Whole lung lavage and GM-CSF use for pulmonary alveolar proteinosis in an infant with lysinuric protein intolerance: a case report.

BACKGROUND: Lysinuric protein intolerance (LPI) is a multi-organ metabolic disorder characterized by the imbalance in absorption and excretion of cationic amino acids like lysine, ornithine and arginine. Infants with LPI typically present with recurrent vomiting, poor growth, interstitial lung disease or renal impairment. The early onset of pulmonary alveolar proteinosis (PAP) has been reported to be associated with a severe form of LPI. Treatment of PAP most commonly consists of whole-lung lavage (WLL) and in autoimmune PAP, granulocyte-macrophage colony stimulating factor (GM-CSF) administration. Nevertheless, GM-CSF therapy in LPI-associated PAP has not been scientifically justified. CASE PRESENTATION: We describe the case of an 8-month-old infant presenting with respiratory failure due to LPI associated with PAP, who was twice treated with WLL; firstly, while on veno-venous ECMO assistance and then by the use of a selective bronchial blocker. After the two treatments with WLL, she was weaned from daytime respiratory support while on initially subcutaneous, then on inhaled GM-CSF therapy. CONCLUSIONS: This case supports the notion that GM-CSF therapy might be of benefit in patients with LPI-associated PAP. Further studies are needed to clarify the exact mechanism of GM-CSF in patients with LPI-associated PAP.

Perinatal asphyxia leads to acute kidney damage and increased renal susceptibility in adulthood.

Perinatal asphyxia (PA) poses a significant threat to multiple organs, particularly the kidneys. Diagnosing PA-associated kidney injury remains challenging, and treatment options are inadequate. Furthermore, there is a lack of long-term follow-up data regarding the renal implications of PA. In this study, 7-day-old male Wistar rats were exposed to PA using a gas mixture (4% O2; 20% CO2 in N2 for 15 min) to investigate molecular pathways linked to renal tubular damage, hypoxia, angiogenesis, heat shock response, inflammation, and fibrosis in the kidney. In a second experiment, adult rats with a history of PA were subjected to moderate renal ischemia-reperfusion (IR) injury to test the hypothesis that PA exacerbates renal susceptibility. Our results revealed an increased gene expression of renal injury markers (kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin), hypoxic and heat shock factors (hypoxia-inducible factor-1α, heat shock factor-1, and heat shock protein-27), proinflammatory cytokines (interleukin-1ß, interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1), and fibrotic markers (transforming growth factor-ß, connective tissue growth factor, and fibronectin) promptly after PA. Moreover, a machine learning model was identified through random forest analysis, demonstrating an impressive classification accuracy (95.5%) for PA. Post-PA rats showed exacerbated functional decline and tubular injury and more intense hypoxic, heat shock, proinflammatory, and profibrotic response after renal IR injury compared with controls. In conclusion, PA leads to subclinical kidney injury, which may increase the susceptibility to subsequent renal damage later in life. In addition, the parameters identified through random forest analysis provide a robust foundation for future biomarker research in the context of PA.NEW & NOTEWORTHY This article demonstrates that perinatal asphyxia leads to subclinical kidney injury that permanently increases renal susceptibility to subsequent ischemic injury. We identified major molecular pathways involved in perinatal asphyxia-induced renal complications, highlighting potential targets of therapeutic approaches. In addition, random forest analysis revealed a model that classifies perinatal asphyxia with 95.5% accuracy that may provide a strong foundation for further biomarker research. These findings underscore the importance of multiorgan follow-up for perinatal asphyxia-affected patients.

Human peritoneal tight junction, transporter and channel expression in health and kidney failure, and associated solute transport.

