Myocardial contrast echocardiography in mice: technical and physiological aspects.

Myocardial contrast echocardiography (MCE) offers the opportunity to study myocardial perfusion defects in mice in detail. The value of MCE compared with single-photon emission computed tomography, positron emission tomography, and computed tomography consists of high spatial resolution, the possibility of quantification of blood volume, and relatively low costs. Nevertheless, a number of technical and physiological aspects should be considered to ensure reproducibility among research groups. The aim of this overview is to describe technical aspects of MCE and the physiological parameters that influence myocardial perfusion data obtained with this technique. First, technical aspects of MCE discussed in this technical review are logarithmic compression of ultrasound data by ultrasound systems, saturation of the contrast signal, and acquisition of images during different phases of the cardiac cycle. Second, physiological aspects of myocardial perfusion that are affected by the experimental design are discussed, including the anesthesia regimen, systemic cardiovascular effects of vasoactive agents used, and fluctuations in body temperature that alter myocardial perfusion. When these technical and physiological aspects of MCE are taken into account and adequately standardized, MCE is an easily accessible technique for mice that can be used to study the control of myocardial perfusion by a wide range of factors.

FGF23 impairs peripheral microvascular function in renal failure.

Cardiovascular diseases account for ~50% of mortality in patients with chronic kidney disease (CKD). Fibroblast growth factor 23 (FGF23) is independently associated with endothelial dysfunction and cardiovascular mortality. We hypothesized that CKD impairs microvascular endothelial function and that this can be attributed to FGF23. Mice were subjected to partial nephrectomy (5/6Nx) or sham surgery. To evaluate the functional role of FGF23, non-CKD mice received FGF23 injections and CKD mice received FGF23-blocking antibodies after 5/6Nx surgery. To examine microvascular function, myocardial perfusion in vivo and vascular function of gracilis resistance arteries ex vivo were assessed in mice. 5/6Nx surgery blunted ex vivo vasodilator responses to acetylcholine, whereas responses to sodium nitroprusside or endothelin were normal. In vivo FGF23 injections in non-CKD mice mimicked this endothelial defect, and FGF23 antibodies in 5/6Nx mice prevented endothelial dysfunction. Stimulation of microvascular endothelial cells with FGF23 in vitro did not induce ERK phosphorylation. Increased plasma asymmetric dimethylarginine concentrations were increased by FGF23 and strongly correlated with endothelial dysfunction. Increased FGF23 concentration did not mimic impaired endothelial function in the myocardium of 5/6Nx mice. In conclusion, impaired peripheral endothelium-dependent vasodilatation in 5/6Nx mice is mediated by FGF23 and can be prevented by blocking FGF23. These data corroborate FGF23 as an important target to combat cardiovascular disease in CKD. NEW & NOTEWORTHY In the present study, we provide the first evidence that fibroblast growth factor 23 (FGF23) is a cause of peripheral endothelial dysfunction in a model of early chronic kidney disease (CKD) and that endothelial dysfunction in CKD can be prevented by blockade of FGF23. This pathological effect on endothelial cells was induced by long-term exposure of physiological levels of FGF23. Mechanistically, increased plasma asymmetric dimethylarginine concentrations were strongly associated with this endothelial dysfunction in CKD and were increased by FGF23.

Microcirculatory perfusion is preserved during off-pump but not on-pump cardiac surgery.

OBJECTIVE: This study investigated the perioperative course of microcirculatory perfusion in off-pump compared with on-pump surgery. Additionally, the impact of changes in systemic hemodynamics, hematocrit, and body temperature was studied. DESIGN: Prospective, nonrandomized, observational study. SETTING: Tertiary university hospital. PARTICIPANTS: Patients undergoing coronary artery bypass grafting with (n = 13) or without (n = 13) use of cardiopulmonary bypass. INTERVENTIONS: Microcirculatory measurements were obtained at 5 time points ranging from induction of anesthesia to ICU admission. MEASUREMENTS AND MAIN RESULTS: Microcirculatory recordings were performed with sublingual sidestream dark field imaging. Despite a comparable reduction in intraoperative blood pressure between groups, the perfused vessel density decreased more than 20% after onset of extracorporeal circulation but remained stable in the off-pump group. The reduction in microvascular perfusion in the on-pump group was further paralleled by decreased hematocrit and temperature. Although postbypass hematocrit levels and body temperature were restored to similar levels as in the off-pump group, the median microvascular flow index remained reduced after bypass (2.4 [2.3-2.7]) compared with baseline (2.8 [2.7-2.9]; p = 0.021). CONCLUSIONS: Microcirculatory perfusion remained unaltered throughout off-pump surgery. In contrast, microvascular perfusion declined after initiation of cardiopulmonary bypass and did not recover in the early postoperative phase.

High Fibroblast Growth Factor 23 concentrations in experimental renal failure impair calcium handling in cardiomyocytes.

