Lung-Kidney Axis in Cystic Fibrosis: Early Urinary Markers of Kidney Injury Correlate with Neutrophil Activation and Worse Lung Function.

Background: Adult people with cystic fibrosis (PwCF) have a higher risk of end-stage kidney disease than the general population. The nature and mechanism of kidney disease in CF are unknown. This study quantifies urinary kidney injury markers and examines the hypothesis that neutrophil activation and lung infection are associated with early kidney injury in CF. Methods: Urinary total protein, albumin, and markers of kidney injury and neutrophil activation, normalized to creatinine, as well as urinary immune cells, were quantified in CF (n = 48) and healthy (n = 33) cohorts. Infection burden and chronicity were defined by sputum culture and serum titers of anti-bacterial antibodies. Results: PwCF had increased urinary protein levels, consisting of low-molecular-weight tubular injury markers, independent of glomerular filtration rate (eGFR). This finding suggests subclinical renal injury processes. Urinary analysis of the CF cohort identified different associations of urinary injury markers with aminoglycoside exposure, lung function, and neutrophil activation. High urinary KIM-1 levels and increased prevalence of neutrophils among urine immune cells correlated with decreased lung function in PwCF. The relationship between tubular injury and decreased lung function was most prominent in patients harboring chronic Pseudomonas aeruginosa infection. Conclusions: Increased urinary tubular injury markers in PwCF suggest early subclinical renal injury not readily detected by eGFR. The strong association of high urinary KIM-1 and neutrophils with diminished lung function and high Pseudomonas aeruginosa burden suggests that pulmonary disease may contribute to renal injury in CF.

LMTK2 switches on canonical TGF-ß1 signaling in human bronchial epithelial cells.

Transforming growth factor (TGF-ß1) is a critical profibrotic mediator in chronic lung disease, and there are no specific strategies to mitigate its adverse effects. Activation of TGF-ß1 signaling is a multipart process involving ligands, transmembrane receptors, and transcription factors. In addition, an intricate network of adaptor proteins fine-tunes the signaling strength, duration, and activity. Namely, Smad7 recruits growth arrest and DNA damage (GADD34) protein that then interacts with the catalytic subunit of phosphoprotein phosphatase 1 (PP1c) to inactivate TGF-ß receptor (TßR)-I and downregulate TGF-ß1 signaling. Little is known about how TGF-ß1 releases TßR-I from the GADD34-PP1c inhibition to activate its signaling. Transmembrane lemur tyrosine kinase 2 (LMTK2) is a PP1c inhibitor, and our published data showed that TGF-ß1 recruits LMTK2 to the cell surface. Here, we tested the hypothesis that TGF-ß1 recruits LMTK2 to inhibit PP1c, allowing activation of TßR-I. First, LMTK2 interacted with the TGF-ß1 pathway in the human bronchial epithelium at multiple checkpoints. Second, TGF-ß1 inhibited PP1c by an LMTK2-dependent mechanism. Third, TGF-ß1 used LMTK2 to activate canonical Smad3-mediated signaling. We propose a model whereby the LMTK2-PP1c and Smad7-GADD34-PP1c complexes serve as on-and-off switches in the TGF-ß1 signaling in human bronchial epithelium.NEW & NOTEWORTHY Activation of the transforming growth factor (TGF)-ß1 signaling pathway is complex, involving many ligands, transmembrane receptors, transcription factors, and modulating proteins. The mechanisms of TGF-ß1 signaling activation/inactivation are not fully understood. We propose for the first time a model by which transmembrane lemur tyrosine kinase 2 (LMTK2) forms a complex with phosphoprotein phosphatase 1 (PP1c) to activate TGF-ß1 signaling and Smad7, growth arrest and DNA damage (GADD34), and PP1C form a complex to inactivate TGF-ß1 signaling in human bronchial epithelium.

Beyond Borders of the Cell: How Extracellular Vesicles Shape COVID-19 for People with Cystic Fibrosis.

