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Children with Alport syndrome develop renal failure early in life. Since the safety and efficacy of preemptive nephroprotective therapy are uncertain we conducted a randomized, placebo-controlled, double-blind trial in 14 German sites of pediatric patients with ramipril for three to six years plus six months follow-up to determine these parameters. Pretreated children and those whose parents refused randomization became an open-arm control, which were compared to prospective real-world data from untreated children. The co-primary endpoints were safety (adverse drug reactions) and efficacy (time to progression). Out of 66 oligosymptomatic children, 22 were randomized and 44 joined the open-arm comparison. Ramipril therapy showed no safety issues (total of 216.4 patient-years on ramipril; adverse event rate-ratio 1.00; 95% confidence interval 0.66-1.53). Although not significant, our results cautiously showed that ramipril therapy was effective: in the randomized arm, Ramipril decreased the risk of disease progression by almost half (hazard ratio 0.51 (0.12-2.20)), diminished the slope of albuminuria progression and the decline in glomerular filtration. In adjusted analysis, indications of efficacy were supported by prospective data from participants treated open label compared with untreated children, in whom ramipril again seemed to reduce progression by almost half (0.53 (0.22-1.29)). Incorporating these results into the randomized data by Bayesian evidence synthesis resulted in a more precise estimate of the hazard-ratio of 0.52 (0.19-1.39). Thus, our study shows the safety of early initiation of therapy and supports the hope to slow renal failure by many years, emphasizing the value of preemptive therapy. Hence, screening programs for glomerular hematuria in children and young adults could benefit from inclusion of genetic testing for Alport-related gene-variants., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

In order to investigate the hypothesis that the mammalian target of rapamycin inhibitor everolimus (EVR) shows anticytomegalovirus (CMV) activity in pediatric patients, we analyzed the impact of EVR-based immunosuppressive therapy on CMV replication and disease in a large cohort (n = 301) of pediatric kidney allograft recipients. The EVR cohort (n = 59), who also received low-dose cyclosporin, was compared with a control cohort (n = 242), who was administered standard-dose cyclosporin or tacrolimus and an antimetabolite, mostly mycophenolate mofetil (91.7%). Multivariate analysis revealed an 83% lower risk of CMV replication in the EVR cohort than in the control cohort (p = 0.005). In CMV high-risk (donor+/recipient-) patients (n = 88), the EVR-based regimen was associated with a significantly lower rate of CMV disease (0% vs. 14.3%, p = 0.046) than the standard regimen. In patients who had received chemoprophylaxis with (val-)ganciclovir (n = 63), the CMV-free survival rates at 1 year and 3 years posttransplant (100%) were significantly (p = 0.015) higher in the EVR cohort (n = 15) than in the control cohort (n = 48; 1 year, 75.0%; 3 years, 63.3%). Our data suggest that in pediatric patients at high risk of CMV, an EVR-based immunosuppressive regimen is associated with a lower risk of CMV disease than a standard-dose calcineurin inhibitor-based regimen., (© Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Administration of recombinant human keratinocyte growth factor (rHuKGF, Delta23N-KGF, palifermin) protects the lung against a variety of injurious stimuli. The exact mechanisms leading to lung protection are unknown. Alterations in the non-neuronal cholinergic system of the lung might be involved, as vital pulmonary functions are regulated by acetylcholine. Here, we investigated the effect of KGF on the expression of nicotinic acetylcholine receptor subunits alpha7, alpha9 and alpha10 in rat lungs. Adult rats were treated via intratracheal instillation with rHuKGF or with an equivalent volume of PBS. The expression of nicotinic acetylcholine receptor subunits was analyzed by real-time RT-PCR, immunoblotting and immunohistochemistry. Treatment with rHuKGF led to a decreased expression of nicotinic receptor subunit alpha7 in the total lung. In contrast, the expression of the receptor subunits alpha9 and alpha10 was up-regulated. In conclusion, nicotinic acetylcholine receptors are differentially regulated by KGF treatment in vivo, which might result in changes in the biological effects of acetylcholine.