Next to the skin, the peritoneum is the largest human organ, essentially involved in abdominal health and disease states, but information on peritoneal paracellular tight junctions and transcellular channels and transporters relative to peritoneal transmembrane transport is scant. We studied their peritoneal localization and quantity by immunohistochemistry and confocal microscopy in health, in chronic kidney disease (CKD) and on peritoneal dialysis (PD), with the latter allowing for functional characterizations, in a total of 93 individuals (0-75 years). Claudin-1 to -5, and -15, zonula occludens-1, occludin and tricellulin, SGLT1, PiT1/SLC20A1 and ENaC were consistently detected in mesothelial and arteriolar endothelial cells, with age dependent differences for mesothelial claudin-1 and arteriolar claudin-2/3. In CKD mesothelial claudin-1 and arteriolar claudin-2 and -3 were more abundant. Peritonea from PD patients exhibited increased mesothelial and arteriolar claudin-1 and mesothelial claudin-2 abundance and reduced mesothelial and arteriolar claudin-3 and arteriolar ENaC. Transperitoneal creatinine and glucose transport correlated with pore forming arteriolar claudin-2 and mesothelial claudin-4/-15, and creatinine transport with mesothelial sodium/phosphate cotransporter PiT1/SLC20A1. In multivariable analysis, claudin-2 independently predicted the peritoneal transport rates. In conclusion, tight junction, transcellular transporter and channel proteins are consistently expressed in peritoneal mesothelial and endothelial cells with minor variations across age groups, specific modifications by CKD and PD and distinct associations with transperitoneal creatinine and glucose transport rates. The latter deserve experimental studies to demonstrate mechanistic links.Clinical Trial registration: The study was performed according to the Declaration of Helsinki and is registered at www.clinicaltrials.gov (NCT01893710).

Neonatal Screening for Cystic Fibrosis in Hungary-First-Year Experiences.

The aim of this study is to evaluate the strategy of the cystic fibrosis newborn screening (CFNBS) programme in Hungary based on the results of the first year of screening. A combined immunoreactive trypsinogen (IRT) and pancreatitis-associated protein (PAP) CFNBS protocol (IRT/IRT×PAP/IRT) was applied with an IRT-dependent safety net (SN). Out of 88,400 newborns, 256 were tested screen-positive. Fourteen cystic fibrosis (CF) and two cystic fibrosis-positive inconclusive diagnosis (CFSPID) cases were confirmed from the screen-positive cases, and two false-negative cases were diagnosed later. Based on the obtained results, a sensitivity of 88% and a positive predictive value (PPV) of 5.9% were calculated. Following the recognition of false-negative cases, the calculation method of the age-dependent cut-off was changed. In purely biochemical CFNBS protocols, a small protocol change, even after a short period, can have a significant positive impact on the performance. CFNBS should be monitored continuously in order to fine-tune the screening strategy and define the best local practices.

How to define and assess the clinically significant causes of hematuria in childhood.

Given the wide diversity of causes of hematuria, ranging from simple urinary tract infections with rapid recovery to severe glomerulonephritis with fast decline in kidney function, it is essential to recognize the underlying disease. The first objective of the assessment is to determine whether the cause of the hematuria is medically significant. The combination of hematuria with proteinuria, the presence of hypertension, or worsening kidney function can represent signs of progressive kidney disease. Differentiating the various causes of hematuria is often simple and obvious based on the clinical signs and gross appearance of the urine. However, in some instances, additional non-invasive investigations, such as ultrasound imaging, urinary red cell morphology, measurement of calcium and other solutes in the urine, evaluation of kidney function, and protein excretion, are needed to elucidate the nature of the hematuria. Taking a detailed family history can help in establishing the underlying cause in cases of familial hematuria. On the other hand, the decision to perform a kidney biopsy in children with asymptomatic hematuria remains a challenging issue for clinicians. Ultimately, the frequency of diagnosis of glomerular involvement causing hematuria may depend on the threshold for performing a kidney biopsy. The following review will focus on the diagnostics of hematuria, starting with difficulties regarding its definition, followed by various means to differentiate between urinary, glomerular, and other causes, and finally reviewing the most common diseases that, due to their frequency or their effect on kidney function, present a diagnostic challenge in everyday practice.

Long-term survival benefit of male and multimorbid COVID-19 patients with 5-day remdesivir treatment.

