is a mechanism in which Na reabsorption in the connecting tubule (CNT) causes afferent arteriole (Af-Art) dilation. CTGF is mediated by eicosanoids, including prostaglandins and epoxyeicosatrienoic acids; however, their exact nature and source remain unknown. We hypothesized that during CTGF, the CNT releases prostaglandin E2, which binds its type 4 receptor (EP4) and dilates the Af-Art. Rabbit Af-Arts with the adherent CNT intact were microdissected, perfused, and preconstricted with norepinephrine. CTGF was elicited by increasing luminal NaCl in the CNT from 10 to 80 mmol/L. We induced CTGF with or without the EP4 receptor blocker ONO-AE3-208 added to the bath in the presence of the epoxyeicosatrienoic acid synthesis inhibitor MS-PPOH. ONO-AE3-208 abolished CTGF (control, 9.4 ± 0.5; MS-PPOH+ONO-AE3-208, -0.6 ± 0.2 µm; P<0.001; n=6). To confirm these results, we used a different, specific EP4 blocker, L161982 (10(-5) mol/L), that also abolished CTGF (control, 8.5 ± 0.9; MS-PPOH+L161982, 0.8 ± 0.4 µm; P<0.001; n=6). To confirm that the eicosanoids that mediate CTGF are released from the CNT rather than the Af-Art, we first disrupted the Af-Art endothelium with an antibody and complement. Endothelial disruption did not affect CTGF (7.9 ± 0.9 versus 8.6 ± 0.6 µm; P=NS; n=7). We then added arachidonic acid to the lumen of the CNT while maintaining zero NaCl in the perfusate. Arachidonic acid caused dose-dependent dilation of the attached Af-Art (from 8.6 ± 1.2 to 15.3 ± 0.7 µm; P<0.001; n=6), and this effect was blocked by ONO-AE3-208 (10(-7) mol/L). We conclude that during CTGF, the CNT releases prostaglandin E2, which acts on EP4 on the Af-Art inducing endothelium-independent dilation.

Connecting tubule glomerular feedback in hypertension.

In Dahl salt-sensitive rats (Dahl SS), glomerular capillary pressure increases in response to high salt intake and this is accompanied by significant glomerular injury compared with spontaneously hypertensive rats with similar blood pressure. Glomerular capillary pressure is controlled mainly by afferent arteriolar resistance, which is regulated by the vasoconstrictor tubule glomerular feedback (TGF) and the vasodilator connecting TGF (CTGF). We hypothesized that Dahl SS have a decreased TGF response and enhanced TGF resetting compared with spontaneously hypertensive rats, and that these differences are attributable in part to an increase in CTGF. In vivo, using micropuncture we measured stop-flow pressure (a surrogate of glomerular capillary pressure). TGF was calculated as the maximal decrease in stop-flow pressure caused by increasing nephron perfusion, TGF resetting as the attenuation in TGF induced by high salt diet, and CTGF as the difference in TGF response before and during CTGF inhibition with benzamil. Compared with spontaneously hypertensive rats, Dahl SS had (1) lower TGF responses in normal (6.6±0.1 versus 11.0±0.2 mm Hg; P<0.001) and high-salt diets (3.3±0.1 versus 10.1±0.3 mm Hg; P<0.001), (2) greater TGF resetting (3.3±0.1 versus 1.0±0.3 mm Hg; P<0.001), and (3) greater CTGF (3.4±0.4 versus 1.2±0.1 mm Hg; P<0.001). We conclude that Dahl SS have lower TGF and greater CTGF than spontaneously hypertensive rats, and that CTGF antagonizes TGF. Furthermore, CTGF is enhanced by a high-salt diet and contributes significantly to TGF resetting. Our findings may explain in part the increase in vasodilatation, glomerular capillary pressure, and glomerular damage in SS hypertension during high salt intake.

Mechanism of inhibition of tubuloglomerular feedback by CO and cGMP.

