Divergent and conserved roles of Dll1 signaling in development of craniofacial and trunk muscle.

Craniofacial and trunk skeletal muscles are evolutionarily distinct and derive from cranial and somitic mesoderm, respectively. Different regulatory hierarchies act upstream of myogenic regulatory factors in cranial and somitic mesoderm, but the same core regulatory network - MyoD, Myf5 and Mrf4 - executes the myogenic differentiation program. Notch signaling controls self-renewal of myogenic progenitors as well as satellite cell homing during formation of trunk muscle, but its role in craniofacial muscles has been little investigated. We show here that the pool of myogenic progenitor cells in craniofacial muscle of Dll1(LacZ/Ki) mutant mice is depleted in early fetal development, which is accompanied by a major deficit in muscle growth. At the expense of progenitor cells, supernumerary differentiating myoblasts appear transiently and these express MyoD. The progenitor pool in craniofacial muscle of Dll1(LacZ/Ki) mutants is largely rescued by an additional mutation of MyoD. We conclude from this that Notch exerts its decisive role in craniofacial myogenesis by repression of MyoD. This function is similar to the one previously observed in trunk myogenesis, and is thus conserved in cranial and trunk muscle. However, in cranial mesoderm-derived progenitors, Notch signaling is not required for Pax7 expression and impinges little on the homing of satellite cells. Thus, Dll1 functions in satellite cell homing and Pax7 expression diverge in cranial- and somite-derived muscle.

Adverse skin reactions to iodinated x-ray contrast agents in healthy rats.

PURPOSE: The aim of this preclinical study on healthy Sprague-Dawley rats was to determine whether differences exist in the induction of adverse skin reactions after the intravenous administration of a monomeric and 2 dimeric iodinated nonionic contrast agents. MATERIALS AND METHODS: After intravenous injection of iopromide (monomeric), iodixanol (dimeric), and iotrolan (dimeric) at a dose of 4 g iodine/kg of body weight, mechanical ear volume measurements (10 minute after injection) and intravital microscopy (baseline, 5 minutes after injection) of the ear with the near-infrared dye indocyanine green were performed to determine the volume change and plasma extravasation. Histopathological analysis (20 minutes, 1 hour, and 3 hours after injection) was performed to diagnose alterations in the skin. Blood plasma was analyzed to identify elevated levels of histamine (5 minutes after injection) and inflammatory markers (a multianalyte profile of 58 markers; 1 hour and 3 hours after injection). RESULTS: Only iodixanol induced immediate angioedema formation, with a 100% incidence rate and with slight mast cell infiltration in the ear, muzzle, and paws. The ear showed a 53% volume increase and strong extravasation of plasma proteins into the interstitium, which correlated with highly (11-fold) increased plasma histamine levels 5 minutes after injection. Elevated levels of tumor necrosis factor-α (7.1-fold), macrophage inflammatory protein (MIP)-1α (3.2-fold), and MIP-2 (7.7-fold) were identified 1 hour after the iodixanol injection. Increased levels (fold-change) of MIP-1ß (14; 6.3), monocyte chemotactic protein-1 (3.3; 3.7), monocyte chemotactic protein-3 (2.4; 3.0), stem cell factor (1.7; 2), vascular endothelial growth factor (2; 2.1), and interferon gamma-induced protein-10 (4.1; 39.1) were identified 1 hour and 3 hours after the iodixanol administration, respectively. The level of these molecules remained unchanged after the iopromide and iotrolan injections (except for stem cell factor). CONCLUSIONS: A reversible anaphylactoid-like reaction in healthy Sprague-Dawley rats was observed after the iodixanol administration but not after the monomeric iopromide or dimeric iotrolan injections. Therefore, we conclude that the induction of adverse skin reactions is not per se because of a class effect of dimeric contrast agent.

Contrast media viscosity versus osmolality in kidney injury: lessons from animal studies.

Iodinated contrast media (CM) can induce acute kidney injury (AKI). CM share common iodine-related cytotoxic features but differ considerably with regard to osmolality and viscosity. Meta-analyses of clinical trials generally failed to reveal renal safety differences of modern CM with regard to these physicochemical properties. While most trials' reliance on serum creatinine as outcome measure contributes to this lack of clinical evidence, it largely relies on the nature of prospective clinical trials: effective prophylaxis by ample hydration must be employed. In everyday life, patients are often not well hydrated; here we lack clinical data. However, preclinical studies that directly measured glomerular filtration rate, intrarenal perfusion and oxygenation, and various markers of AKI have shown that the viscosity of CM is of vast importance. In the renal tubules, CM become enriched, as water is reabsorbed, but CM are not. In consequence, tubular fluid viscosity increases exponentially. This hinders glomerular filtration and tubular flow and, thereby, prolongs intrarenal retention of cytotoxic CM. Renal cells become injured, which triggers hypoperfusion and hypoxia, finally leading to AKI. Comparisons between modern CM reveal that moderately elevated osmolality has a renoprotective effect, in particular, in the dehydrated state, because it prevents excessive tubular fluid viscosity.

