Dissociation of Hypertension and Renal Damage After Cessation of High-Salt Diet in Dahl Rats.

BACKGROUND: Every year, thousands of patients with hypertension reduce salt consumption in an effort to control their blood pressure. However, hypertension has a self-sustaining character in a significant part of the population. We hypothesized that chronic hypertension leads to irreversible renal damage that remains after removing the trigger, causing an elevation of the initial blood pressure. METHODS: Dahl salt-sensitive rat model was used for chronic, continuous observation of blood pressure. Rats were fed a high salt diet to induce hypertension, and then the diet was switched back to normal sodium content. RESULTS: We found that developed hypertension was irreversible by salt cessation: after a short period of reduction, blood pressure grew even higher than in the high-salt phase. Notably, the self-sustaining phase of hypertension was sensitive to benzamil treatment due to sustaining epithelial sodium channel hyperactivity, as shown with patch-clamp analysis. Glomerular damage and proteinuria were also irreversible. In contrast, some mechanisms, contributing to the development of salt-sensitive hypertension, normalized after salt restriction. Thus, flow cytometry demonstrated that dietary salt reduction in hypertensive animals decreased the number of total CD45+, CD3+CD4+, and CD3+CD8+ cells in renal tissues. Also, we found tubular recovery and improvement of glomerular filtration rate in the postsalt period versus a high-salt diet. CONCLUSIONS: Based on earlier publications and current data, poor response to salt restriction is due to the differential contribution of the factors recognized in the developmental phase of hypertension. We suggest that proteinuria or electrolyte transport can be prioritized over therapeutic targets of inflammatory response.

Role of Alström syndrome 1 in the regulation of glomerular hemodynamics.

Inactivating mutations in the ALMS1 gene in humans cause Alström syndrome, characterized by the early onset of obesity, insulin resistance, and renal dysfunction. However, the role of ALMS1 in renal function and hemodynamics is unclear. We previously found that ALMS1 is expressed in thick ascending limbs, where it binds and decreases Na+-K+-2Cl- cotransporter activity. We hypothesized that ALMS1 is expressed in macula densa cells and that its deletion enhances tubuloglomerular feedback (TGF) and reduces glomerular filtration rate (GFR) in rats. To test this, homozygous ALMS1 knockout (KO) and littermate wild-type Dahl salt-sensitive rats were studied. TGF sensitivity was higher in ALMS1 KO rats as measured by in vivo renal micropuncture. Using confocal microscopy, we confirmed immunolabeling of ALMS1 in macula densa cells (nitric oxide synthase 1 positive), supporting a role for ALMS1 in TGF regulation. Baseline glomerular capillary pressure was higher in ALMS1 KO rats, as was mean arterial pressure. Renal interstitial hydrostatic pressure was lower in ALMS1 KO rats, which is linked to increased Na+ reabsorption and hypertension. GFR was reduced in ALMS1 KO rats. Seven-week-old ALMS1 KO rats were not proteinuric, but proteinuria was present in 18- to 22-wk-old ALMS1 KO rats. The glomerulosclerosis index was higher in 18-wk-old ALMS1 KO rats. In conclusion, ALMS1 is involved in the control of glomerular hemodynamics in part by enhancing TGF sensitivity, and this may contribute to decreased GFR. Increased TGF sensitivity, enhanced glomerular capillary pressure, and hypertension may lead to glomerular damage in ALMS1 KO rats. These are the first data supporting the role of ALMS1 in TGF and glomerular hemodynamics.NEW & NOTEWORTHY ALMS1 is a novel protein involved in regulating tubuloglomerular feedback (TGF) sensitivity, glomerular capillary pressure, and blood pressure, and its dysfunction may reduce renal function and cause glomerular damage.

Knockout of ACE-N facilitates improved cardiac function after myocardial infarction.

