Correction to: The urinary microbiome shows different bacterial genera in renal transplant recipients and non-transplant patients at time of acute kidney injury - a pilot study.

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The urinary microbiome shows different bacterial genera in renal transplant recipients and non-transplant patients at time of acute kidney injury - a pilot study.

BACKGROUND: In the past urine was considered sterile. Through the introduction of next generation sequencing, it has become clear that a urinary microbiome exists. Acute kidney injury (AKI) represents a major threat to kidney transplant recipients. Remarkable changes in the urinary metabolome occur during AKI, which may influence the urinary microbiome. To our knowledge, this is the first study that examines the urinary microbiome in renal transplant recipients (RTX) and non-transplant recipients (nRTX) at time of AKI. METHODS: In this cross-sectional pilot-study the urinary microbiome of 21 RTX and 9 nRTX with AKI was examined. Clean catch morning urine samples were obtained from all patients on the first day of AKI diagnosis. AKI was defined according to KDIGO guidelines. Urinary microbiota and the urinary metabolome during AKI were assessed in one patient. 16S rRNA sequencing was performed. Sequences were processed using UPARSE-pipeline for operational taxonomic units (OTU) and taxon finding. RESULTS: We successfully extracted and sequenced bacterial DNA from 100% of the urine samples. All 30 patients revealed at least 106,138 reads. 319 OTU and 211 different genera were identified. The microbiotic diversity richness in the RTX group was no different from the nRTX group. Eighteen genera were solely present in nRTX and 7 in RTX. CONCLUSIONS: The urinary microbiome at time of AKI showed different bacterial genera in RTX compared to nRTX. The nRTX group exhibited no different diversity to the RTX group. Irrespective of the status of a previous renal transplantation, the urinary microbiome comprised > 210 different genera. An intraindividual change in microbiota diversity and richness was observed in one study patient during recovery from AKI.

Schistosomiasis-associated pulmonary arterial hypertension: a systematic review.

Schistosomiasis-associated pulmonary arterial hypertension (Sch-PAH) is a life-threatening complication of chronic hepatosplenic schistosomiasis. It is suggested to be the leading cause of pulmonary arterial hypertension (PAH) worldwide. However, pathophysiological data on Sch-PAH are scarce. We examined the hypothesis that there are pronounced similarities in pathophysiology, haemodynamics, and survival of Sch-PAH and idiopathic PAH (iPAH).This systematic review and meta-analysis was registered in the PROSPERO database (identifier CRD42018104066). A systematic search and review of the literature was performed according to PRISMA guidelines for studies published between 01 January 1990 and 29 June 2018.For Sch-PAH, 18 studies evaluating pathophysiological mechanisms, eight studies on haemodynamics (n=277), and three studies on survival (n=191) were identified. 16 clinical registries reporting data on haemodynamics and survival including a total of 5792 patients with iPAH were included for comparison. Proinflammatory molecular pathways are involved in both Sch-PAH and iPAH. The transforming growth factor (TGF)-ß signalling pathway is upregulated in Sch-PAH and iPAH. While there was no difference in mean pulmonary artery pressure (54±17 mmHg versus 55±15 mmHg, p=0.29), cardiac output (4.4±1.3 L·min-1 versus 4.1±1.4 L·min-1, p=0.046), and cardiac index (2.6±0.7 L·min-1·m-2 versus 2.3±0.8 L·min-1·m-2, p<0.001) were significantly higher in Sch-PAH compared to iPAH, resulting in a lower pulmonary vascular resistance in Sch-PAH (10±6 Woods units versus 13±7 Woods units, p<0.001). 1- and 3-year survival were significantly better in the Sch-PAH group (p<0.001).Sch-PAH and iPAH share common pathophysiological mechanisms related to inflammation and the TGF-ß signalling pathway. Patients with Sch-PAH show a significantly better haemodynamic profile and survival than patients with iPAH.

A novel approach to immunoapheresis of C3a/C3 and proteomic identification of associates.