Keratinocyte growth factor protects the lung against various injurious stimuli. The protective mechanisms, however, are not yet fully understood. The aim of this study is to determine the influence of keratinocyte growth factor on the pulmonary capacity to synthesize acetylcholine, a potent regulator of pulmonary functions which is potentially involved in lung damage. Rats were treated twice (days 1 and 2) intratracheally with keratinocyte growth factor and analyzed at day 4. The mRNA expression of choline acetyltransferase - the acetylcholine synthesizing enzyme - was analyzed by real-time RT-PCR in the lung and in isolated alveolar epithelial type II cells. Choline acetyltransferase protein was assessed by immunoblotting and immunohistochemistry. Finally, pulmonary acetylcholine content was assessed biochemically. Keratinocyte growth factor-treatment led to decreased levels of choline acetyltransferase mRNA in the lung and in isolated alveolar epithelial type II cells. Accordingly, pulmonary choline acetyltransferase protein levels were reduced and pulmonary acetylcholine content declined from 2.8 nmol (control) to 0.4 nmol acetylcholine per gram of wet weight. In conclusion, the present data show that the potent regulator of pulmonary functions, acetylcholine, is produced by the major pulmonary target cell of keratinocyte growth factor, that is alveolar epithelial type II cells. Acetylcholine synthesis is down-regulated by keratinocyte growth factor administration which might contribute to lung protection and to harmonize surfactant homeostasis under conditions of keratinocyte growth factor-induced alveolar epithelial type II cell hyperplasia.

Exogenous application of keratinocyte growth factor protects the lung against a variety of injurious stimuli. KGF-treatment leads to pronounced hyperplasia of alveolar epithelial type II cells and to stabilization of surfactant homeostasis after lung injury. Epidermal fatty acid-binding protein is involved in the synthesis of surfactant phospholipids and acts as an antioxidant scavenging reactive lipids. We treated adult rats with recombinant human keratinocyte growth factor (Palifermin) via intratracheal instillation and analyzed the expression of epidermal fatty acid-binding protein mRNA and protein by quantitative RT-PCR, immunoblotting as well as immunohistochemistry. Keratinocyte growth factor-treatment in vivo leads to an increased expression of epidermal fatty acid-binding protein mRNA and protein in the total lung. Epidermal fatty acid-binding protein mRNA expression per alveolar epithelial type II cell remains constant as shown in isolated type II cells. Epidermal fatty acid-binding protein immunoreactivity is seen in most if not all hyperplastic alveolar epithelial type II cells, and is mainly localized to the cytoplasm. The increase in epidermal fatty acid-binding protein gene expression associated with type II cell hyperplasia might contribute to the molecular mechanisms mediating lung protection by keratinocyte growth factor.

Background: After clinical lung transplantation, the amount of vascular endothelial growth factor (VEGF) was found to be decreased in the bronchoalveolar lavage from lungs with acute lung injury. Since Type II pneumocytes are a major site of VEGF synthesis, VEGF depression may be an indicator of pulmonary epithelial damage after ischemia and reperfusion., Methods: Using an established rat lung model, we investigated the relationship between VEGF protein expression, oxygenation capacity and structural integrity after extracorporeal ischemia and reperfusion (ischemia 6 hours at 10 degrees C, reperfusion 50 minutes) and preservation with either low-potassium dextran solution (Perfadex 40 kD, n = 8) or Celsior (n = 6). Untreated, non-ischemic lungs served as controls (n = 5 per group). Perfusate oxygenation was recorded during reperfusion. An enzyme-linked immunoassay (ELISA) for VEGF protein and reverse transcription-polymerase chain reaction (RT-PCR) for mRNA splice variants were determined on tissue collected from the left lungs, whereas the right lungs were fixed by vascular perfusion for VEGF immunohistochemistry as well as structural analysis by light and electron microscopy. Tissue collection by systematic uniform random sampling was representative for the whole organ and allowed for quantification of structures by stereological means., Results: After ischemia and reperfusion, the 3 major VEGF isoforms, VEGF(120), VEGF(164) and VEGF(188), were present. VEGF protein expression was reduced, which correlated significantly with perfusate oxygenation (r = 0.736; p = 0.002) at the end of reperfusion. It was inversely related to Type II cell volume (r = 0.600; p = 0.047). VEGF protein was localized by immunohistochemistry in Type II pneumocytes, alveolar macrophages as well as bronchial epithelium, and staining intensity of Type II cells was reduced after ischemia and reperfusion. Alveolar edema did not occur but significant interstitial edema accumulated around vessels and in the blood-gas barrier, which showed a higher degree of epithelial damage after preservation with Celsior compared with the other groups., Conclusions: Depression in VEGF protein expression can be considered an indicator for increased alveolar epithelial damage. Preservation with low-potassium dextran solution resulted in improved oxygenation and tissue integrity compared with Celsior.