Angiotensin-converting enzyme (ACE) hydrolyzes N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) into inactive fragments through its N-terminal site (ACE-N). We previously showed that Ac-SDKP mediates ACE inhibitors' cardiac effects. Whether increased bioavailability of endogenous Ac-SDKP caused by knocking out ACE-N also improves cardiac function in myocardial infarction (MI)-induced heart failure (HF) is unknown. Wild-type (WT) and ACE-N knockout (ACE-NKO) mice were subjected to MI by ligating the left anterior descending artery and treated with vehicle or Ac-SDKP (1.6 mg/kg/day, s.c.) for 5 weeks, after which echocardiography was performed and left ventricles (LV) were harvested for histology and molecular biology studies. ACE-NKO mice showed increased plasma Ac-SDKP concentrations in both sham and MI group compared to WT. Exogenous Ac-SDKP further increased its circulating concentrations in WT and ACE-NKO. Shortening (SF) and ejection (EF) fractions were significantly decreased in both WT and ACE-NKO mice post-MI, but ACE-NKO mice exhibited significantly lesser decrease. Exogenous Ac-SDKP ameliorated cardiac function post-MI only in WT but failed to show any additive improvement in ACE-NKO mice. Sarcoendoplasmic reticulum calcium transport ATPase (SERCA2), a marker of cardiac function and calcium homeostasis, was significantly decreased in WT post-MI but rescued with Ac-SDKP, whereas ACE-NKO mice displayed less loss of SERCA2 expression. Our study demonstrates that gene deletion of ACE-N resulted in improved LV cardiac function in mice post-MI, which is likely mediated by increased circulating Ac-SDKP and minimally reduced expression of SERCA2. Thus, future development of specific and selective inhibitors for ACE-N could represent a novel approach to increase endogenous Ac-SDKP toward protecting the heart from post-MI remodeling.

Decreased tubuloglomerular feedback response in high-fat diet-induced obesity.

Obesity increases the risk of renal damage, but the mechanisms are not clear. Normally, kidneys autoregulate to keep the glomerular capillary pressure (PGC), renal blood flow, and glomerular filtration rate in a steady state. However, in obesity, higher PGC, renal blood flow, and glomerular filtration rate are noted. Together, these may lead to glomerular damage. PGC is controlled mainly by afferent arteriole resistance, which, in turn, is regulated by tubuloglomerular feedback (TGF), a vasoconstrictor mechanism. High fat-induced obesity causes renal damage, and this may be related to increased PGC. However, there are no studies as to whether high-fat diet (HFD)-induced obesity affects TGF. We hypothesized that TGF would be attenuated in obesity caused by HFD feeding (60% fat) in Sprague-Dawley rats. Sprague-Dawley rats fed a normal-fat diet (NFD; 12% fat) served as the control. We studied 4 and 16 wk of HFD feeding using in vivo renal micropuncture of individual rat nephrons. We did not observe significant differences in body weight, TGF response, and mean arterial pressure at 4 wk of HFD feeding, but after 16 wk of HFD, rats were heavier and hypertensive. The maximal TGF response was smaller in HFD-fed rats than in NFD-fed rats, indicating an attenuation of TGF in HFD-induced obesity. Baseline PGC was higher in HFD-fed rats than in NFD-fed rats and was associated with higher glomerulosclerosis. We conclude that attenuated TGF and higher PGC along with hypertension in HFD-fed obese Sprague-Dawley rats could explain the higher propensity of glomerular damage observed in obesity.NEW & NOTEWORTHY Reduced tubuloglomerular feedback, higher glomerular capillary pressure, and hypertension in combination may explain the higher glomerular damage observed in high-fat diet-induced obesity.

High SARS-CoV-2 Viral Load in Urine Sediment Correlates with Acute Kidney Injury and Poor COVID-19 Outcome.