BACKGROUND: Complement factor C3 represents the central component of the complement cascade and its activation split product C3a plays an important role in inflammation and disease. Many human disorders are linked to dysregulation of the complement system and alteration in interaction molecules. Therefore, various therapeutic approaches to act on the complement system have been initiated. METHODS AND RESULTS: Aiming to develop a tool to eliminate C3a/C3 from the circulation, in a first step a high affine murine monoclonal antibody (mAb) (3F7E2-mAb) was generated against complement factor C3 and selected for binding to the C3a region to serve as immunoaffinity reagent. Functional testing of the 3F7E2-mAb revealed an inhibition of Zymosan-induced cleavage of C3a from C3. Subsequently, a C3a/C3 specific 3F7E2-immunoaffinity column was developed and apheresis of C3a/C3 and associates was performed. Finally, a proteomic analysis was carried out for identification of apheresis products. C3a/C3 was liberated from the 3F7E2-column together with 278 proteins. C3a/C3 interaction specificity was validated by using a haptoglobin immunoaffinity column as control and biostatistic analysis revealed 39 true C3a/C3 interactants. CONCLUSION: A novel and functionally active mAb was developed against complement factor C3a/C3 and used in a specific immunoaffinity column that allows apheresis of C3a/C3 and associates and their identification by proteomic analysis. This methodological approach of developing specific antibodies that can be used as immunoaffinity reagents to design immunoaffinity columns for elimination and further identification of associated proteins could open new avenues for the development of tailored immunotherapy in various complement-mediated or autoimmune diseases.

Structural analysis of urinary light chains and proteomic analysis of hyaline tubular casts in light chain associated kidney disorders.

BACKGROUND: Monoclonal overproduction of kappa and/or lambda light chains might result in renal light chain deposition disease. Light chain associated cast nephropathy and renal AL-amyloidosis represent two further pathologies going along with monoclonal gammopathy of renal significance and multiple myeloma. While cast nephropathy often manifests with acute kidney injury, AL-amyloidosis is rather accompanied with chronic kidney disease. METHODS: Urine samples were collected from 17 patients with multiple myeloma or monoclonal gammopathy. The urine sediment was stained for cast morphology by H/E and light chain immunofluorescence. Following micro-selection of casts under microscope, proteomic analysis of casts was performed by mass spectrometry. Sucrose gradient sedimentation was employed and light chain architecture examined by immunoblotting. Uromodulin was measured by ELISA in sucrose gradient fractions. RESULTS: Urinary casts were observed of about 30 µm in diameter by H/E staining and under immunofluorescence microscopy. Casts with a diameter of 20 µm were observed as a novel variant. Proteome analysis showed that in addition to the expected light chain variants produced by the malignant clone of plasma cells, also histones such as H2B and cathepsin B were contained. Uromodulin was not detectable in urinary casts of all patients. All eleven patients with lambda light chains showed predominant dimerized light chains in the urine immunoblot. Six patients with kappa light chains presented with predominantly monomeric forms of light chains in the immunoblot. The densitometric evaluated ratio of lambda dimers vs. monomers was significantly higher (2.12 ± 0.75) when compared with the ratio of kappa dimers vs. monomers (0.64 ± 0.47), p = 0.00001. Aggregates of light chains separated in part into denser sucrose fractions. CONCLUSION: This work on urinary casts and light chains demonstrates that hyaline tubular casts represent a complex formation of protein-protein aggregates with histones and cathepsin B identified as novel cast components. Apart from the proteomic composition of the casts, also the formation of the light chains and aggregates is of relevance. Dimerized light chains, which are typical for lambda paraproteins, might be less dialyzable than monomeric forms and may therefore identify patients less responsive to high cut-off dialysis.

Urinary nephrospheres indicate recovery from acute kidney injury in renal allograft recipients - a pilot study.