Background: Although administration of nitric oxide (NO) has been suggested to reduce pulmonary reimplantation response, concerns remain about cytotoxic side effects., Methods: Using light and electron microscopy, we examined the effects of the NO donor nitroglycerin (NTG) (0.1 mg/ml) as a supplement to the preservation solution Celsior on the structural integrity of rat lungs after extracorporeal ischemia (4 hours at 10 degrees C) and reperfusion (50 minutes) (IR). We performed evaluation in comparison with Celsior alone after IR using either standard antegrade perfusion through the pulmonary artery or retrograde perfusion through the left atrium as an alternative way to improve the preservation quality. Untreated, non-ischemic lungs served as controls (n = 5 per group). We recorded respiratory and hemodynamic parameters during reperfusion. Tissue collection using systematic uniform random sampling was representative for the whole organ and allowed stereologic quantification of structures., Results: After IR, histochemistry revealed no breaks in the alveolo-capillary barrier and we detected no alveolar flooding. Edema formed in the peribronchovascular cuffs, of which the volume fraction was increased (p =.008). Vasoconstriction of the smaller arteries accompanied antegrade flush, which occurred neither after administration of NTG nor after retrograde flush, as shown by immunostaining for alpha-smooth muscle actin. Treatment with NTG was associated with focal disintegration of Type II cells, which displayed edematous swelling of distinct cell compartments and lysis of mitochondria and cells. Nitroglycerin prevented alveolar collapse, which was increased in the other IR groups (p = 0.013). We observed alterations in intra-alveolar surfactant components., Conclusion: These findings indicate pathologic effects of NTG treatment on alveolar epithelial integrity. Therefore, we suggest further critical evaluation of NTG/NO for therapeutic use in lung transplantation.

The effect of the carbonyl compound glyoxal on the induction of advanced glycation end products (AGEs) in the fetal epithelial lung cells L132 was investigated using immunohistochemical, immunoelectron microscopic, and biochemical methods. It was found that glyoxal treatment resulted in morphological changes of the cells and in the membranous and cytosolic localization of AGEs such as methyl-glyoxal-derived compounds, N-(carboxymethyllysine) (CML) and imidazolone. The formation of AGEs was accompanied with a change in the intracellular expression of cathepsin D and a loss of enzymatic activity., (Copyright 1999 Academic Press.)

Background: The poor tolerance of the lung to ischemia and reperfusion (IR) still represents one of the limitations in clinically successful lung transplantation. Modified Euro-Collins (EC) is routinely used in lung preservation, but alternative solutions have been developed for improvement of pulmonary preservation. Celsior is an extracellular solution that has significantly reduced the IR-induced pulmonary damage in animal studies. So far, no extensive experimental studies exist concerning the influence of Celsior on pulmonary gas exchange following IR., Methods: In an extracorporeal rat lung model 10 lungs, each, were preserved with Celsior (CE) and Celsior/prostacyclin (CEPC, 6 microg/100 ml) at 4 degrees and 15 degrees C, each, and compared to low-potassium Euro-Collins (EC-40, 40 mmol/liter potassium). After 2 hours of ischemia lungs were reventilated and reperfused using a roller pump. Oxygenation in terms of oxygen partial tension in the left atrial effluent, pulmonary vascular resistance (PVR), peak inspiratory pressure, and wet/dry ratio were monitored for 50 minutes. Furthermore, edema formation was evaluated by light microscopy. Statistical analysis was performed using ANOVA models., Results: Compared to the EC-40 group, oxygenation was increased and amount of edema was reduced in most Celsior-preserved organs (p<0.032) with exception of the CEPC group at 4 degrees C (p = 0.06). Additional application of prostacyclin did not have any significant effect on oxygenation in the Celsior group. However, after temperature elevation of the CEPC perfusate to 15 degrees C, a superior partial tension of oxygen was observed (p<0.023) in contrast to the 4 degrees C groups CE and CEPC. The lowest PVR was found in the CE 4 degrees C group (p<0.02)., Conclusions: Celsior provides better lung preservation than EC-40 solution. Application of prostacyclin at higher perfusate temperatures results in additional functional improvement. In vivo experiments and ultrastructural analysis are warranted for further evaluation of Celsior in lung preservation.