BACKGROUND: AKI is a complication of coronavirus disease 2019 (COVID-19) that is associated with high mortality. Despite documented kidney tropism of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there are no consistent reports of viral detection in urine or correlation with AKI or COVID-19 severity. Here, we hypothesize that quantification of the viral load of SARS-CoV-2 in urine sediment from patients with COVID-19 correlates with occurrence of AKI and mortality. METHODS: The viral load of SARS-CoV-2 in urine sediments (U-viral load) was quantified by qRT-PCR in 52 patients with PCR-confirmed COVID-19 diagnosis, who were hospitalized between March 15 and June 8, 2020. Immunolabeling of SARS-CoV-2 proteins Spike and Nucleocapsid was performed in two COVID-19 kidney biopsy specimens and urine sediments. Viral infectivity assays were performed from 32 urine sediments. RESULTS: A total of 20 patients with COVID-19 (39%) had detectable SARS-CoV-2 U-viral load, of which 17 (85%) developed AKI with an average U-viral load four-times higher than patients with COVID-19 who did not have AKI. U-viral load was highest (7.7-fold) within 2 weeks after AKI diagnosis. A higher U-viral load correlated with mortality but not with albuminuria or AKI stage. SARS-CoV-2 proteins partially colocalized with the viral receptor ACE2 in kidney biopsy specimens in tubules and parietal cells, and in urine sediment cells. Infective SARS-CoV-2 was not detected in urine sediments. CONCLUSION: Our results further support SARS-CoV-2 kidney tropism. A higher SARS-CoV-2 viral load in urine sediments from patients with COVID-19 correlated with increased incidence of AKI and mortality. Urinary viral detection could inform the medical care of patients with COVID-19 and kidney injury to improve prognosis.

Renal release of N-acetyl-seryl-aspartyl-lysyl-proline is part of an antifibrotic peptidergic system in the kidney.

The antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is released from thymosin-ß4 (Tß4) by the meprin-α and prolyl oligopeptidase (POP) enzymes and is hydrolyzed by angiotensin-converting enzyme (ACE). Ac-SDKP is present in urine; however, it is not clear whether de novo tubular release occurs or if glomerular filtration is the main source. We hypothesized that Ac-SDKP is released into the lumen of the nephrons and that it exerts an antifibrotic effect. We determined the presence of Tß4, meprin-α, and POP in the kidneys of Sprague-Dawley rats. The stop-flow technique was used to evaluate Ac-SDKP formation in different nephron segments. Finally, we decreased Ac-SDKP formation by inhibiting the POP enzyme and evaluated the long-term effect in renal fibrosis. The Tß4 precursor and the releasing enzymes meprin-α and POP were expressed in the kidneys. POP enzyme activity was almost double that in the renal medulla compared with the renal cortex. With the use of the stop-flow technique, we detected the highest Ac-SDKP concentrations in the distal nephron. The infusion of a POP inhibitor into the kidney decreased the amount of Ac-SDKP in distal nephron segments and in the proximal nephron to a minor extent. An ACE inhibitor increased the Ac-SDKP content in all nephron segments, but the increase was highest in the distal portion. The chronic infusion of a POP inhibitor increased kidney medullary fibrosis, which was prevented by Ac-SDKP. We conclude that Ac-SDKP is released by the nephron and is part of an important antifibrotic system in the kidney.

Role of Alström syndrome 1 in the regulation of blood pressure and renal function.

Elevated blood pressure (BP) and renal dysfunction are complex traits representing major global health problems. Single nucleotide polymorphisms identified by genome-wide association studies have identified the Alström syndrome 1 (ALMS1) gene locus to render susceptibility for renal dysfunction, hypertension, and chronic kidney disease (CKD). Mutations in the ALMS1 gene in humans causes Alström syndrome, characterized by progressive metabolic alterations including hypertension and CKD. Despite compelling genetic evidence, the underlying biological mechanism by which mutations in the ALMS1 gene lead to the above-mentioned pathophysiology is not understood. We modeled this effect in a KO rat model and showed that ALMS1 genetic deletion leads to hypertension. We demonstrate that the link between ALMS1 and hypertension involves the activation of the renal Na+/K+/2Cl- cotransporter NKCC2, mediated by regulation of its endocytosis. Our findings establish a link between the genetic susceptibility to hypertension, CKD, and the expression of ALMS1 through its role in a salt-reabsorbing tubular segment of the kidney. These data point to ALMS1 as a potentially novel gene involved in BP and renal function regulation.

Renal Protective Effects of N-Acetyl-Seryl-Aspartyl-Lysyl-Proline (Ac-SDKP) in Obese Rats on a High-Salt Diet.