BACKGROUND: Acute kidney injury represents a major threat to the transplanted kidney. Nevertheless, these kidneys have the potential to fully recover. Tubular regeneration following acute kidney injury is driven by the regenerative potential of tubular cells originating from a tubular stem cell pool. We investigated urinary sediments of acute kidney injury transplanted patients and compared it to those of non-transplanted patients. Thereby we discovered tubular cell agglomerates, which have not been described in vivo. We hypothesized that these so-called nephrospheres were associated with recovery from acute kidney injury. METHODS: Urine sediment of 45 kidney-transplanted and 19 non-transplanted individuals was investigated. Nephrospheres were isolated and stained for several molecular markers including aquaporin 1 (AQP1) and calcium sensing receptor (CASR). Nephrospheres were cultured to examine their growth behavior in vitro. In addition, quantitative PCR for CASR, AQP1, and podocin (NPHS2) was performed. RESULTS: Nephrospheres were excreted in the urine of 17 kidney-transplant recipients 7 days after onset of acute kidney injury and were detectable over several days until kidney function was recovered to baseline creatinine levels. None were found in the urine of non-transplanted individuals. Nephrospheres were either AQP1+/CASR+ or AQP1-/CASR+ and could be cultured for 27 days. Mitotic cells could still be visualized after 17 days in culture. Quantitative PCR detected AQP1 in both kidney-transplanted and non-transplanted individuals during the phase of creatinine decline. As a limitation qPCR was only performed for the entire urinary sediment. CONCLUSIONS: Nephrospheres are three dimensional tubular cell agglomerates which appeared in urine of kidney transplant recipients recovering from acute kidney injury. Appearance of nephrospheres in urine was independent of the duration after kidney transplantation. Nephrospheres proliferated in cell culture and kept expressing kidney specific marker. Presence of nephrospheres in urine showed a specificity of 100% and a sensitivity of 60.71% for recovery.

Hypoalbuminemia as predictor of recurrence of Clostridium difficile infection.

BACKGROUND: Novel drugs for Clostridium difficile (C. difficile) infections have been proven to reduce recurrent infections. Because of their high financial costs, identification of patients at high risk for recurrence is essential to provide optimal treatment. The ATLAS score's ability to predict 90-day recurrence, disease complications and 1­year all-cause mortality was evaluated. METHODS: 144 consecutive symptomatic patients with positive stool test for C. difficile were enrolled. The ATLAS score (consisting of the variables age, temperature, leukocyte count, albumin, systemic antibiotics, serum creatinine) was calculated and patients were stratified into 4 subgroups according to their scores. A Cox regression model was used to estimate the extent to which ATLAS was associated with 90-day recurrence. Furthermore, the score was correlated with disease complications and one-year all-cause mortality. RESULTS: ATLAS was unable to predict 90-day recurrence (p = 0.064, HR 1.134 [0.993;1.295]), but performed well for disease complications (D = 0.382, p < 0.001, HR 1.547 [1.266;1.889]) and mortality (p < 0.001, HR 1.374 [1.194;1.583]). Serum albumin was the only parameter able to predict 90-day recurrence (p = 0.016, HR 0.958 [0.926;0.992]) and was also a predictor of disease complications (p < 0.001, HR 0.865[0.809;0.924]) and one-year all-cause mortality (p < 0.001, HR 0.923 [0.896;0.950]). A threshold of 33.1g/L (sensitivity = 56%, specificity = 80%, AUC 0.683) and 29.2g/L (sensitivity = 75%, specificity = 70%, AUC 0.763) of serum albumin could be identified to be predictive for 90-day recurrence and one-year all-cause mortality, respectively. CONCLUSIONS: Serum albumin and ATLAS are predictors of disease complications and mortality, while only serum albumin is significantly associated with 90-day disease recurrence.

The urine biomarker panel [IGFBP7]x[TIMP-2] (NephroCheck® parameter) does not correlate with IGFBP7 and TIMP-2 gene expression in urinary sediment.