Background: Low-potassium solutions have been shown to improve lung preservation. The optimal potassium concentration, however, has not been investigated systematically. The purpose of this study was to evaluate the effect of solutions with different potassium concentrations on functional and structural preservation after flush-perfusion and ischemia. We used our established extracorporeal working heart-lung model and a modification of this model with isolated pulmonary perfusion at defined flow rates., Methods: In two sets of experiments 42 rat heart-lung blocks (experiment I and II: n=7/group) were used. Lungs were flush-preserved with 20 ml Euro-Collins solution (EC115; K+ 115 mmol/L), potassium-reduced Euro-Collins solution (EC40; K+ 40 mmol/L), or low-potassium Euro-Collins solution (EC10; K+ 10 mmol/L) and stored for 2 hours at 10 degrees C. Reperfusion was performed for 40 minutes with Krebs-Henseleit solution containing washed bovine red blood cells (38%) while the lungs were ventilated with room air. In experiment I pulsatile perfusion of the lungs was achieved by the working right side of the heart. In experiment II lungs were perfused at defined flow rates by a roller pump. Postischemic function was assessed by means of oxygenation capacity and pulmonary vascular resistance. The degree of structural damage to the air-blood barrier was assessed by quantitative stereologic light and electron microscopic evaluation., Results: In both experiments after 40 minutes reperfusion oxygenation capacity was significantly higher in EC40 than in EC115 and EC10, whereas pulmonary vascular resistance was significantly higher in EC115 than in EC40 and EC10. Quantitative histologic examination showed surprisingly modest damage to the endothelial side of the air-blood barrier but a considerable degree of damage to the epithelium in both experiments. The alterations in the pump-perfused isolated lung experiments exceeded those of the pulsatile perfused heart-lung experiments. The comparative analysis of the study groups revealed a minor degree of epithelial swelling and fragmentation in EC40 than in EC115 and EC10, respectively., Conclusions: The results obtained with two modifications of an extracorporeal model indicate that flush perfusion of the lung with a potassium-reduced solution results in better functional and structural preservation than flush perfusion with either high- or low-potassium solutions. The optimum may lie in the vicinity of 40 mmol/L. Further studies are necessary to verify these initial findings.

The aim of this study was to relate changes in rat lung functions caused by the endotoxin lipopolysaccharide (LPS) to alterations in structure. The following four experimental groups were used: 1), control in vitro, perfusion for 150 minutes; 2), LPS in vitro, perfusion for 150 minutes and infusion of 5 mg of LPS after 40 minutes; 3), control ex vivo, perfusion for 10 minutes; and 4), LPS ex vivo, lungs perfused for 10 minutes from rats treated for 110 minutes with 20 mg/kg LPS intraperitoneally. Histologically, blood-derived leukocytes were detectable only in lungs from group 4, where neutrophils were found in capillaries, interstitium, and endothelial pouches. LPS treatment increased pulmonary resistance and decreased pulmonary compliance in group 4 (ex vivo), and, to a greater extent, in group 2 (in vitro). In these two groups, formation of giant lamellar bodies in the type II pneumocytes was observed. By histological examination, the bronchoconstriction induced by LPS in vitro was localized to the terminal bronchioles. At 2 hours after LPS treatment, no edema and no change in precapillary and postcapillary resistance, capillary pressure, vascular compliance, capillary permeability, and the wet/dry ratio was observed. Thus, our major findings are that LPS induced constriction of the terminal bronchioles in vitro, formation of giant lamellar bodies in type II pneumocytes ex vivo and in vitro, and trapping of neutrophils in endothelial pouches in vivo.

The quality of pulmonary preservation after Euro-Collins flush perfusion was assessed by means of morphometric analysis based on transmission electron microscopy. In five patients undergoing single lung transplantation, the contralateral donor lung that could not be matched for another recipient was studied by means of light microscopy and transmission electron microscopy. While one of the donor lungs was transplanted, the contralateral lung was fixed by airway instillation at the same time and subsequently processed for microscopic examination. Although light microscopy showed an excellent quality of organ preservation, transmission electron microscopy revealed the presence of fine to medium alterations at the level of the air-blood-barrier. In the five contralateral donor lungs, different degrees of the cellular alterations were recorded by morphometric analysis, which correlated with differences observed in the early postoperative course of the patients receiving the other lung, respectively. Although in four of five patients the clinical course showed no complications and extubation was performed within 36 hours after the operation, one patient required artificial ventilation over a period of 10 days because of impaired oxygenation of the transplanted lung. In this patient, morphometric analysis of the air-blood-barrier showed a significantly (p < 0.02) smaller surface fraction of normal type 1 pneumocytes, a significantly (p < 0.05) smaller volume density of the capillary endothelial cells, and a significantly (p < 0.01) higher volume density of type 2 pneumocytes. The alterations of the alveolar epithelium have to be interpreted as a result of influences occurring during the donor's medical history rather than being an effect of preservation and/or ischemia.