BACKGROUND: Obesity is a public health problem, associated with salt sensitive hypertension, kidney inflammation, and fibrosis. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a tetra peptide with anti-inflammatory and anti-fibrotic properties. However, its effect on preventing kidney damage in obesity is unknown. We hypothesized that Zucker obese (ZO) rats on a high-salt (HS) diet develop renal damage, inflammation, fibrosis, and this is prevented with Ac-SDKP treatment. METHODS: Zucker lean (ZL) and ZO rats (8 weeks old) were treated with Ac-SDKP (1.6 mg/kg/day) while maintained on either a normal-salt (NS; 0.4%) or HS (4%) diet for 8 weeks. Systolic blood pressure (SBP), albuminuria, renal inflammation, and fibrosis were evaluated. RESULTS: HS diet increased macrophage infiltration in the kidneys of both ZL and ZO rats but was significantly higher in ZO rats receiving the HS diet (ZL + NS, 13.9 ± 1.3 vs. ZL + HS, 19.14 ± 1.5 and ZO + NS, 25.5 ± 1.4 vs. ZO + HS, 87.8 ± 10.8 cells/mm2; P < 0.05). Ac-SDKP prevented macrophage infiltration in ZO rats (ZO + HS + Ac-SDKP, 32.18 ± 2.4 cells/mm2; P < 0.05). Similarly, glomerulosclerosis, cortical, and medullary interstitial fibrosis were increased in ZO rats fed the HS diet, and Ac-SDKP attenuated these alterations (P < 0.05). SBP was increased in ZO rats fed the HS diet (ZO + NS, 121.3 ± 8.9 vs. ZO + HS, 164 ± 6.9 mm Hg; P < 0.05), and it was significantly decreased with Ac-SDKP treatment (ZO + HS + Ac-SDKP, 144.05 ± 14.1 mm Hg; P = 0.004). Albuminuria was higher in ZO rats than in ZL rats; however, neither HS nor Ac-SDKP treatment affected it. CONCLUSIONS: Ac-SDKP treatment in ZO rats fed a HS diet prevented renal damage by reducing inflammation, fibrosis, and SBP.

Thymosin ß4 Deficiency Exacerbates Renal and Cardiac Injury in Angiotensin-II-Induced Hypertension.

Thymosin ß4 (Tß4), a ubiquitous peptide, regulates several cellular processes that include cell morphology, wound healing, and inflammatory response. Administration of exogenous Tß4 is protective in diabetic nephropathy and in a unilateral ureteral obstruction model. However, the role of endogenous Tß4 in health and disease conditions remains unclear. To elucidate the pathophysiological role of endogenous Tß4 in hypertension, we examined angiotensin-II (Ang-II)-induced renal and cardiac damage in Tß4 knockout (Tß4 KO) mice. Tß4 KO and wild-type C57BL/6 mice were infused continuously for 6 weeks with either vehicle or Ang-II (980 ng/kg per minute). At baseline, Tß4 deficiency did not affect renal and cardiac function. Systolic blood pressure in the Ang-II group was similar in wild-type and Tß4 KO mice (wild-type Ang-II, 179.25±10.11 mm Hg; Tß4 KO Ang-II, 169.81±6.54 mm Hg). Despite the similar systolic blood pressure after Ang-II infusion, Tß4-deficient mice had dramatically increased albuminuria and decreased nephrin expression in the kidney (P<0.005). In the heart of Tß4 KO mice, Ang-II reduced ejection fraction and shortening fraction (ejection fraction: wild-type Ang-II 77.95%±1.03%; Tß4 KO Ang-II 62.58%±3.25%; P<0.005), which was accompanied by cardiac hypertrophy and left ventricular dilatation. In addition, renal and cardiac infiltration of CD68 macrophages, intercellular adhesion molecule-1, and total collagen content were increased after Ang-II infusion in Tß4 KO mice (P<0.005). Overall, our data indicate that endogenous Tß4 is crucial in preventing tissue injury from Ang-II-induced hypertension. This study gives new insights into the protective role of endogenous Tß4 in hypertensive end-organ damage.