The interaction between extracellular vesicles (EVs) and SARS-CoV-2, the virus causing COVID-19, especially in people with cystic fibrosis (PwCF) is insufficiently studied. EVs are small membrane-bound particles involved in cell-cell communications in different physiological and pathological conditions, including inflammation and infection. The CF airway cells release EVs that differ from those released by healthy cells and may play an intriguing role in regulating the inflammatory response to SARS-CoV-2. On the one hand, EVs may activate neutrophils and exacerbate inflammation. On the other hand, EVs may block IL-6, a pro-inflammatory cytokine associated with severe COVID-19, and protect PwCF from adverse outcomes. EVs are regulated by TGF-ß signaling, essential in different disease states, including COVID-19. Here, we review the knowledge, identify the gaps in understanding, and suggest future research directions to elucidate the role of EVs in PwCF during COVID-19.

A revised nomenclature for the lemur family of protein kinases.

The lemur family of protein kinases has gained much interest in recent years as they are involved in a variety of cellular processes including regulation of axonal transport and endosomal trafficking, modulation of synaptic functions, memory and learning, and they are centrally placed in several intracellular signalling pathways. Numerous studies have also implicated role of the lemur kinases in the development and progression of a wide range of cancers, cystic fibrosis, and neurodegenerative diseases. However, parallel discoveries and inaccurate prediction of their kinase activity have resulted in a confusing and misleading nomenclature of these proteins. Herein, a group of international scientists with expertise in lemur family of protein kinases set forth a novel nomenclature to rectify this problem and ultimately help the scientific community by providing consistent information about these molecules.

Post-transplant recurrence of focal segmental glomerular sclerosis: consensus statements.

Focal segmental glomerular sclerosis (FSGS) is 1 of the primary causes of nephrotic syndrome in both pediatric and adult patients, which can lead to end-stage kidney disease. Recurrence of FSGS after kidney transplantation significantly increases allograft loss, leading to morbidity and mortality. Currently, there are no consensus guidelines for identifying those patients who are at risk for recurrence or for the management of recurrent FSGS. Our work group performed a literature search on PubMed/Medline, Embase, and Cochrane, and recommendations were proposed and graded for strength of evidence. Of the 614 initially identified studies, 221 were found suitable to formulate consensus guidelines for recurrent FSGS. These guidelines focus on the definition, epidemiology, risk factors, pathogenesis, and management of recurrent FSGS. We conclude that additional studies are required to strengthen the recommendations proposed in this review.

TGF-ß1 Inhibition of ACE2 Mediated by miRNA Uncovers Novel Mechanism of SARS-CoV-2 Pathogenesis.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for COVID-19, utilizes receptor binding domain (RBD) of spike glycoprotein to interact with angiotensin (Ang)-converting enzyme 2 (ACE2). Altering ACE2 levels may affect entry of SARS-CoV-2 and recovery from COVID-19. Decreased cell surface density of ACE2 leads to increased local levels of Ang II and may contribute to mortality resulting from acute lung injury and fibrosis during COVID-19. Studies published early during the COVID-19 pandemic reported that people with cystic fibrosis (PwCF) had milder symptoms, compared to people without CF. This finding was attributed to elevated ACE2 levels and/or treatment with the high efficiency CFTR modulators. Subsequent studies did not confirm these findings reporting variable effects of CFTR gene mutations on ACE2 levels. Transforming growth factor (TGF)-ß signaling is essential during SARS-CoV-2 infection and dominates the chronic immune response in severe COVID-19, leading to pulmonary fibrosis. TGF-ß1 is a gene modifier associated with more severe lung disease in PwCF but its effects on the COVID-19 course in PwCF is unknown. To understand whether TGF-ß1 affects ACE2 levels in the airway, we examined miRNAs and their gene targets affecting SARS-CoV-2 pathogenesis in response to TGF-ß1. Small RNAseq and micro(mi)RNA profiling identified pathways uniquely affected by TGF-ß1, including those associated with SARS-CoV-2 invasion, replication, and the host immune responses. TGF-ß1 inhibited ACE2 expression by miR-136-3p and miR-369-5p mediated mechanism in CF and non-CF bronchial epithelial cells. ACE2 levels were higher in two bronchial epithelial cell models expressing the most common CF-causing mutation in CFTR gene F508del, compared to controls without the mutation. After TGF-ß1 treatment, ACE2 protein levels were still higher in CF, compared to non-CF cells. TGF-ß1 prevented the modulator-mediated rescue of F508del-CFTR function while the modulators did not prevent the TGF-ß1 inhibition of ACE2 levels. Finally, TGF-ß1 reduced the interaction between ACE2 and the recombinant spike RBD by lowering ACE2 levels and its binding to RBD. Our data demonstrate novel mechanism whereby TGF-ß1 inhibition of ACE2 in CF and non-CF bronchial epithelial cells may modulate SARS-CoV-2 pathogenicity and COVID-19 severity. By reducing ACE2 levels, TGF-ß1 may decrease entry of SARS-CoV-2 into the host cells while hindering the recovery from COVID-19 due to loss of the anti-inflammatory and regenerative effects of ACE2. The above outcomes may be modulated by other, miRNA-mediated effects exerted by TGF-ß1 on the host immune responses, leading to a complex and yet incompletely understood circuitry.

