Levels of Cell-Free DNA in Kidney Failure Patients before and after Renal Transplantation.

Circulating cell-free DNA (cfDNA) has diverse applications in oncological, prenatal, toxicological, cardiovascular, and autoimmune diseases, diagnostics, and organ transplantation. In particular, mitochondrial cfDNA (mt-cfDNA) is associated with inflammation and linked to early vascular ageing (EVA) in end-stage kidney failure (ESKF), which could be a noninvasive marker for graft rejection and organ damage. Plasma samples from 44 ESKF patients, of whom half (n = 22) underwent either conservative therapy (non-HD) or hemodialysis (HD) before kidney transplantation (KT). These samples were analyzed at baseline and two years after KT. cfDNA was extracted from plasma and quantified using the fluorometric method. qPCR was used to quantify and differentiate the fractions of mt-cfDNA and nuclear cfDNA (nc-cfDNA). mt-cfDNA levels in KT patients decreased significantly from baseline to two years post-KT (p < 0.0268), while levels of total cfDNA and nc-cfDNA did not differ. Depending on therapy modality (HD vs. non-HD) before KT, total cfDNA levels were higher in HD patients at both baseline (p = 0.0133) and two years post-KT (p = 0.0421), while nc-cfDNA levels were higher in HD only at baseline (p = 0.0079). Males showed a nonsignificant trend of higher cfDNA levels. Patients with assessed vascular fibrosis (p = 0.0068), either alone or in combination with calcification plus fibrosis, showed reduced mt-cfDNA post-KT (p = 0.0195). Changes in mt-cfDNA levels suggests the impact of KT on the inflammatory state of ESKF, as evidenced via its correlation with high sensitivity C-reactive protein after KT. Further studies are warranted to assess if cfDNA could serve as a noninvasive method for monitoring the response to organ transplantation and even for amelioration of EVA status per se.

Kidney outcomes in early adolescence following perinatal asphyxia and hypothermia-treated hypoxic-ischaemic encephalopathy.

BACKGROUND: Acute kidney injury (AKI) remains common among infants with hypothermia-treated hypoxic-ischaemic encephalopathy (HIE). Little is known about long-term kidney outcomes following hypothermia treatment. We recently reported that 21% of survivors of hypothermia-treated HIE had decreased estimated glomerular filtration rate (eGFR) based on plasma creatinine in early adolescence. Here, we assessed kidney functions more comprehensively in our population-based cohort of children born in Stockholm 2007-2009 with a history of hypothermia-treated HIE. METHODS: At 10-12 years of age, we measured cystatin C (cyst C) to estimate GFR. Children with decreased cyst C eGFR also underwent iohexol clearance examination. We measured urine-albumin/creatinine ratio, blood pressure (BP) and kidney volume on magnetic resonance imaging. Fibroblast growth factor 23 (FGF 23) levels in plasma were assessed by enzyme-linked immunosorbent assay (ELISA). Outcomes were compared between children with and without a history of neonatal AKI. RESULTS: Forty-seven children participated in the assessment. Two children (2/42) had decreased cyst C eGFR, for one of whom iohexol clearance confirmed mildly decreased GFR. One child (1/43) had Kidney Disease Improving Global Outcomes (KDIGO) category A2 albuminuria, and three (3/45) had elevated office BP. Subsequent ambulatory 24-h BP measurement confirmed high normal BP in one case only. No child had hypertension. Kidney volume and FGF 23 levels were normal in all children. There was no difference in any of the parameters between children with and without a history of neonatal AKI. CONCLUSION: Renal sequelae were rare in early adolescence following hypothermia-treated HIE regardless of presence or absence of neonatal AKI. A higher resolution version of the Graphical abstract is available as Supplementary information.

Cardiac biomarkers in pediatric CKD-a prospective follow-up study.