Background: Treatment of the coronavirus disease (COVID-19) is still challenging due to the lack of evidence-based treatment protocols and continuously changing epidemiological situations and vaccinations. Remdesivir (RDV) is among the few antiviral medications with confirmed efficacy for specific patient groups. However, real-world data on long-term outcomes for a short treatment course are scarce. Methods: This retrospective observational cohort study included real-life data collected during the second and third wave of the COVID-19 pandemic in Hungary (September 1, 2020-April 30, 2021) from inpatients at a University Center (n = 947). Participants consisted of two propensity score-matched cohorts (370/370 cases): Group RDV including patients receiving RDV and supplementary oxygen and Group standard of care (SOC) as control. The primary outcome was the effect of 5-day RDV treatment on 30- and 60-day all-cause mortality. Multivariate analyses were performed to assess the effect of RDV by different covariates. Results: Group RDV included significantly more patients from the alpha variant wave, with greater frequency of comorbidities diabetes and anemia, and larger degree of parenchymal involvement. All-cause mortality at 30- and 60-day were significantly lower in Group RDV compared to Group SOC. Significant risk reduction of 60-day all-cause mortality was observed for RDV treatment in men and patients with COPD or multiple comorbidities. Conclusions: Hospitalized COVID-19 patients with 5-day RDV treatment had significantly lower 30- and 60-day all-cause mortality, despite their more severe clinical condition. Men and patients with multiple comorbidities, including COPD, profited the most from RDV treatment in the long term. Due to the ongoing COVID-19 pandemic, effective treatment regimens are needed for hospitalized patients.

Transient Agarose Spot (TAS) Assay: A New Method to Investigate Cell Migration.

Fibroblasts play a central role in diseases associated with excessive deposition of extracellular matrix (ECM), including idiopathic pulmonary fibrosis. Investigation of different properties of fibroblasts, such as migration, proliferation, and collagen-rich ECM production is unavoidable both in basic research and in the development of antifibrotic drugs. In the present study we developed a cost-effective, 96-well plate-based method to examine the migration of fibroblasts, as an alternative approach to the gold standard scratch assay, which has numerous limitations. This article presents a detailed description of our transient agarose spot (TAS) assay, with instructions for its routine application. Advantages of combined use of different functional assays for fibroblast activation in drug development are also discussed by examining the effect of nintedanib-an FDA approved drug against IPF-on lung fibroblasts.

Atypical HUS and Crohn's disease-interference of intestinal disease activity with complement-blocking treatment.

BACKGROUND: In atypical hemolytic-uremic syndrome (aHUS), various defects of the complement system have been reported to explain pathophysiology. Therapeutic options for complement inhibition are well-recognized; however, the links between various immune-derived diseases and aHUS are unclear, and their interference with treatment efficacy during long-term complement-blocking therapy is scarcely known. CASE-DIAGNOSIS/TREATMENT: We present a pediatric patient who developed aHUS with acute kidney injury in parallel with the onset of Crohn's disease (CD), and who required long-term complement-blocking therapy with eculizumab (ECU). Unexpectedly, during the 6-year ECU treatment, an important intra-patient variation of the degree of complement inhibition was observed. In spite of continuous and stable doses of complement-blocking therapy, periods of incomplete blockade were observed in strong association with relapses of CD. When conventional and later biological therapy with adalimumab was introduced, with CD going into remission, complement blockade became complete again. Despite periodically low ECU levels and insufficient complement inhibition, no clinical or hematological signs of aHUS recurrence were detected during CD relapses. CONCLUSION: In aHUS cases secondary to CD, close monitoring of both complement inhibition and serum ECU levels is needed as intestinal disease can interfere with complement-blocking treatment. Increased doses of ECU may be necessary to maintain therapeutic blood levels of ECU and full complement blockade, especially if the intestinal disease is not under control.

Immunomodulatory role of Parkinson's disease 7 in inflammatory bowel disease.