Cell signaling and regulation of CFTR expression in cystic fibrosis cells in the era of high efficiency modulator therapy.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP- and protein kinase A (PKA)-regulated channel, expressed on the luminal surface of secretory and absorptive epithelial cells. CFTR has a complex, cell-specific regulatory network playing a major role in cAMP- and Ca2+-activated secretion of electrolytes. It secretes intracellular Cl- and bicarbonate and regulates absorption of electrolytes by differentially controlling the activity of the epithelial Na+ channel (ENaC) in colon, airways, and sweat ducts. The CFTR gene expression is regulated by cell-specific, time-dependent mechanisms reviewed elsewhere [1]. This review will focus on the transcriptional, post-transcriptional, and translational regulation of CFTR by cAMP-PKA, non-coding (nc)RNAs, and TGF-ß signaling pathways in cystic fibrosis (CF) cells.

Differential Gene Expression Analysis Reveals Global LMTK2 Regulatory Network and Its Role in TGF-ß1 Signaling.

Lemur tyrosine kinase 2 (LMTK2) is a transmembrane Ser/Thr kinase whose role has been increasingly recognized; however, when compared to other kinases, understanding of the LMTK2 networks and biological functions is still limited. Recent data have shown that transforming growth factor (TGF)-ß1 plays a role in modulating LMTK2 function by controlling its endocytic trafficking in human bronchial epithelial cells. Here, we aimed to unveil the LMTK2 regulatory network and elucidate how it affects cellular functions and disease pathways in either TGF-ß1 dependent or independent manner. To understand how the LMTK2 and TGF-ß1 pathways interconnect, we knocked down (KD) LMTK2 using small(si)RNA-mediated silencing in human bronchial epithelial CFBE41o- cells, treated cells with TGF-ß1 or vehicle control, and performed differential gene expression analysis by RNA sequencing (RNAseq). In vehicle-treated cells, LMTK2 KD affected expression of 2,506 genes while it affected 4,162 genes after TGF-ß1 stimulation. Bioinformatics analysis shows that LMTK2 is involved in diverse cellular functions and disease pathways, such as cell death and survival, cellular development, and cancer susceptibility. In summary, our study increases current knowledge about the LMTK2 network and its intersection with the TGF-ß1 signaling pathway. These findings will serve as basis for future exploration of the predicted LMTK2 interactions and signaling pathways.

Therapeutic Response to Corticosteroids Remains a Valid Approach to Initial Management of Children With Idiopathic Nephrotic Syndrome.