The overwhelming majority of patients with chronic kidney disease (CKD) die prematurely before reaching end-stage renal disease, mainly due to cardiovascular causes, of which heart failure is the predominant clinical presentation. We hypothesized that CKD-induced increases of plasma FGF23 impair cardiac diastolic and systolic function. To test this, mice were subjected to 5/6 nephrectomy (5/6Nx) or were injected with FGF23 for seven consecutive days. Six weeks after surgery, plasma FGF23 was higher in 5/6Nx mice compared to sham mice (720 ± 31 vs. 256 ± 3 pg/mL, respectively, P = 0.034). In cardiomyocytes isolated from both 5/6Nx and FGF23 injected animals the rise of cytosolic calcium during systole was slowed (-13% and -19%, respectively) as was the decay of cytosolic calcium during diastole (-15% and -21%, respectively) compared to controls. Furthermore, both groups had similarly decreased peak cytosolic calcium content during systole. Despite lower cytosolic calcium contents in CKD or FGF23 pretreated animals, no changes were observed in contractile parameters of cardiomyocytes between the groups. Expression of calcium handling proteins and cardiac troponin I phosphorylation were similar between groups. Blood pressure, the heart weight:tibia length ratio, α-MHC/ß-MHC ratio and ANF mRNA expression, and systolic and diastolic function as measured by MRI did not differ between groups. In conclusion, the rapid, CKD-induced rise in plasma FGF23 and the similar decrease in cardiomyocyte calcium transients in modeled kidney disease and following 1-week treatment with FGF23 indicate that FGF23 partly mediates cardiomyocyte dysfunction in CKD.

Deregulated Renal Calcium and Phosphate Transport during Experimental Kidney Failure.

Impaired mineral homeostasis and inflammation are hallmarks of chronic kidney disease (CKD), yet the underlying mechanisms of electrolyte regulation during CKD are still unclear. Here, we applied two different murine models, partial nephrectomy and adenine-enriched dietary intervention, to induce kidney failure and to investigate the subsequent impact on systemic and local renal factors involved in Ca(2+) and Pi regulation. Our results demonstrated that both experimental models induce features of CKD, as reflected by uremia, and elevated renal neutrophil gelatinase-associated lipocalin (NGAL) expression. In our model kidney failure was associated with polyuria, hypercalcemia and elevated urinary Ca(2+) excretion. In accordance, CKD augmented systemic PTH and affected the FGF23-αklotho-vitamin-D axis by elevating circulatory FGF23 levels and reducing renal αklotho expression. Interestingly, renal FGF23 expression was also induced by inflammatory stimuli directly. Renal expression of Cyp27b1, but not Cyp24a1, and blood levels of 1,25-dihydroxy vitamin D3 were significantly elevated in both models. Furthermore, kidney failure was characterized by enhanced renal expression of the transient receptor potential cation channel subfamily V member 5 (TRPV5), calbindin-D28k, and sodium-dependent Pi transporter type 2b (NaPi2b), whereas the renal expression of sodium-dependent Pi transporter type 2a (NaPi2a) and type 3 (PIT2) were reduced. Together, our data indicates two different models of experimental kidney failure comparably associate with disturbed FGF23-αklotho-vitamin-D signalling and a deregulated electrolyte homeostasis. Moreover, this study identifies local tubular, possibly inflammation- or PTH- and/or FGF23-associated, adaptive mechanisms, impacting on Ca(2+)/Pi homeostasis, hence enabling new opportunities to target electrolyte disturbances that emerge as a consequence of CKD development.

Membrane-bound Klotho is not expressed endogenously in healthy or uraemic human vascular tissue.

AIMS: Cardiovascular disease (CVD) is the leading cause of death in patients with chronic kidney disease (CKD), a disease state that is strongly associated with loss of renal and systemic (alpha-)Klotho. Reversely, murine Klotho deficiency causes marked medial calcification. It is therefore thought that Klotho conveys a vasculoprotective effect. Klotho expression in the vessel wall, however, is disputed. METHODS AND RESULTS: We assessed Klotho expression in healthy human renal donor arteries (n = 9), CKD (renal graft recipient) arteries (n = 10), carotid endarterectomy specimens (n = 8), other elastic arteries (three groups of n = 3), and cultured human aortic smooth muscle cells (HASMCs) (three primary cell lines), using immunohistochemistry (IHC), immunofluorescence, quantitative reverse transcriptase-polymerase chain reaction, and western blotting (WB). We have extensively validated anti-Klotho antibody KM2076 by comparing staining patterns with other anti-Klotho antibodies (SC-22220, SC-22218, and AF1819), competition assays with recombinant Klotho, IHC on Klotho-deficient kl/kl mouse kidney, and WB with recombinant Klotho. Using KM2076, we could not detect full-length Klotho in vascular tissues or HASMCs. On the mRNA level, using primers against all four exon junctions, klotho expression could not be detected either. Fibroblast growth factor 23 (FGF23) injections in mice induced FGF23 signalling in kidneys but not in the aorta, indicating the absence of Klotho-dependent FGF23 signalling in the aorta. CONCLUSION: Using several independent and validated methods, we conclude that full-length, membrane-bound Klotho is not expressed in healthy or uraemic human vascular tissue.