Recently the role of Parkinson's disease 7 (PARK7) was studied in gastrointestinal diseases, however, the complex role of PARK7 in the intestinal inflammation is still not completely clear. Expression and localization of PARK7 were determined in the colon biopsies of children with inflammatory bowel disease (IBD), in the colon of dextran sodium sulphate (DSS) treated mice and in HT-29 colonic epithelial cells treated with interleukin (IL)-17, hydrogen peroxide (H2O2), tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-ß or lipopolysaccharide (LPS). Effect of PARK7 on the synthesis of IBD related cytokines was determined using PARK7 gene silenced HT-29 cells and 3,4,5-trimethoxy-N-(4-(8-methylimidazo(1,2-a)pyridine-2-yl)phenyl)benzamide (Comp23)-compound increasing PARK7 activity-treated mice with DSS-colitis. PARK7 expression was higher in the mucosa of children with Crohn's disease compared to that of controls. While H2O2 and IL-17 treatment increased, LPS, TNF-α or TGF-ß treatment decreased the PARK7 synthesis of HT-29 cells. PARK7 gene silencing influenced the synthesis of IL1B, IL6, TNFA and TGFB1 in vitro. Comp23 treatment attenuated the ex vivo permeability of colonic sacs, the clinical symptoms, and mucosal expression of Tgfb1, Il1b, Il6 and Il10 of DSS-treated mice. Our study revealed the role of PARK7 in the regulation of IBD-related inflammation in vitro and in vivo, suggesting its importance as a future therapeutic target.

PARK7 Diminishes Oxidative Stress-Induced Mucosal Damage in Celiac Disease.

Coeliac disease (CD) is a chronic, immune-mediated small intestinal enteropathy, accompanied with gluten-triggered oxidative damage of duodenal mucosa. Previously, our research group reported an increased mucosal level of the antioxidant protein Parkinson's disease 7 (PARK7) in children with CD. In the present study, we investigated the role of increased PARK7 level on the epithelial cell and mucosal integrity of the small intestine. The presence of PARK7 was investigated using immunofluorescent staining on duodenal mucosa of children with CD and on FHs74Int duodenal epithelial cells. To investigate the role of oxidative stress, FHs74Int cells were treated with H2O2 in the absence or presence of Comp23, a PARK7-binding compound. Intracellular accumulation of reactive oxygen species (ROS) was determined by DCFDA-based assay. Cell viability was measured by MTT, LDH, and Annexin V apoptosis assays. Disruption of cytoskeleton and cell adhesion was investigated by immunofluorescence staining and by real-time RT PCR. Effect of PARK7 on mucosal permeability was investigated ex vivo using intestinal sacs derived from control and Comp-23-pretreated mice. Comp23 treatment reduced the H2O2-induced intracellular accumulation of ROS, thus preserving the integrity of the cytoskeleton and also the viability of the FHs74Int cells. Accordingly, Comp23 treatment increased the expression of antioxidants (NRF2, TRX1, GCLC, HMOX1, NQO1), cell-cycle regulators (TP53, CDKN1A, PCNA, BCL2, BAX), and cell adhesion molecules (ZO1, CDH1, VCL, ITGB5) of H2O2-treated cells. Pretreatment with Comp23 considerably decreased the small intestinal permeability. In this study, we demonstrate that PARK7-binding Comp23 reduces the oxidative damage of duodenal epithelial cells, via increased expression of NRF2- and P53-regulated genes. Our results suggest that PARK7 plays a significant role in the maintenance of mucosal integrity in CD.

[The immunomodulatory role of sodium]. / A nátrium immunmoduláns szerepe.

High salt intake, which is common in the Western world, is the cause of several lifestyle diseases. Recent investigations shed light on novel extrarenal processes, which play role in the maintenance of sodium balance. In the short term, sodium storage of the skin may serve as a buffer against volume overload arising from the osmotic properties of sodium. Increased tissue sodium concentration may also potentiate immune response against infections. In the long run, however, tissue sodium concentration over a certain limit may initiate pathophysiological processes by provoking inflammatory response. Due to the immune modulating role of sodium, the effector cells of the innate as well as the adaptive immune system are activated, while certain regulator cells of the same systems are repressed, ultimately resulting in a proinflammatory state characterized by the imbalance of the immune system. Experiments applying dietary salt overload/salt depletion imply the role of sodium in the initiation/exacerbation of several diseases. Thus the relationship between sodium and the immune system may give an explanation to the pathomechanism of diseases with so far unknown origin such as hypertonia (primary, salt sensitive) or autoimmune diseases - all these putting tremendous pressure on the healthcare system due to their increasing incidence. Orv Hetil. 2019; 160(17): 646-653.