Tubuloglomerular feedback (TGF) is a mechanism that senses NaCl in the macula densa (MD) and causes constriction of the afferent arteriole. CO, either endogenous or exogenous, inhibits TGF at least in part via cGMP. We hypothesize that CO in the MD, acting via both cGMP-dependent and -independent mechanisms, attenuates TGF by acting downstream from depolarization and calcium entry into the MD cells. In vitro, microdissected rabbit afferent arterioles and their MD were simultaneously perfused and TGF was measured as the decrease in afferent arteriole diameter. MD depolarization was induced with ionophores, while adding the CO-releasing molecule-3 to the MD perfusate at nontoxic concentrations. CO-releasing molecule-3 blunted depolarization-induced TGF at 50 µmol/L, from 3.6±0.4 to 2.5±0.4 µm (P<0.01), and abolished it at 100 µmol/L, to 0.1±0.1 µm (P<0.001; n=6). When cGMP generation was blocked by guanylyl cyclase inhibitor LY83583 added to the MD, CO-releasing molecule-3 no longer affected depolarization-induced TGF at 50 µmol/L (2.9±0.4 versus 3.0±0.4 µm) but partially inhibited TGF at 100 µmol/L (to 1.3±0.2 µm; P<0.05; n=9). Experiments using eicosatetraynoic acid and indomethacin suggest arachidonic acid metabolites do not mediate the cGMP-independent effect of CO. We then added the calcium ionophore A23187 to the MD, which caused TGF (4.1±0.6 µmol/L); A23187-induced TGF was inhibited by CO-releasing molecule-3 at 50 µmol/L (1.9±0.6 µmol/L; P<0.01) and 100 µmol/L (0.2±0.5 µmol/L; P<0.001; n=6). We conclude that CO inhibits TGF acting downstream from depolarization and calcium entry, acting via cGMP at low concentrations, but additional mechanisms of action may be involved at higher concentrations.

Mechanisms of carbon monoxide attenuation of tubuloglomerular feedback.

Carbon monoxide (CO) is a physiological messenger with diverse functions in the kidney, including controlling afferent arteriole tone both directly and via tubuloglomerular feedback (TGF). We have reported that CO attenuates TGF, but the mechanisms underlying this effect remain unknown. We hypothesized that CO, acting via cGMP, cGMP-dependent protein kinase, and cGMP-stimulated phosphodiesterase 2, reduces cAMP in the macula densa, leading to TGF attenuation. In vitro, microdissected rabbit afferent arterioles and their attached macula densa were simultaneously perfused. TGF was measured as the decrease in afferent arteriole diameter elicited by switching macula densa NaCl from 10 to 80 mmol/L. Adding a CO-releasing molecule (CORM-3, 5 × 10(-5) mol/L) to the macula densa blunted TGF from 3.3 ± 0.3 to 2.0 ± 0.3 µm (P<0.001). The guanylate cyclase inhibitor LY-83583 (10(-6) mol/L) enhanced TGF (5.8 ± 0.6 µm; P<0.001 versus control) and prevented the effect of CORM-3 on TGF (LY-83583+CORM-3, 5.5 ± 0.3 µm). Similarly, the cGMP-dependent protein kinase inhibitor KT-5823 (2 × 10(-6) mol/L) enhanced TGF and prevented the effect of CORM-3 on TGF (KT-5823, 6.0 ± 0.7 µm; KT-5823+CORM-3, 5.9 ± 0.8 µm). However, the phosphodiesterase 2 inhibitor BAY-60-7550 (10(-6) mol/L) did not prevent the effect of CORM-3 on TGF (BAY-60-7550, 4.07 ± 0.31 µm; BAY-60-7550+CORM-3, 1.84 ± 0.31 µm; P<0.001). Finally, the degradation-resistant cAMP analog dibutyryl-cAMP (10(-3) mol/L) prevented the attenuation of TGF by CORM-3 (dibutyryl-cAMP, 4.6 ± 0.5 µm; dibutyryl-cAMP+CORM-3, 5.0 ± 0.6 µm). We conclude that CO attenuates TGF by reducing cAMP via a cGMP-dependent pathway mediated by cGMP-dependent protein kinase rather than phosphodiesterase 2. Our results will lead to a better understanding of the mechanisms that control the renal microcirculation.

Mechanisms of angiotensin II-enhanced connecting tubule glomerular feedback.

Increasing Na delivery to the connecting tubule (CNT) causes afferent arteriole (Af-Art) dilation, a process we call CNT glomerular feedback (CTGF). Angiotensin II (ANG II) in the CNT lumen enhances CTGF via PKC. We hypothesized that luminal ANG II stimulates CTGF via activation of protein kinase C (PKC), NADPH oxidase 2 (NOX2), and enhanced production of superoxide (O(2)(-)). Rabbit Af-Arts and adherent CNTs were microdissected and microperfused in vitro. Dilation of the Af-Art was induced by increasing luminal CNT NaCl from 0 to 5, 10, 30, 45, and 80 mM, and the concentration of NaCl that elicited a half-maximal response (EC(50)) was calculated. Compared with vehicle, adding ANG II (10(-9) M) to the CNT lumen reduced EC(50) from 37 ± 3 to 14 ± 1 mM (P < 0.001), indicating ANG II potentiates CTGF. In the presence of ANG II, the O(2)(-) scavenger tempol (10(-4) M) increased EC(50) from 20 ± 4 to 41 ± 3 mM (P < 0.01), the NOX inhibitor apocynin (10(-5) M) increased EC(50) from 17 ± 2 to 39 ± 4 mM (P < 0.01), and the specific NOX2 inhibitor gp91ds-tat (10(-5) M) increased EC(50) from 19 ± 2 to 34 ± 2 mM (P < 0.01). However, tempol, apocynin, and gp91ds-tat had no effect on CTGF in the absence of ANG II. Compared with vehicle, the PKC activator PMA (2 × 10(-7) M) decreased EC(50) from 35 ± 2 to 14 ± 1 (P < 0.001). In the presence of PMA, tempol increased EC(50) from 14 ± 2 to 35 ± 2 mM (P < 0.01). We conclude the PKC/NOX2/O(2)(-) pathway mediates the enhancement of CTGF by luminal ANG II but it does not participate in CTGF in the absence of ANG II.