Effects of N-acetyl-seryl-asparyl-lysyl-proline on blood pressure, renal damage, and mortality in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease with a high prevalence of hypertension. NZBWF1 (SLE-Hyp) mice develop hypertension that can be prevented by modulating T cells. The peptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) decreases renal damage and improves renal function in a model of SLE without hypertension (MRL/lpr). However, it is not known whether Ac-SDKP prevents hypertension in NZBWF1 mice. We hypothesized that in SLE-Hyp, Ac-SDKP prevents hypertension and renal damage by modulating T cells. Animals were divided into four groups: (1) control + vehicle, (2) control + Ac-SDKP, (3) SLE + vehicle, and (4) SLE + Ac-SDKP Systolic blood pressure (SBP), albuminuria, renal fibrosis, and T-cell phenotype were analyzed. SBP was higher in SLE compared to control mice and was not decreased by Ac-SDKP treatment. Half of SLE mice developed an acute and severe form of hypertension accompanied by albuminuria followed by death. Ac-SDKP delayed development of severe hypertension, albuminuria, and early mortality, but this delay did not reach statistical significance. Ac-SDKP prevented glomerulosclerosis, but not interstitial fibrosis in SLE-Hyp mice. SLE-Hyp mice showed a decrease in helper and cytotoxic T cells as well as an increase in double negative lymphocytes and T helper 17 cells, but these cells were unaffected by Ac-SDKP In conclusion, Ac-SDKP prevents kidney damage, without affecting blood pressure in an SLE animal model. However, during the acute relapse of SLE, Ac-SDKP might also delay the manifestation of an acute and severe form of hypertension leading to early mortality. Ac-SDKP is a potential tool to treat renal damage in SLE-Hyp mice.

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.

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.

N-acetyl-seryl-aspartyl-lysyl-proline reduces cardiac collagen cross-linking and inflammation in angiotensin II-induced hypertensive rats.

We have reported previously that Ac-SDKP (N-acetyl-seryl-aspartyl-lysyl-proline) reduces fibrosis and inflammation (in macrophages and mast cells). However, it is not known whether Ac-SDKP decreases collagen cross-linking and lymphocyte infiltration; lymphocytes modulate both collagen cross-linking and ECM (extracellular matrix) formation in hypertension. Thus we hypothesized that (i) in AngII (angiotensin II)-induced hypertension, Ac-SDKP prevents increases in cross-linked and total collagen by down-regulating LOX (lysyl oxidase), the enzyme responsible for cross-linking, and (ii) these effects are associated with decreased pro-fibrotic cytokine TGFß (transforming growth factor ß) and the pro-inflammatory transcription factor NF-κB (nuclear factor κB) and CD4+/CD8+ lymphocyte infiltration. We induced hypertension in rats by infusing AngII either alone or combined with Ac-SDKP for 3 weeks. Whereas Ac-SDKP failed to lower BP (blood pressure) or LV (left ventricular) hypertrophy, it did prevent AngII-induced increases in (i) cross-linked and total collagen, (ii) LOX mRNA expression and LOXL1 (LOX-like 1) protein, (iii) TGFß expression, (iv) nuclear translocation of NF-κB, (v) CD4+/CD8+ lymphocyte infiltration, and (vi) CD68+ macrophages infiltration. In addition, we found a positive correlation between CD4+ infiltration and LOXL1 expression. In conclusion, the effect of Ac-SDKP on collagen cross-linking and total collagen may be due to reduced TGFß1, LOXL1, and lymphocyte and macrophage infiltration, and its effect on inflammation could be due to lower NF-κB.

Treatment with N-acetyl-seryl-aspartyl-lysyl-proline prevents experimental autoimmune myocarditis in rats.