The osmolality of nonionic, iodinated contrast agents as an important factor for renal safety.

OBJECTIVE: Nonionic iodinated contrast agents (CAs) can be divided into monomeric, low-osmolar, and dimeric, iso-osmolar classes. In clinical practice, renal tolerance of CAs is a concern, especially in patients with impaired renal function. With regard to renal safety, we wanted to evaluate the role of osmolality and viscosity in renal tolerance. MATERIAL AND METHODS: We generated a formulation (iodixanol/mannitol) consisting of the dimeric iodixanol with an osmolality of the monomeric iopromide. Male Han-Wistar rats were intravenously injected with low-osmolar iopromide 300, iso-osmolar iodixanol 320, and iodixanol/mannitol. Saline and diatrizoate were used as controls. A total number of 227 rats were used in the following experiments. We compared the impact of osmolality on renal iodine retention using computed tomography 2 and 24 hours postinjection (p.i.). The animals were killed 2, 24, and 72 hours after injection, and the kidneys were excised for further investigations. Changes in renal cell proliferation were analyzed by 5-bromo-2'-deoxyuridine incorporation 48 hours p.i. as a degree of tissue regeneration after induced injury. To specify potential renal injury, we quantified the expression of acute kidney injury (AKI) markers (kidney injury marker-1 [KIM-1], neutrophil gelatinase-associated lipocalin [NGAL], and plasminogen activator inhibitor-1 [PAI-1]) by quantitative real-time polymerase chain reaction. Furthermore, the kidneys were analyzed histologically, including immunofluorescence analysis. RESULTS: After intravenous application of the CAs into Han-Wistar rats, renal iodine concentration was increased (3-fold) for iodixanol 2 hours p.i. and iodine retention was detected to be prolonged 24 hours p.i. compared with iopromide injection (iodixanol, 520 ± 50 Hounsfield Units [HU] vs iopromide, 42 ± 5 HU). The higher iodine concentration 2 hours p.i. upon iodixanol injection was reduced almost to the level of iopromide when injecting iodixanol/mannitol (iopromide: 289 ± 68 HU vs iodixanol/mannitol: 343 ± 68 HU). In addition, iodixanol application induced increased renal cell proliferation (2.7-fold vs saline), indicating renal injury, which was significantly lower in iopromide-treated animals (1.6-fold vs saline). More detailed analysis of markers for AKI revealed that iodixanol significantly induced the expression of PAI-1 (7.7-fold at 2 hours) as well as KIM-1 (2.1-fold) and NGAL (3.2-fold) at 2 and 24 hours when compared with saline treatment. In contrast, the expression of markers for AKI was low after iopromide (1.4-fold NGAL, 1.7-fold PAI-1, KIM-1 not significant) and iodixanol/mannitol (1.6-fold NGAL, 2.6-fold PAI-1, KIM-1 not significant) injection. CONCLUSION: The present results clearly show that prolonged iodine retention and the enhanced expression of kidney injury markers are caused mainly by the explicitly higher urine viscosity induced by iodixanol. We conclude that the osmolality of low-osmolar CAs such as iopromide induces a positive diuretic effect that is responsible for rapid iodine clearance and prevents increased expression of acute injury markers in the kidney.

Changes of renal water diffusion coefficient after application of iodinated contrast agents: effect of viscosity.

OBJECTIVE: X-ray contrast agents (CA) possess specific physicochemical properties and are excreted renally by glomerular filtration. Thereby, they may affect the diffusion of water molecules within the kidney. The aim of our preclinical study was to investigate potential changes in the apparent diffusion coefficient (ADC) of the kidney after administration of monomeric, low-osmolar, and dimeric, iso-osmolar CA by using diffusion-weighted magnetic resonance imaging (DWI). MATERIAL AND METHODS: First, the relationship between CA viscosity and the ADC of water was assessed by phantom measurements. Subsequently, Han Wistar rats (8 per group) received an intravenous injection of iso-osmolar CA (iodixanol) or low-osmolar CA (iopromide) at a dosage of 4 gI/kg body weight. The control group received saline (0.9% NaCl) at the same volume. The renal ADC was dynamically monitored up to 40 minutes postinjection (p.i.) by DWI using a 1.5-T clinical MR unit. After DWI, the animals were killed and the kidneys were removed for iodine measurements by x-ray fluorescence analysis. RESULTS: The in vitro measurements yielded an inverse relationship between increasing viscosity and decreasing water diffusion. In vivo, a slight increase in ADC was observed immediately after administration of the low-osmolar iopromide (ΔADC=80±78 µm²/s) and saline (ΔADC=89±53 µm²/s), which normalized to the baseline level at 40 minutes p.i. In contrast, a strong decrease in ADC was observed after administration of the iso-osmolar iodixanol. This was most prominent 12 minutes p.i. (ΔADC=-555±194 µm²/s) and persisted throughout the investigation. Concomitantly, the kidney iodine concentration 50 minutes p.i. was significantly higher after iodixanol (58.6±5.3 mgI/g kidney) compared with iopromide injection (18.4±4.5 mgI/g kidney). CONCLUSION: A significant difference in the renal ADC was observed between the low-osmolar CA/saline and the iso-osmolar CA. The in vitro measurements suggest that the substantial decrease in ADC observed after administration of the iso-osmolar CA is based on the high viscosity of the agent during renal passage. This, in turn, may explain the delayed iodine retention after administration of iso-osmolar CA and demonstrates the importance of the physicochemical properties of CA during their renal elimination.