RAAS inhibitors directly reduce diabetes-induced renal fibrosis via growth factor inhibition.

KEY POINTS: Increased activation of the renin-angiotensin-aldosterone system (RAAS) and elevated growth factor production are of crucial importance in the development of renal fibrosis leading to diabetic kidney disease. The aim of this study was to provide evidence for the antifibrotic potential of RAAS inhibitor (RAASi) treatment and to explore the exact mechanism of this protective effect. We found that RAASi ameliorate diabetes-induced renal interstitial fibrosis and decrease profibrotic growth factor production. RAASi prevents fibrosis by acting directly on proximal tubular cells, and inhibits hyperglycaemia-induced growth factor production and thereby fibroblast activation. These results suggest a novel therapeutic indication and potential of RAASi in the treatment of renal fibrosis. ABSTRACT: In diabetic kidney disease (DKD) increased activation of renin-angiotensin-aldosterone system (RAAS) contributes to renal fibrosis. Although RAAS inhibitors (RAASi) are the gold standard therapy in DKD, the mechanism of their antifibrotic effect is not yet clarified. Here we tested the antifibrotic and renoprotective action of RAASi in a rat model of streptozotocin-induced DKD. In vitro studies on proximal tubular cells and renal fibroblasts were also performed to further clarify the signal transduction pathways that are directly altered by hyperglycaemia. After 5 weeks of diabetes, male Wistar rats were treated for two more weeks per os with the RAASi ramipril, losartan, spironolactone or eplerenone. Proximal tubular cells were cultured in normal or high glucose (HG) medium and treated with RAASi. Platelet-derived growth factor (PDGF) or connective tissue growth factor (CTGF/CCN2)-induced renal fibroblasts were also treated with various RAASi. In diabetic rats, reduced renal function and interstitial fibrosis were ameliorated and elevated renal profibrotic factors (TGFß1, PDGF, CTGF/CCN2, MMP2, TIMP1) and alpha-smooth muscle actin (αSMA) levels were decreased by RAASi. HG increased growth factor production of HK-2 cells, which in turn induced activation and αSMA production of fibroblasts. RAASi decreased tubular PDGF and CTGF expression and reduced production of extracellular matrix (ECM) components in fibroblasts. In proximal tubular cells, hyperglycaemia-induced growth factor production increased renal fibroblast transformation, contributing to the development of fibrosis. RAASi, even in non-antihypertensive doses, decreased the production of profibrotic factors and directly prevented fibroblast activation. All these findings suggest a novel therapeutic role for RAASi in the treatment of renal fibrosis.

The role of Sigma-1 receptor in sex-specific heat shock response in an experimental rat model of renal ischaemia/reperfusion injury.

We previously showed that female rats are more protected against renal ischaemia/reperfusion (I/R) injury than males, which is partly attributed to their more pronounced heat shock response. We recently described that Sigma-1 receptor (S1R) activation improves postischaemic survival and renal function. 17ß-estradiol activates S1R, thus here we investigated the role of sex-specific S1R activation and heat shock response in severe renal I/R injury. Proximal tubular cells were treated with 17ß-estradiol, which caused direct S1R activation and subsequent induction of heat shock response. Uninephrectomized female, male and ovariectomized female (Ovx) Wistar rats were subjected to 50-min renal ischaemia followed by 2 (T2) and 24 (T24) hours of reperfusion. At T24 renal functional, impairment was less severe and structural damage was less prominent in females versus males or Ovx. Postischaemic increase in S1R, pAkt, HSF-1, HSP72 levels were detected as early as at T2, while pHSP27 was elevated later at T24. Abundance of heat shock proteins was higher in healthy female rats and remained higher at T2 and T24 (female versus male or Ovx; resp.). We propose a S1R-dependent mechanism, which contributes to the relative renoprotection of females after I/R injury by enhancing the heat shock response.

Association between interferon-induced helicase (IFIH1) rs1990760 polymorphism and seasonal variation in the onset of type 1 diabetes mellitus.