Myocarditis is commonly associated with cardiotropic infections and has been linked to development of autoimmunity. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a naturally occurring tetrapeptide that prevents inflammation and fibrosis in hypertension and other cardiovascular diseases; however, its effect on autoimmune-mediated cardiac diseases remains unknown. We studied the effects of Ac-SDKP in experimental autoimmune myocarditis (EAM), a model of T cell-mediated autoimmune disease. This study was conducted to test the hypothesis that Ac-SDKP prevents autoimmune myocardial injury by modulating the immune responses. Lewis rats were immunized with porcine cardiac myosin and treated with Ac-SDKP or vehicle. In EAM, Ac-SDKP prevented both systolic and diastolic cardiac dysfunction, remodeling as shown by hypertrophy and fibrosis, and cell-mediated immune responses without affecting myosin-specific autoantibodies or antigen-specific T cell responses. In addition, Ac-SDKP reduced cardiac infiltration by macrophages, dendritic cells, and T cells, pro-inflammatory cytokines [interleukin (IL)-1α, tumor necrosis factor-α, IL-2, IL-17] and chemokines (cytokine-induced neutrophil chemoattractant-1, interferon-γ-induced protein 10), cell adhesion molecules (intercellular adhesion molecule-1, L-selectin), and matrix metalloproteinases (MMP). Ac-SDKP prevents autoimmune cardiac dysfunction and remodeling without reducing the production of autoantibodies or T cell responses to cardiac myosin. The protective effects of Ac-SDKP in autoimmune myocardial injury are most likely mediated by inhibition of 1) innate and adaptive immune cell infiltration and 2) expression of proinflammatory mediators such as cytokines, chemokines, adhesion molecules, and MMPs.

Arterial injury promotes medial chondrogenesis in Sm22 knockout mice.

AIMS: Expression of SM22 (also known as SM22alpha and transgelin), a vascular smooth muscle cells (VSMCs) marker, is down-regulated in arterial diseases involving medial osteochondrogenesis. We investigated the effect of SM22 deficiency in a mouse artery injury model to determine the role of SM22 in arterial chondrogenesis. METHODS AND RESULTS: Sm22 knockout (Sm22(-/-)) mice developed prominent medial chondrogenesis 2 weeks after carotid denudation as evidenced by the enhanced expression of chondrogenic markers including type II collagen, aggrecan, osteopontin, bone morphogenetic protein 2, and SRY-box containing gene 9 (SOX9). This was concomitant with suppression of VSMC key transcription factor myocardin and of VSMC markers such as SM α-actin and myosin heavy chain. The conversion tendency from myogenesis to chondrogenesis was also observed in primary Sm22(-/-) VSMCs and in a VSMC line after Sm22 knockdown: SM22 deficiency altered VSMC morphology with compromised stress fibre formation and increased actin dynamics. Meanwhile, the expression level of Sox9 mRNA was up-regulated while the mRNA levels of myocardin and VSMC markers were down-regulated, indicating a pro-chondrogenic transcriptional switch in SM22-deficient VSMCs. Furthermore, the increased expression of SOX9 was mediated by enhanced reactive oxygen species production and nuclear factor-κB pathway activation. CONCLUSION: These findings suggest that disruption of SM22 alters the actin cytoskeleton and promotes chondrogenic conversion of VSMCs.

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.

Local angiotensin II aggravates cardiac remodeling in hypertension.

Angiotensin II (ANG II) contributes to hypertension, cardiac hypertrophy, fibrosis, and dysfunction; however, it is difficult to separate the cardiac effect of ANG II from its hemodynamic action in vivo. To overcome the limitations, we used transgenic mice with cardiac-specific expression of a transgene fusion protein that releases ANG II from cardiomyocytes (Tg-ANG II) and treated them with deoxycorticosterone acetate (DOCA)-salt to suppress their systemic renin-angiotensin system. Using this unique model, we tested the hypothesis that cardiac ANG II, acting on the angiotensin type 1 receptor (AT(1)R), increases inflammation, oxidative stress, and apoptosis, accelerating cardiac hypertrophy and fibrosis. Male Tg-ANG II mice and their nontransgenic littermates (n-Tg) were uninephrectomized and divided into the following three groups: 1) vehicle-treated normotensive controls; 2) DOCA-salt; and 3) DOCA-salt + valsartan (AT(1)R blocker).Under basal conditions, systolic blood pressure (SBP) and cardiac phenotypes were similar between strains. In DOCA-salt hypertension, SBP increased similarly in both n-Tg and Tg-ANG II, and cardiac function did not differ between strains; however, Tg-ANG II had 1) greater ventricular hypertrophy as well as interstitial and perivascular fibrosis; 2) a higher number of deoxynucleotidyl-transferase-mediated dUTP nick end labeling-positive cells and infiltrating macrophages; 3) increased protein expression of NADPH oxidase 2 and transforming growth factor-ß(1); and 4) downregulation of phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase B (Akt) phosphorylation. Valsartan partially reversed these effects in Tg-ANG II but not in n-Tg. We conclude that, when hemodynamic loading conditions remain unchanged, cardiac ANG II does not alter heart size or cardiac functions. However, in animals with hypertension, cardiac ANG II, acting via AT(1)R, enhances inflammation, oxidative stress, and cell death (most likely via downregulation of PI 3-kinase and Akt), contributing to cardiac hypertrophy and fibrosis.