BACKGROUND: Acute kidney injury (AKI) is frequently observed in serious infections, following nephrotoxic medication, surgery and trauma. Here we tested whether the detection of two recently identified biomarkers for AKI, Tissue Inhibitor of Metalloproteinase-2 (TIMP-2) and Insulin-Like Growth Factor Binding Protein 7 (IGFBP7), depends on the expression of these proteins in cells of the urinary sediment. METHOD: We collected urine samples of 33 kidney transplant recipients and 14 non-transplanted patients who all had AKI (stages 1-3 according to KDIGO), and measured [IGFBP7]x[TIMP-2] using the NephroCheck® Astute1 40 ™ meter. Concomitantly, we analyzed IGFBP7 and TIMP-2 mRNA expression by quantitative polymerase chain reaction (qPCR) from urinary sediment of the same patients, and correlated the results with [IGFBP7]x[TIMP-2] (protein), by linear regression analysis. We also determined the association between [IGFBP7]x[TIMP-2] and estimated glomerular filtration rate (eGFR), and between IGFBP7 and TIMP-2 mRNA expression and markers of inflammation. Light microscopy and confocal immunofluorescence served to illustrate changes in the urinary sediment over the time course of renal function improvement. RESULTS: Of the 47 analyzed AKI patients, 14 presented with ascending urinary tract infection. Serum creatinine (sCr), blood urea nitrogen (BUN) and eGFR in all patients were 3.9±2.28 mg/dL, 47.59±23.1 mg/dL and 22.88±16.0 mL/min/1.73m2, respectively, on average ±standard deviation. [IGFBP7]x[TIMP-2] was 2.33±9.95 (ng/ml)2/1000, and did not associate with IGFBP7 and TIMP-2 gene expression (r = -0.0220, p = 0.4216; respectively r = 0.0972, p = 0.1909). [IGFBP7]x[TIMP-2] did not associate with eGFR; IGFBP7 and TIMP-2 mRNA expression. Improvement of renal function went along with disappearance of casts, decrease in aquaporin1 positive renal epithelial cells and leukocytes from the urinary sediment. CONCLUSION: The gene expression pattern of IGFBP7 and TIMP-2 from urinary sediment, which contains desquamated renal tubular epithelial cells, did not correlate with [IGFBP7]x[TIMP-2] protein, indicating that IGFBP7 and TIMP-2 measured in the NephroCheck® test originated predominantly from intact but stressed cells of the kidney itself.

In-vitro release pharmacokinetics of amikacin, teicoplanin and polyhexanide in a platelet rich fibrin-layer (PRF)-a laboratory evaluation of a modern, autologous wound treatment.

OBJECTIVES: Platelet rich fibrin (PRF) is an autologous fibrin glue, produced from patients' blood, which, besides intraoperative use, has applications in the treatment of infected wounds. The combination with antimicrobial agents results in a prolonged antibacterial effect allowing for wound dressing change intervals of seven days even in infected wounds. The aim of this study was to evaluate release kinetics of amikacin, teicoplanin or polyhexanide from a PRF-layer. METHODS: PRF mixed with teicoplanin, amikacin or polyhexanide was sprayed on a silicon gauze patch and put on a colombia agar with bacteria with known minimal inhibitory concentration (MIC) and incubated for 24 hours and afterwards transferred to another agar with the same bacterial strain. Inhibition zones were measured every 24 hours. This was repeated on 7 consecutive days. Antibiotic concentrations were calculated by interpolation. RESULTS: More than 1000 mg/L teicoplanin were released within the first 24 hours and 28.22 mg/L after 168 hours. Amikacin release was above 10,000 mg/L within the first 24 hours and still 120.8 mg/L after 120 hours. A release of polyhexanide could be verified for the first 24 hours only. Consequently teicoplanin and amikacin released from PRF showed antimicrobial in-vitro effects for almost a week, whereas an antimicrobial effect of polyhexanide could only be verified for the first 24 hours. CONCLUSIONS: Our Results show that a weekly dressing regimen may be justified in wounds treated with PRF plus amikacin or teicoplanin, since bacteria will be eradicated over a considerable period of time after a single application of PRF.