BACKGROUND: The N-terminal pro-B-type natriuretic peptide (NT-proBNP) and high-sensitive cardiac-specific troponin T (hs-cTnT) are associated with abnormal cardiac structure and function and an increased risk of cardiovascular death in chronic kidney disease (CKD) patients. There is limited knowledge about these cardiac markers in pediatric CKD patients. METHODS: Longitudinal levels of NT-proBNP and hs-cTnT were analyzed in 48 pediatric patients, 22 with CKD (GFR range 8.8-68 mL/min/1.73 m2) and 26 transplanted patients (CKD-T; GFR range 30-99 mL/min/1.73 m2). Follow-up was scheduled after 1 and 3 years. Longitudinal patterns and associations to kidney function, cardiovascular risk markers, and echocardiographic parameters were assessed. RESULTS: High NT-proBNP was present in 27% of CKD and 11% of CKD-T patients. Similarly 32% of CKD and 8% of CKD-T patients had elevated hs-cTnT levels. In longitudinal multivariate analyses, high log NT-proBNP was associated with low GFR (ß = - 0.01, p = 0.01) and elevated left ventricular mass index (LVMI; ß = 0.02, p = 0.05). The strong association to LVMI remained when using GFR-adjusted NT-proBNP in similar analysis. Patients with left ventricular hypertrophy (LVH) also had higher NT-proBNP (235 [146-301] ng/L) than patients without LVH (86 [11-477] ng/L), p = 0.02. High hs-cTnT over-time was also associated with low GFR (ß = - 0.007, p = 0.01) and a low cc-TDI e´/a´, indicating a worse LV diastolic function (ß = - 0.09, p = 0.05). This association did not persist for GFR-adjusted hs-cTnT. CONCLUSIONS: NT-proBNP and hs-cTnT are elevated in pediatric CKD and CKD-T patients. GFR-adjusted NT-proBNP was associated with longitudinal levels of elevated LVMI suggesting this might be a marker for early subclinical myocardial damage. A higher resolution version of the Graphical abstract is available as Supplementary information.

Hyponatraemia despite isotonic maintenance fluid therapy: a time series intervention study.

OBJECTIVE: To examine the prevalence of dysnatraemias among children admitted for paediatric surgery before and after a change from hypotonic to isotonic intravenous maintenance fluid therapy. DESIGN: Retrospective consecutive time series intervention study. SETTING: Paediatric surgery ward at the Children's Hospital in Lund, during a 7-year period, 2010-2017. PATIENTS: All children with a blood sodium concentration measurement during the study period were included. Hypotonic maintenance fluid (40 mmol/L NaCl and 20 mmol/L KCl) was used during the first 3 years of the study (646 patients), and isotonic solution (140 mmol/L NaCl and 20 mmol/L KCl) was used during the following period (807 patients). MAIN OUTCOME MEASURES: Primary outcomes were sodium concentration and occurrence of hyponatraemia (<135 mmol/L) or hypernatraemia (>145 mmol/L). RESULTS: Overall, the change from hypotonic to isotonic intravenous maintenance fluid therapy was associated with a decreased prevalence of hyponatraemia from 29% to 22% (adjusted OR 0.65 (0.51-0.82)) without a significantly increased odds for hypernatraemia (from 3.4% to 4.3%, adjusted OR 1.2 (0.71-2.1)). Hyponatraemia <130 mmol/L decreased from 6.2% to 2.6%, and hyponatraemia <125 mmol/L decreased from 2.0% to 0.5%. CONCLUSIONS: Routine use of intravenous isotonic maintenance fluids was associated with lower prevalence of hyponatraemia, although hyponatraemia still occurred in over 20% of patients. We propose that the composition and the volume of administered fluid need to be addressed.

Shiga toxin signals via ATP and its effect is blocked by purinergic receptor antagonism.

Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli (EHEC), that cause gastrointestinal infection leading to hemolytic uremic syndrome. The aim of this study was to investigate if Stx signals via ATP and if blockade of purinergic receptors could be protective. Stx induced ATP release from HeLa cells and in a mouse model. Toxin induced rapid calcium influx into HeLa cells, as well as platelets, and a P2X1 receptor antagonist, NF449, abolished this effect. Likewise, the P2X antagonist suramin blocked calcium influx in Hela cells. NF449 did not affect toxin intracellular retrograde transport, however, cells pre-treated with NF449 exhibited significantly higher viability after exposure to Stx for 24 hours, compared to untreated cells. NF449 protected HeLa cells from protein synthesis inhibition and from Stx-induced apoptosis, assayed by caspase 3/7 activity. The latter effect was confirmed by P2X1 receptor silencing. Stx induced the release of toxin-positive HeLa cell- and platelet-derived microvesicles, detected by flow cytometry, an effect significantly reduced by NF449 or suramin. Suramin decreased microvesicle levels in mice injected with Stx or inoculated with Stx-producing EHEC. Taken together, we describe a novel mechanism of Stx-mediated cellular injury associated with ATP signaling and inhibited by P2X receptor blockade.

Early Terminal Complement Blockade and C6 Deficiency Are Protective in Enterohemorrhagic Escherichia coli-Infected Mice.

Complement activation occurs during enterohemorrhagic Escherichia coli (EHEC) infection and may exacerbate renal manifestations. In this study, we show glomerular C5b-9 deposits in the renal biopsy of a child with EHEC-associated hemolytic uremic syndrome. The role of the terminal complement complex, and its blockade as a therapeutic modality, was investigated in a mouse model of E. coli O157:H7 infection. BALB/c mice were treated with monoclonal anti-C5 i.p. on day 3 or 6 after intragastric inoculation and monitored for clinical signs of disease and weight loss for 14 d. All infected untreated mice (15 of 15) or those treated with an irrelevant Ab (8 of 8) developed severe illness. In contrast, only few infected mice treated with anti-C5 on day 3 developed symptoms (three of eight, p < 0.01 compared with mice treated with the irrelevant Ab on day 3) whereas most mice treated with anti-C5 on day 6 developed symptoms (six of eight). C6-deficient C57BL/6 mice were also inoculated with E. coli O157:H7 and only 1 of 14 developed disease, whereas 10 of 16 wild-type mice developed weight loss and severe disease (p < 0.01). Complement activation via the terminal pathway is thus involved in the development of disease in murine EHEC infection. Early blockade of the terminal complement pathway, before the development of symptoms, was largely protective, whereas late blockade was not. Likewise, lack of C6, and thereby deficient terminal complement complex, was protective in murine E. coli O157:H7 infection.

Uropathogenic Escherichia coli modulates immune responses and its curli fimbriae interact with the antimicrobial peptide LL-37.

Bacterial growth in multicellular communities, or biofilms, offers many potential advantages over single-cell growth, including resistance to antimicrobial factors. Here we describe the interaction between the biofilm-promoting components curli fimbriae and cellulose of uropathogenic E. coli and the endogenous antimicrobial defense in the urinary tract. We also demonstrate the impact of this interplay on the pathogenesis of urinary tract infections. Our results suggest that curli and cellulose exhibit differential and complementary functions. Both of these biofilm components were expressed by a high proportion of clinical E. coli isolates. Curli promoted adherence to epithelial cells and resistance against the human antimicrobial peptide LL-37, but also increased the induction of the proinflammatory cytokine IL-8. Cellulose production, on the other hand, reduced immune induction and hence delayed bacterial elimination from the kidneys. Interestingly, LL-37 inhibited curli formation by preventing the polymerization of the major curli subunit, CsgA. Thus, even relatively low concentrations of LL-37 inhibited curli-mediated biofilm formation in vitro. Taken together, our data demonstrate that biofilm components are involved in the pathogenesis of urinary tract infections by E. coli and can be a target of local immune defense mechanisms.

Cytotoxic necrotizing factor 1 (CNF1) induces an inflammatory response in the urinary tract in vitro but not in vivo.

Cytotoxic necrotizing factor 1 (CNF1) is a well-defined virulence factor of uropathogenic Escherichia coli. We studied the role of CNF1 in uroepithelial cells as well as in children and adults with sporadic and recurrent UTI. Our study suggests that CNF1 may promote bacterial attachment and invasion and can induce an inflammatory response in the urinary tract in vitro but that its role in vivo is possibly minor in comparison with other virulence factors of the uropathogenic E. coli.

The antimicrobial peptide cathelicidin protects the urinary tract against invasive bacterial infection.