Role of prolylcarboxypeptidase in angiotensin II type 2 receptor-mediated bradykinin release in mouse coronary artery endothelial cells.

Activation of angiotensin II type 2 receptors (AT(2)R) causes the release of kinins, which have beneficial effects on the cardiovascular system. However, it is not clear how AT(2)R interact with the kallikrein-kinin system to generate kinins. Prolylcarboxypeptidase is an endothelial membrane-bound plasma prekallikrein activator that converts plasma prekallikrein to kallikrein, leading to generation of bradykinin from high-molecular-weight kininogen. We hypothesized that AT(2)R-induced bradykinin release is at least in part mediated by activation of prolylcarboxypeptidase. Cultures of mouse coronary artery endothelial cells were transfected with an adenoviral vector containing the AT(2)R gene (Ad-AT(2)R) or green fluorescent protein only (Ad-GFP) as control. We found that overexpression of AT(2)R increased prolylcarboxypeptidase mRNA by 1.7-fold and protein 2.5-fold compared with Ad-GFP controls. AT(2)R overexpression had no effect on angiotensin II type 1 receptor mRNA. Bradykinin release was increased 2.2-fold in AT(2)R-transfected cells. Activation of AT(2)R by CGP42112A, a specific AT(2)R agonist, increased bradykinin further in AT(2)R-transfected cells. These effects were diminished or abolished by AT(2)R blockade or a plasma kallikrein inhibitor. Furthermore, blocking prolylcarboxypeptidase with a small interfering RNA partially but significantly reduced bradykinin release by transfected AT(2)R cells either at the basal condition or when stimulated by the AT(2)R agonist CGP42112A. These findings suggest that overexpression of AT(2)R in mouse coronary artery endothelial cells increases expression of prolylcarboxypeptidase, which may contribute to kinin release.

Lack of glutathione peroxidase 1 accelerates cardiac-specific hypertrophy and dysfunction in angiotensin II hypertension.

Glutathione peroxidase 1 (Gpx1) plays an important role in cellular defense by converting hydrogen peroxide and organic hydroperoxides to nonreactive products, and Gpx1(-/-) mice, which are characterized by reduced tissue glutathione peroxidase activity, are known to exhibit enhanced oxidative stress. Peroxides participate in tissue injury, as well as the hypertrophy of cultured cells, yet the role of Gpx1 to prevent end organ damage in cardiovascular tissue is not clear. We postulated that Gpx1 deletion would potentiate both aortic and cardiac hypertrophy, as well as mean arterial blood pressure, in response to angiotensin II (AngII). Our results show that short-term AngII markedly increased left ventricular mass, myocyte cross-sectional area, and interventricular septum thickness and decreased shortening fraction in Gpx1(-/-) mice as compared with wild-type animals. On the other hand, AngII resulted in a similar increase in mean arterial blood pressure in wild-type and Gpx1(-/-) mice. Collagen deposition increased in response to AngII, but no differences were found between strains. Vascular hypertrophy increased to the same extent in Gpx1(-/-) and wild-type mice. Collectively, our results indicate that Gpx1 deficiency accelerates cardiac hypertrophy and dysfunction but has no effect on vascular hypertrophy and mean arterial blood pressure and suggest a major role for Gpx1 in cardiac dysfunction in AngII-dependent hypertension.