BACKGROUND: Infections, mostly of viral origin, may contribute to the seasonal variation in the onset of type 1 diabetes mellitus (T1DM). The rs1990760 (A>G, Ala946Thr) polymorphism (GG genotype) of the interferon induced helicase (IFIH1), a virus recognition receptor, confers a modest protection for T1DM. The aim of our study was to evaluate a possible association between this IFIH1 polymorphism and the seasonal variation in the onset of T1DM. MATERIALS AND METHODS: The IFIH1 rs1990760 polymorphism was genotyped in 1055 patients of Central-Eastern European ancestry with T1DM (median age at diagnosis: 8.2 [interquartile range, IQR 4.8-11.8] years). T1DM onset was recorded in monthly intervals. RESULTS: The IFIH1 genotype distribution was the following: 436 patients (41.3%) had AA genotype, 483 patients (45.8%) had AG genotype, and 136 patients (12.9%) had GG genotype. Significant seasonal variation in manifestation of T1DM (highest rate in winter and lowest rate in summer period) was observed in the total cohort (n = 1055), irrespective of gender. The disease predisposing AA genotype was more frequently found among new cases with onset in summer vs in those with onset in winter (44.3% vs 37.9%); conversely, the protective GG genotype was less frequent (9.3% vs 12.9%, respectively; P = .0268 for trend). Significant effect of genotype (P = .0418) was found on the seasonal variability of T1DM onset in the total cohort. CONCLUSIONS: The IFIH1 rs1990760 polymorphism seems to be associated with the seasonal manifestation of T1DM. Our findings suggest that this virus receptor gene may contribute to T1DM manifestation primarily in the summer period.

Specific MicroRNA Pattern in Colon Tissue of Young Children with Eosinophilic Colitis.

Eosinophilic colitis (EC) is a common cause of haematochezia in infants and young children. The exact pathomechanism is not understood, and the diagnosis is challenging. The role of microRNAs as key class of regulators of mRNA expression and translation in patients with EC has not been explored. Therefore, the aim of the present study was to explore the miRNA profile in EC with respect to eosinophilic inflammation. Patients enrolled in the study (n = 10) had persistent rectal bleeding, and did not respond to elimination dietary treatment. High-throughput microRNA sequencing was carried out on colonic biopsy specimens of children with EC (EC: n = 4) and controls (C: n = 4) as a preliminary screening of the miRNA profile. Based on the next-generation sequencing (NGS) results and literature data, a potentially relevant panel of miRNAs were selected for further measurements by real-time reverse transcription (RT)-PCR (EC: n = 14, C: n = 10). Validation by RT-PCR resulted in significantly altered expression of miR-21, -31, -99b, -125a, -146a, -184, -221, -223, and -559 compared to controls (p ≤ 0.05). Elevation in miR-21, -99b, -146a, -221, and -223 showed statistically significant correlation to the extent of tissue eosinophilia. Based on our results, we conclude that the dysregulated miRNAs have a potential role in the regulation of apoptosis by targeting Protein kinase B/Mechanistic target of rapamycin (AKT/mTOR)-related pathways in inflammation by modulating Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-related signalling and eosinophil cell recruitment and activation, mainly by regulating the expression of the chemoattractant eotaxin and the adhesion molecule CD44. Our results could serve as a basis for further extended research exploring the pathomechanism of EC.

Increased duodenal expression of miR-146a and -155 in pediatric Crohn's disease.