Connecting tubule glomerular feedback mediates acute tubuloglomerular feedback resetting.

Tubuloglomerular feedback (TGF) and connecting tubule glomerular feedback (CTGF) are mechanisms that control afferent arteriole (Af-Art) tone. TGF, initiated by increased NaCl at the macula densa, causes Af-Art constriction. Prolonged activation of TGF leads to an attenuation or "resetting" of its constrictor effect. The mechanisms of TGF resetting remain incompletely understood. CTGF is initiated by increased NaCl in the connecting tubule and Na(+) entry via epithelial sodium channels (ENaC). Contrary to TGF, CTGF dilates the Af-Art. Here, we hypothesize that CTGF, in part, mediates TGF resetting. We performed micropuncture of individual rat nephrons while measuring stop-flow pressure (P(SF)), an index of glomerular filtration pressure and Af-Art tone. Increases in Af-Art tone cause P(SF) to decrease. TGF responses, measured as the decrease in P(SF) induced by switching late proximal tubule perfusion from 5 to 40 nl/min, were elicited before and after a 30-min period of sustained perfusion of the late proximal tubule at a rate of 40 nl/min designed to induce TGF resetting. TGF responses were 7.3 ± 0.3 and 4.9 ± 0.2 mmHg before and after resetting was induced (P < 0.001, n = 6). When CTGF was inhibited with the ENaC blocker benzamil (1 µM), TGF responses were 9.5 ± 0.3 and 8.8 ± 0.6 mmHg (NS, n = 6), thus resetting was abolished. In the presence of the carbonic anhydrase inhibitor acetazolamide (10 mM), TGF responses were 8.8 ± 0.6 and 3.3 ± 0.4 mmHg before and after resetting (P < 0.001, n = 6). With both acetazolamide and benzamil, TGF responses were 10.4 ± 0.2 and 8.4 ± 0.5 mmHg (P < 0.01, n = 6), thus resetting was attenuated. We conclude that CTGF, in part, mediates acutely induced TGF resetting.

Angiotensin II-induced dilated cardiomyopathy in Balb/c but not C57BL/6J mice.

Balb/c mice, which are T-helper lymphocyte 2 (Th2) responders, are highly susceptible to infectious and non-infectious heart diseases, whereas C57BL/6 mice (Th1 responders) are not. Angiotensin II (Ang II) is not only a vasopressor but also a pro-inflammatory factor that leads to cardiac hypertrophy, fibrosis and dysfunction. We hypothesized that Ang II exacerbates cardiac damage in Balb/c but not in C57BL/6 mice even though both strains have a similar level of hypertension. Twelve-week-old male C57BL/6J and Balb/c mice received either vehicle or Ang II (1.4 mg kg(-1) day(-1), s.c. via osmotic minipump) for 8 weeks. At baseline, Balb/c mice exhibited the following: (1) a lower heart rate; (2) an enlarged left ventricular chamber; (3) a lower ejection fraction and shortening fraction; and (4) twice the left ventricular collagen deposition of age-matched C57BL/6J mice. Angiotensin II raised systolic blood pressure (to ∼150 mmHg) and induced cardiomyocyte hypertrophy in a similar manner in both strains. While C57BL/6J mice developed compensatory concentric hypertrophy and fibrosis in response to Ang II, Balb/c mice demonstrated severe left ventricular chamber dilatation, wall thinning and fibrosis, leading to congestive heart failure as evidenced by dramatically decreased ejection fraction and lung congestion (significant increase in lung weight), which are both characteristic of dilated cardiomyopathy. Our study suggests that the Th phenotype plays an active role in cardiac remodelling and function both in basal conditions and in hypertension. Angiotensin II-induced dilated cardiomyopathy in Balb/c mice is an ideal animal model for studying the impact of the adaptive immune system on cardiac remodelling and function and for testing strategies to prevent or treat hypertension-associated heart failure.

Heme oxygenase metabolites inhibit tubuloglomerular feedback in vivo.