The urinary tract functions in close proximity to the outside environment, yet must remain free of microbial colonization to avoid disease. The mechanisms for establishing an antimicrobial barrier in this area are not completely understood. Here, we describe the production and function of the cathelicidin antimicrobial peptides LL-37, its precursor hCAP-18 and its ortholog CRAMP in epithelial cells of human and mouse urinary tract, respectively. Bacterial contact with epithelial cells resulted in rapid production and secretion of the respective peptides, and in humans LL-37/hCAP-18 was released into urine. Epithelium-derived cathelicidin substantially contributed to the protection of the urinary tract against infection, as shown using CRAMP-deficient and neutrophil-depleted mice. In addition, clinical E. coli strains that were more resistant to LL-37 caused more severe urinary tract infections than did susceptible strains. Thus, cathelicidin seems to be a key factor in mucosal immunity of the urinary tract.

Non-infected preterm parturition is related to increased concentrations of IL-6, IL-8 and MCP-1 in human cervix.

BACKGROUND: Human cervical ripening is an inflammatory process. In labour at term the mRNA-levels and protein concentrations for interleukin-6 (IL-6) and IL-8 in cervix significantly increase. The aim of this study was to investigate if there are differences in the inflammatory process of preterm and term cervical ripening. METHODS: Cervical biopsies from 50 singleton pregnant women without clinical signs of infection were allocated to four groups: preterm labour, term labour, preterm not in labour and term not in labour. The protein levels of IL-8, IL-6, monocyte chemotactic protein-1 (MCP-1), regulated upon activation normal t cells expressed and secreted (RANTES) and tumour necrosis factor-alpha (TNF-alpha) were quantified in tissue homogenates by ELISA or Immulite. The mRNA expression of IL-8, MCP-1 and RANTES was studied using RT-PCR. White blood cell count (WBC) and C-reactive protein (CRP) in the blood were determined. For determination of statistically significant differences between study groups Mann-Whitney U test or Kruskal-Wallis test were applied. RESULTS: Protein concentrations of IL-8, IL-6, and MCP-1 were significantly increased during labour compared to non-labouring groups, whereas no changes were observed for RANTES and TNF-alpha. The mRNA levels of representative cytokines such as IL-8 and MCP-1 increased significantly during labour whereas RANTES mRNA expression remained unchanged. WBC and CRP were significantly higher in the labouring groups as compared to groups not in labour. For neither of the analysed cytokines, WBC or CRP levels were there any changes between preterm and term respective groups. CONCLUSION: Our findings indicate that non-infected preterm cervical ripening is an inflammatory process, just as cervical ripening at term, with cytokines as important mediators.

Interleukin-8 response in cells from the human urinary tract induced by lipopolysaccharides of Proteus mirabilis O3 and O18.

PURPOSE: Proteus mirabilis is a common pathogen associated mainly with complicated urinary tract infections and sometimes with septicemia. There is great serological diversity of the microorganism. While P. mirabilis O3 is commonly found in patients with infections, the serotype O18 rarely occurs. The O18 lipopolysaccharide contains a phosphocholine substitute, which makes it unique among Proteus strains. To explain different clinical significance of the strains we evaluated the biological activity of the lipopolysaccharides of P. mirabilis O3 and O18, as measured by interleukin-8 (IL-8) production. MATERIALS AND METHODS: Three cell lines were used, namely uroepithelial cells, renal epithelial cells and monocytes. IL-8 production was determined on the protein and mRNA levels using enzyme-linked immunosorbent assay and real-time polymerase chain reaction, respectively, and CD14 expression on the cell surface was studied using flow cytometry. RESULTS: Uroepithelial cells and monocytes reacted to lipopolysaccharides by higher IL-8 production compared with renal epithelial cells. Cell specific IL-8 response corresponded to the cell expression of CD14. Renal epithelial cells produced more IL-8 after stimulation with the phosphocholine rich lipopolysaccharide O18, although adding phosphocholine alone did not induce or increase IL-8 production. CONCLUSIONS: Our data suggest that different cells within the human urinary tract have different roles during urinary tract infection. The biological activity and pathogenicity of P. mirabilis lipopolysaccharides might be determined by their specific chemical structure.