AIM: To evaluate the role of microRNA (miR)-146a, -155 and -122 in the duodenal mucosa of pediatric patients with Crohn's disease (CD) and the effect of transforming growth factor-ß (TGF-ß) on these miRs in duodenal epithelial and fibroblast cells. METHODS: Formalin-fixed, paraffin-embedded biopsies derived from the macroscopically inflamed (CD inflamed: n = 10) and intact (CD intact: n = 10) duodenal mucosa of pediatric CD patients and control children (C: n = 10) were examined. Expression of miR-146a, -155 and -122 was determined by real-time polymerase-chain reaction (PCR). The expression of the above miRs was investigated in recombinant human TGF-ß (1 nmol/L, 24 h) or vehicle treated small intestinal epithelial cells (CCL-241) and primary duodenal fibroblast cells derived from healthy children as well. RESULTS: Expression of miR-146a was significantly higher in the inflamed duodenal mucosa compared to the intact duodenal mucosa of children with CD (CD inflamed: 3.21 ± 0.50 vs CD intact: 0.62 ± 0.26, P ≤ 0.01) and to the control group (CD inflamed: 3.21 ± 0.50 vs C: 1.00 ± 0.33, P ≤ 0.05). The expression of miR-155 was significantly increased in the inflamed region of the duodenum compared to the control group (CD inflamed: 4.87 ± 1.02 vs CONTROL: 1.00 ± 0.40, P ≤ 0.001). The expression of miR-122 was unchanged in the inflamed or intact mucosa of CD patients compared to controls. TGF-ß treatment significantly decreased the expression of miR-155 in small intestinal epithelial cells (TGF-ß: 0.7 ± 0.083 vs CONTROL: 1 ± 0.09, P ≤ 0.05) and also the expression of miR-146a (TGF-ß: 0.67 ± 0.04 vs CONTROL: 1 ± 0.15, P ≤ 0.01) and miR-155 (TGF-ß: 0.72 ± 0.09 vs CONTROL: 1 ± 0.06, P ≤ 0.05) in primary duodenal fibroblasts compared to corresponding vehicle treated controls. TGF-ß treatment did not influence the expression of miR-122. CONCLUSION: The elevated expression of miR-146a and -155 in the inflamed duodenal mucosa of CD patients suggests the role of these miRs in the pathomechanism of inflammatory bowel disease. Anti-inflammatory TGF-ß plays an important role in the regulation of the expression of these miRs.

Role of O-linked N-acetylglucosamine modification in diabetic nephropathy.

Increased O-linked ß-N-acetylglucosamine glycosylation (O-GlcNAcylation) is a known contributor to diabetes; however, its relevance in diabetic nephropathy (DN) is poorly elucidated. Here, we studied the process and enzymes of O-GlcNAcylation with a special emphasis on Akt-endothelial nitric oxide synthase (eNOS) and heat shock protein (HSP)72 signaling. Since tubular injury is the prominent site of DN, the effect of hyperglycemia was first measured in proximal tubular (HK2) cells cultured in high glucose. In vivo O-GlcNAcylation and protein levels of O-GlcNAc transferase (OGT), O-GlcNAcase (OGA), phosphorylated (p)Akt/Akt, peNOS/eNOS, and HSP72 were assessed in the kidney cortex of streptozotocin-induced diabetic rats. The effects of various renin-angiotensin-aldosterone system (RAAS) inhibitors were also evaluated. In proximal tubular cells, hyperglycemia-induced OGT expression led to increased O-GlcNAcylation, which was followed by a compensatory increase of OGA. In parallel, peNOS and pAkt levels decreased, whereas HSP72 increased. In diabetic rats, elevated O-GlcNAcylation was accompanied by decreased OGT and OGA. RAAS inhibitors ameliorated diabetes-induced kidney damage and prevented the elevation of O-GlcNAcylation and the decrement of pAkt, peNOS, and HSP72. In conclusion, hyperglycemia-induced elevation of O-GlcNAcylation contributes to the progression of DN via inhibition of Akt/eNOS phosphorylation and HSP72 induction. RAAS blockers successfully inhibit this process, suggesting a novel pathomechanism of their renoprotective action in the treatment of DN.

Role of Altered Expression of miR-146a, miR-155, and miR-122 in Pediatric Patients with Inflammatory Bowel Disease.