Tubuloglomerular feedback (TGF) is a renal autoregulatory mechanism that constricts the afferent arteriole in response to increases in distal NaCl. Heme oxygenases (HO-1 and HO-2) release carbon monoxide (CO) and biliverdin, which may help control renal function. We showed in vitro that HO products inhibit TGF; however, we do not know whether this also occurs in vivo or the mechanism(s) involved. We hypothesized that in vivo HO-1 and HO-2 in the nephron inhibit TGF via release of CO and biliverdin. We first performed laser capture microdissection followed by real-time PCR and found that both HO-1 and HO-2 are expressed in the macula densa. We next performed micropuncture experiments in vivo on individual rat nephrons, adding different compounds to the perfusate, and found that an HO inhibitor, stannous mesoporphyrin (SnMP), potentiated TGF (P < 0.05, SnMP vs. control). The CO-releasing molecule (CORM)-3 partially inhibited TGF at 50 µmol/l (P < 0.01, CORM-3 vs. control) and blocked it completely at higher doses. A soluble guanylyl cyclase (sGC) inhibitor, LY83583, blocked the inhibitory effect of CORM-3 on TGF. Biliverdin also partially inhibited TGF (P < 0.01, biliverdin vs. control), most likely attributable to decreased superoxide (O(2)(-)) because biliverdin was rendered ineffective by tempol, a O(2)(-) dismutase mimetic. We concluded that HO-1 and HO-2 in the nephron inhibit TGF by releasing CO and biliverdin. The inhibitory effect of CO on TGF is mediated by the sGC/cGMP signaling pathway, whereas biliverdin probably acts by reducing O(2)(-).

Dose-dependent toxic effects of high-dose estrogen on renal and cardiac injury in surgically postmenopausal mice.

AIMS: We previously found that in mice with experimental myocardial infarction (MI), 17ß-estradiol (E2) increased mortality and worsened cardiac remodeling and these deleterious effects were associated with renal enlargement and hydronephrosis in a dose-dependent manner. In the present study we questioned whether E2-induced renal damage predisposes to rather than results from its adverse effects on the heart. MAIN METHODS: Ovariectomized (ovx) mice received either placebo (P) or E2 at 0.02 (E2-L, low dose), 0.42 (E2-M, moderate dose) or 4.2 µg/d (E2-H, high dose) for 8 weeks. KEY FINDINGS: E2-L partially restored uterine weight and plasma estrogen levels without affecting heart, lung and liver weight, hemodynamic parameters, or heart and kidney morphology and function. E2-M restored normal uterine weight, but this was accompanied by a significant increase in kidney weight, albuminuria, glomerular matrix formation and markers for oxidative stress. E2-H increased uterine weight 4.5-fold and resulted in higher plasma creatinine levels, severe albuminuria, renal tubular dilatation, tubulointerstitial injury, hydronephrosis, glomerulosclerosis and oxidative stress. E2-H also caused ascites, hepatomegaly and fluid retention in the uterine horns but had no significant effect on blood pressure or heart function. SIGNIFICANCE: Our data demonstrated that an excessive dose of E2 that raises uterine weight beyond physiological levels adversely affects the kidney even before it damages the heart. We believe estrogen dosage should be taken into account when considering hormonal replacement therapy, since inappropriate doses of E2 may damage not only the heart but also the kidney.

Angiotensin II enhances connecting tubule glomerular feedback.

Increasing Na delivery to epithelial Na channels (ENaCs) in the connecting tubule (CNT) causes dilation of the afferent arteriole (Af-Art), a process we call CNT glomerular feedback (CTGF). Angiotensin II (Ang II) stimulates ENaC in the collecting duct via Ang II type 1 receptors. We hypothesized that Ang II in the CNT lumen enhances CTGF by activation of Ang II type 1 receptors, protein kinase C and ENaC. Rabbit afferent arterioles and their adherent CNT were microperfused and preconstricted with norepinephrine. Each experiment involved generating 2 consecutive concentration-response curves by increasing NaCl in the CNT lumen. During the control period, the maximum dilation of the afferent arteriole was 7.9±0.4 µm, and the concentration of NaCl in the CNT needed to achieve half maximal response (EC(50)) was 34.7±5.2 mmol/L. After adding Ang II (10(-9) mol/L) to the CNT lumen, the maximal response was 9.5±0.7 µm and the EC(50) was 11.6±1.3 mmol/L (P=0.01 versus control). Losartan, an Ang II type 1 antagonist (10(-6) mol/L) blocked the stimulatory effect of Ang II; PD123319, an Ang II type 2 antagonist (10(-6) mol/L), did not. The protein kinase C inhibitor staurosporine (10(-8) mol/L) added to the CNT inhibited the stimulatory effect of Ang II. The ENaC inhibitor benzamil (10(-6) mol/L) prevented both CTGF and its stimulation by Ang II. We concluded that Ang II in the CNT lumen enhances CTGF via activation of Ang II type 1 and that this effect requires activation of protein kinase C and ENaC. Potentiation of CTGF by Ang II could help preserve glomerular filtration rate in the presence of renal vasoconstriction.