CAPD peritonitis induces the production of a novel peptide, daintain/allograft inflammatory factor-1.

OBJECTIVES: To study the occurrence of a novel macrophage-derived peptide, daintain/allograft inflammatory factor-1 (AIF-1), in dialysate from continuous ambulatory peritoneal dialysis (CAPD) patients at commencement and after a follow-up period of therapy and during peritonitis. In addition, we studied peptide production in response to bacterial stimulation of monocytes and macrophages. DESIGN: Peritoneal fluid and supernatants from cells stimulated with different bacteria were analyzed for daintain/AIF-1. PATIENTS AND SETTING: Peritoneal fluid was obtained from 5 patients at commencement of CAPD therapy and during 8 weeks' follow-up, and from 14 patients (10 males, 4 females) during CAPD peritonitis and during the noninfected steady state. All patients were admitted to the Karolinska Hospital. A human monocyte cell-line, THP-1 was differentiated to macrophages, and both monocytes and macrophages were stimulated with live and heat-inactivated Escherichia coli, Staphylococcus aureus, and S. epidermidis. Cells were also stimulated with interleukin (IL)-1beta and interferon gamma (IFNgamma). Daintain/AIF-1 was analyzed with radioimmunoassay technique and IL-8 with enzyme immunoassay technique. RESULTS: An increased production of daintain/AIF-1 was observed in the first spent dialysate in the newly started CAPD patients, with a decrease during the follow-up period (p < 0.05). During peritonitis, the first spent dialysate revealed significantly higher levels of daintain/AIF-1 (3.9 ng/mL) compared to the noninfected state (0.8 ng/mL), with production normalizing after 9-12 days. Bacterial stimulation with E. coli, S. aureus, or S. epidermidis induced higher daintain/AIF-1 response in monocytes compared to macrophages (p < 0.05). Live bacteria induced higher production of the peptide compared to heat-inactivated bacteria (p < 0.05). Interleukin-1beta and IFNgamma were used to stimulate monocytes and macrophages; however, no daintain/AIF-1 production was found, although increased IL-8 levels were detected. CONCLUSION: CAPD peritonitis induces a high and prominent daintain/AIF-1 response. Bacteria are able to induce a response of the peptide from monocytes and macrophages, and it is likely that the virulent parts of the bacteria are heat-labile structures. The early rise in daintain/ AIF-1 might be used as a marker of CAPD peritonitis.

Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinases-1 in acute pyelonephritis and renal scarring.

The aim of the present study was to elucidate the role of matrix metalloproteinase-9 (MMP-9), and its main inhibitor tissue inhibitor of metalloproteinases-1 (TIMP-1), in acute pyelonephritis and the process of renal scarring. Urine samples from 40 children with acute pyelonephritis, 16 children at 6-wk follow-up and 15 children with nonrenal fever were analyzed using ELISA. MMP-9 and TIMP-1 levels were compared with the outcome of pyelonephritis as measured by renal static scintigraphy. A mouse model of acute ascending pyelonephritis was used to localize the sites of production and the kinetics of MMP-9 and TIMP-1 using immunohistochemistry and ELISA. Human renal epithelial A498 cells, primary mesangial cells and monocytic THP-1 cells were stimulated by Escherichia coli. MMP-9 and TIMP-1 mRNA was analyzed by reverse transcription-PCR (RT-PCR) and protein production by ELISA. We demonstrate a significant increase of MMP-9 and TIMP-1 in the urine of children with acute pyelonephritis. Both proteins were produced mainly by leukocytes, and TIMP-1 also by resident kidney cells. Cells reacted differently after stimulation by bacteria. In mesangial cells and monocytes a decreased constitutive TIMP-1 production was found, which was in contrast to epithelial cells. Out of 40 children with pyelonephritis, 23 had higher urinary TIMP-1 than MMP-9 levels. These children had significantly more severe changes in both acute and follow-up scintigraphy scans indicating higher degree of acute tissue damage and renal scarring. Thus, our findings suggest an association between TIMP-1 and the process of renal scarring.