Viscosity of iodinated contrast agents during renal excretion.

OBJECTIVE: Modern iodinated non-ionic contrast agents (CAs) can be classified based on their molecular structure into monomeric and dimeric CAs and have at comparable iodine concentrations a different viscosity and osmolality. During their renal excretion, CAs are concentrated in the renal tubuli which might enhance the viscosity difference between monomeric and dimeric CAs. The viscosity of a CA might have an underestimated importance for renal safety, as suggested by recent publications. In this study, we investigated the viscosities of CAs at the concentrations expected to be present in renal tubules. This concentration process was simulated in vitro using dialysis. Furthermore, we investigated urine viscosity and urine flow in rodents after administration of several non-ionic monomeric and dimeric CAs. MATERIALS AND METHODS: To estimate the viscosity of the CAs in vivo, we performed an in vitro dialysis of monomeric and dimeric CAs at various physiological osmolalities of the renal tubulus (290, 400, 500, 700 and 1000 mOsm/kg H2O). Following the dialysis, the iodine concentrations and the viscosities of the CAs were determined. Furthermore, to investigate the concentration process in vivo, we measured the urine viscosity and the urine flow in Han Wister rats after the administration of Iopromide, Iohexol, Ioversol, Iomeprol, Iodixanol, and Iosimenol at comparable iodine concentrations. As a control, saline was injected at the same volume. RESULTS: In vitro dialysis of the dimeric CA increased the iodine concentration and strongly increased the viscosity at all tested osmolalities. In contrast, for the monomeric agents an increase in concentration and viscosity was observed only at 700 as well 1000 mOsm/kg H2O but to a lesser extent. In summary, dialysis strongly enhanced the viscosity differences between the non-ionic monomeric and dimeric CAs. The administration of dimeric CAs leads to a strong increase in urine viscosity; this was not observed for the monomeric CAs. In contrast, a significantly higher urine flow was measured after the administration of the monomeric CAs as compared to the dimeric CAs. CONCLUSION: We demonstrated that the viscosity differences between monomeric and dimeric CAs are strongly enhanced due to a concentration process of the CAs upon increasing osmolalities, a process which is likely to take place in a similar manner in the tubular system. This result suggests that the viscosity of the dimeric agents increases dramatically in vivo and gives a plausible explanation for measured enhancement of urine viscosity upon dimeric CA administration. On the other hand, the higher osmolality of the monomeric agents causes an osmodiuresis, indicated by a higher urine flow, which leads to a faster elimination of the CAs from the kidney.

Notch function in myogenesis.

Notch genes encode cell surface proteins, which are evolutionary conserved and found in invertebrates like Drosophila melanogaster as well as in all vertebrate species. The transcription factor RBP-J (Rbpsuh) is a primary nuclear mediator of Notch signals. Signals provided by Notch receptors control cell fate decisions, patterning, and they also affect proliferation or the maintenance of progenitor cells. In these Perspectives we highlight the recent findings on the role of Notch/RBP-J signaling in the maintenance of muscle progenitor cells during embryogenesis and in the generation of satellite cells in fetal development.

RBP-J (Rbpsuh) is essential to maintain muscle progenitor cells and to generate satellite cells.

In the developing muscle, a pool of myogenic progenitor cells is formed and maintained. These resident progenitors provide a source of cells for muscle growth in development and generate satellite cells in the perinatal period. By the use of conditional mutagenesis in mice, we demonstrate here that the major mediator of Notch signaling, the transcription factor RBP-J, is essential to maintain this pool of progenitor cells in an undifferentiated state. In the absence of RBP-J, these cells undergo uncontrolled myogenic differentiation, leading to a depletion of the progenitor pool. This results in a lack of muscle growth in development and severe muscle hypotrophy. In addition, satellite cells are not formed late in fetal development in conditional RBP-J mutant mice. We conclude that RBP-J is required in the developing muscle to set aside proliferating progenitors and satellite cells.