BACKGROUND: Evidence suggests the central role of tumor necrosis factor (TNF)-α in the pathomechanism of inflammatory bowel disease (IBD); however, its effect on epigenetic factors, including small non-coding microRNAs (miRs), is less known. Our present aim was the comparative investigation of the expression of TNF-α and immune response-related miRs in children with Crohn's disease (CD) and ulcerative colitis (UC). METHODS: Fresh-frozen (FF) and formalin-fixed, paraffin-embedded (FFPE) biopsies were used to analyze the expression of miR-146a, -155, -122, and TNF-α by real-time reverse transcription polymerase chain reaction in macroscopically inflamed (CD: 12 FFPE and 24 FF; UC: 10 FF) and intact (CD: 12 FFPE; 14 FF) colonic biopsies of children with IBD and controls (16 FFPE; 23 FF). The expression of miR-146a, -155, and -122 was also determined in TNF-α-treated HT-29 colonic epithelial cells. RESULTS: Increased expression of TNF-α was observed in the colonic mucosa of children with CD and UC in comparison with controls. Expression of miR-146a and -155 was higher in the inflamed mucosa of children with CD and UC than in the intact mucosa. Expression of miR-122 elevated in the macroscopically intact colonic regions of CD compared with controls and patients with UC. In HT-29 cells, TNF-α treatment increased the expression of miR-146a and -155, but not that of miR-122. CONCLUSIONS: Our results showed altered expression of miR-146a, -155, and -122 in the colonic mucosa of children with IBD and in TNF-α-treated colonic epithelial cells. Our data suggest the TNF-α-related involvement of these miRs in the pathogenesis of IBD.

Calcineurin-inhibition Results in Upregulation of Local Renin and Subsequent Vascular Endothelial Growth Factor Production in Renal Collecting Ducts.

BACKGROUND: Tacrolimus (Tac) and Cyclosporine A (CyA) calcineurin inhibitors (CNIs) are 2 effective immunosuppressants which are essential to prevent allograft rejection. Calcineurin inhibitors are known to be nephrotoxic. However, the precise mechanism of nephrotoxicity is not fully understood. In this study, we investigated the in vivo effects of CNIs on the local renal renin-angiotensin system in the collecting duct (CD). METHODS: Three-week-old mice were treated with either vehicle, CyA (2 mg/kg per day), Tac (0.075 mg/kg per day), CyA + Aliskiren (25 mg/kg per day), or Tac + Aliskiren for 3 weeks. Serum creatinine was measured. Renin and vascular endothelial growth factor (VEGF) contents in CD were evaluated with flow cytometry and multiphoton microscopy. The diameter of vessels was assessed with multiphoton microscopy, and the amount of renal collagen was determined by real-time polymerase chain reaction and Masson staining. RESULTS: The elevated level of serum creatinine in CNI groups was abolished by Aliskiren. Flow cytometric analysis found elevated renin content in principal cells, which was prevented by Aliskiren. This result was further confirmed with multiphoton microscopy. The VEGF content in CD correlated with reduced capillary diameter and with the formation of fibrotic islands. CONCLUSIONS: Calcineurin inhibitors induce production of renin in the CD that may contribute to decreased renal blood flow. In turn, CD responds with increased VEGF production, resulting in disproportional vessel growth, further worsening the local hypoxia and striped fibrosis surrounding the CDs. Aliskiren, a direct renin inhibitor blocks these effects and improves CNI-induced nephropathy by decreasing renin production in the CDs. Our data suggest that Aliskiren may be used for the prevention of CNI nephrotoxicity.

Severe Hyperinsulinemic Hypoglycemia in a Neonate: Response to Sirolimus Therapy.

Hyperinsulinemic hypoglycemia (HH) is one of the most common causes of persistent hypoglycemic episodes in neonates. Current pharmacologic treatment of neonatal HH includes diazoxide and octreotide, whereas for diffuse, unresponsive cases a subtotal pancreatectomy may be the last resort, with questionable efficacy. Here we report a case of congenital diffuse neonatal HH, first suspected when severe hypoglycemia presented with extremely high serum insulin levels immediately after birth. Functional imaging and genetic tests later confirmed the diagnosis. Failure to respond to a sequence of different treatments and to avoid extensive surgery with predictable morbidity prompted us to introduce a recently suggested alternative therapy with sirolimus, a mammalian target of rapamycin inhibitor. Glucose intake could be reduced gradually while euglycemia was maintained, and we were able to achieve exclusively enteral feeding within 6 weeks. Sirolimus was found to be effective and well tolerated, with no major adverse side effects attributable to its administration.