Complete remission of idiopathic nephrotic syndrome (INS) in response to corticosteroids has been widely adopted as an indicator of satisfactory long-term outcomes in pediatric patients. The approach was based on the results of studies conducted in the 1960s and 1970s. The studies found that corticosteroid-responsive minimal change disease (MCD) was the most frequent diagnosis in INS patients. In more recent years, studies have reported increased frequency of focal segmental glomerulosclerosis (FSGS) and primary corticosteroid resistance without a corresponding increase of FSGS. It became unclear whether withholding kidney biopsy before treatment with corticosteroids is still the best management practice. We performed a retrospective chart review at the UPMC Children's Hospital of Pittsburgh and identified patients who were referred for evaluation of edema or proteinuria between 2002 and 2014. We identified 114 pediatric patients with INS who were treated initially with a corticosteroid (prednisone or prednisolone) 2 mg/kg (max 60 mg)/day for 4-6 weeks followed by 2 mg/kg (max 60 mg) every other day for 4-6 weeks and had not received a corticosteroid-sparing agent before completing at least 8 weeks of the initial therapy. Corticosteroid resistance in pediatric INS patients was independently associated with the black race, older age at presentation (>8 years), and female sex. The majority of blacks who were resistant to corticosteroids had a tissue diagnosis of MCD. Among the whites who were steroid-resistant, MCD and FSGS were diagnosed in similar proportions of cases. Thus, the tissue diagnosis in could not predict the response to corticosteroids. Nineteen percent of whites with FSGS were steroid-sensitive and none of the blacks with FSGS responded to corticosteroids. These data suggest that the histologic diagnosis of FSGS could not rule out response to corticosteroids, at least, in the white patient population. In summary, our data demonstrate that at this time, the therapeutic response to corticosteroids continues to be a valid approach for the initial evaluation and therapy of children diagnosed with INS at our center. Future studies should evaluate the mechanisms of changing characteristics of pediatric INS. The specific role of patient demographics, ethnicity, as well as genetic and environmental factors could be evaluated by a prospective, multicenter study.

The Ago2-miRNA-co-IP Assay to Study TGF- ß1 Mediated Recruitment of miRNA to the RISC in CFBE Cells.

Micro(mi)RNAs are short, non-coding RNAs that mediate the RNA interference (RNAi) by post-transcriptional mechanisms. Specific miRNAs are recruited to the cytoplasmic RNA induced silencing complex (RISC). Argonaute2 (Ago2), an essential component of RISC, facilitates binding of miRNA to the target-site on mRNA, followed by cleaving the miRNA-mRNA duplex with its endonuclease activity. RNAi is mediated by a specific pool of miRNAs recruited to RISC, and thus is referred to as the functional pool. The cellular levels of many miRNAs are affected by the cytokine Transforming Growth Factor-ß1 (TGF-ß1). However, little is known about whether the TGF-ß1 affects the functional pools of these miRNAs. The Ago2-miRNA-co-IP assay, discussed in this manuscript, is designed to examine effects of TGF-ß1 on the recruitment of miRNAs to RISC and it helps to determine whether changes in the cellular miRNA levels correlate with changes in the RISC-associated, functional pools. The general principles of the assay are as follows. Cultured cells treated with TGF-ß1 or vehicle control are lysed and the endogenous Ago2 is immunoprecipitated with immobilized anti-Ago2 antibody, and the active miRNAs complexed with Ago2 are isolated with a RISC immunoprecipitation (RIP) assay kit. The miRNAs are identified with quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) using miRNA-specific stem-looped primers during reverse transcription, followed by PCR using miRNA-specific forward and reverse primers, and TaqMan hydrolysis probes.

The Role of MicroRNA in the Airway Surface Liquid Homeostasis.

Mucociliary clearance, mediated by a coordinated function of cilia bathing in the airway surface liquid (ASL) on the surface of airway epithelium, protects the host from inhaled pathogens and is an essential component of the innate immunity. ASL is composed of the superficial mucus layer and the deeper periciliary liquid. Ion channels, transporters, and pumps coordinate the transcellular and paracellular movement of ions and water to maintain the ASL volume and mucus hydration. microRNA (miRNA) is a class of non-coding, short single-stranded RNA regulating gene expression by post-transcriptional mechanisms. miRNAs have been increasingly recognized as essential regulators of ion channels and transporters responsible for ASL homeostasis. miRNAs also influence the airway host defense. We summarize the most up-to-date information on the role of miRNAs in ASL homeostasis and host-pathogen interactions in the airway and discuss concepts for miRNA-directed therapy.

TGF-ß1 Augments the Apical Membrane Abundance of Lemur Tyrosine Kinase 2 to Inhibit CFTR-Mediated Chloride Transport in Human Bronchial Epithelia.

The most common disease-causing mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, F508del, leads to cystic fibrosis (CF), by arresting CFTR processing and trafficking to the plasma membrane. The FDA-approved modulators partially restore CFTR function and slow down the progression of CF lung disease by increasing processing and delivery to the plasma membrane and improving activity of F508del-CFTR Cl- channels. However, the modulators do not correct compromised membrane stability of rescued F508del-CFTR. Transforming growth factor (TGF)-ß1 is a well-established gene modifier of CF associated with worse lung disease in F508del-homozygous patients, by inhibiting CFTR biogenesis and blocking the functional rescue of F508del-CFTR. Lemur tyrosine kinase 2 (LMTK2) is a transmembrane protein localized at the apical and basolateral membrane domain of human bronchial epithelial cells. Phosphorylation of the apical membrane CFTR by LMTK2 triggers its endocytosis and reduces the abundance of membrane-associated CFTR, impairing the CFTR-mediated Cl- transport. We have previously shown that LMTK2 knockdown improves the pharmacologically rescued F508del-CFTR abundance and function. Thus, reducing the LMTK2 recruitment to the plasma membrane may provide a useful strategy to potentiate the pharmacological rescue of F508del-CFTR. Here, we elucidate the mechanism of LMTK2 recruitment to the apical plasma membrane in polarized CFBE41o- cells. TGF-ß1 increased LMTK2 abundance selectively at the apical membrane by accelerating its recycling in Rab11-positive vesicles without affecting LMTK2 mRNA levels, protein biosynthesis, or endocytosis. Our data suggest that controlling TGF-ß1 signaling may attenuate recruitment of LMTK2 to the apical membrane thereby improving stability of pharmacologically rescued F508del-CFTR.

Precise Targeting of miRNA Sites Restores CFTR Activity in CF Bronchial Epithelial Cells.

MicroRNAs that are overexpressed in cystic fibrosis (CF) bronchial epithelial cells (BEC) negatively regulate CFTR and nullify the beneficial effects of CFTR modulators. We hypothesized that it is possible to reverse microRNA-mediated inhibition of CFTR using CFTR-specific target site blockers (TSBs) and to develop a drug-device combination inhalation therapy for CF. Lead microRNA expression was quantified in a series of human CF and non-CF samples and in vitro models. A panel of CFTR 3' untranslated region (UTR)-specific locked nucleic acid antisense oligonucleotide TSBs was assessed for their ability to increase CFTR expression. Their effects on CFTR activity alone or in combination with CFTR modulators were measured in CF BEC models. TSB encapsulation in poly-lactic-co-glycolic acid (PLGA) nanoparticles was assessed as a proof of principle of delivery into CF BECs. TSBs targeting the CFTR 3' UTR 298-305:miR-145-5p or 166-173:miR-223-3p sites increased CFTR expression and anion channel activity and enhanced the effects of ivacaftor/lumacaftor or ivacaftor/tezacaftor in CF BECs. Biocompatible PLGA-TSB nanoparticles promoted CFTR expression in primary BECs and retained desirable biophysical characteristics following nebulization. Alone or in combination with CFTR modulators, aerosolized CFTR-targeting TSBs encapsulated in PLGA nanoparticles could represent a promising drug-device combination therapy for the treatment for CFTR dysfunction in the lung.

Transforming Growth Factor-ß1 Selectively Recruits microRNAs to the RNA-Induced Silencing Complex and Degrades CFTR mRNA under Permissive Conditions in Human Bronchial Epithelial Cells.

Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene lead to cystic fibrosis (CF). The most common mutation F508del inhibits folding and processing of CFTR protein. FDA-approved correctors rescue the biosynthetic processing of F508del-CFTR protein, while potentiators improve the rescued CFTR channel function. Transforming growth factor (TGF-ß1), overexpressed in many CF patients, blocks corrector/potentiator rescue by inhibiting CFTR mRNA in vitro. Increased TGF-ß1 signaling and acquired CFTR dysfunction are present in other lung diseases. To study the mechanism of TGF-ß1 repression of CFTR, we used molecular, biochemical, and functional approaches in primary human bronchial epithelial cells from over 50 donors. TGF-ß1 destabilized CFTR mRNA in cells from lungs with chronic disease, including CF, and impaired F508del-CFTR rescue by new-generation correctors. TGF-ß1 increased the active pool of selected micro(mi)RNAs validated as CFTR inhibitors, recruiting them to the RNA-induced silencing complex (RISC). Expression of F508del-CFTR globally modulated TGF-ß1-induced changes in the miRNA landscape, creating a permissive environment required for degradation of F508del-CFTR mRNA. In conclusion, TGF-ß1 may impede the full benefit of corrector/potentiator therapy in CF patients. Studying miRNA recruitment to RISC under disease-specific conditions may help to better characterize the miRNAs utilized by TGF-ß1 to destabilize CFTR mRNA.

The human nephrin Y1139RSL motif is essential for podocyte foot process organization and slit diaphragm formation during glomerular development.

Nephrin is an immunoglobulin-type cell-adhesion molecule with a key role in the glomerular interpodocyte slit diaphragm. Mutations in the nephrin gene are associated with defects in the slit diaphragm, leading to early-onset nephrotic syndrome, typically resistant to treatment. Although the endocytic trafficking of nephrin is essential for the assembly of the slit diaphragm, nephrin's specific endocytic motifs remain unknown. To search for endocytic motifs, here we performed a multisequence alignment of nephrin and identified a canonical YXXØ-type motif, Y1139RSL, in the nephrin cytoplasmic tail, expressed only in primates. Using site-directed mutagenesis, various biochemical methods, single-plane illumination microscopy, a human podocyte line, and a human nephrin-expressing zebrafish model, we found that Y1139RSL is a novel endocytic motif and a structural element for clathrin-mediated nephrin endocytosis that functions as a phosphorylation-sensitive signal. We observed that Y1139RSL motif-mediated endocytosis helps to localize nephrin to specialized plasma membrane domains in podocytes and is essential for normal foot process organization into a functional slit diaphragm between neighboring foot processes in zebrafish. The importance of nephrin Y1139RSL for healthy podocyte development was supported by population-level analyses of genetic variations at this motif, revealing that such variations are very rare, suggesting that mutations in this motif have autosomal-recessive negative effects on kidney health. These findings expand our understanding of the mechanism underlying nephrin endocytosis and may lead to improved diagnostic tools or therapeutic strategies for managing early-onset, treatment-resistant nephrotic syndrome.

Unraveling the Function of Lemur Tyrosine Kinase 2 Network.

Lemur Tyrosine Kinase 2 (LMTK2) is a recently cloned transmembrane protein, actually a serine/threonine kinase named after the Madagascar primate lemur due to the long intracellular C-terminal tail. LMTK2 is relatively little known, compared to other kinases but its role has been increasingly recognized. Published data show that LMTK2 regulates key cellular events, including endocytic trafficking, nerve growth factor signaling, apoptosis, and Cl- transport. Abnormalities in the expression and function of LMTK2 are associated with human disease, such as neurodegeneration, cancer and infertility. We summarized the current state of knowledge on LMTK2 structure, regulation, interactome, intracellular localization, and tissue expression and point out future research directions to better understand the role of LMTK2.

Extra-renal manifestations of atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) is a rare and complex disease resulting from abnormal alternative complement activation with a wide range of clinical presentations. Extra-renal manifestations of aHUS can involve many organ systems, including the peripheral and central nervous, gastrointestinal, cardiovascular, integumentary, pulmonary, as well as the eye. While some of these extra-renal manifestations occur in the acute phase of aHUS, some can also occur as long-term sequelae of unopposed complement activation. Extra-renal symptoms are observed in approximately 20% of patients with aHUS, with the incidence of specific organ system complications ranging from a few case reports to 50% of described patients. Careful monitoring for extra-renal involvement is critical in patients with aHUS, as prompt evaluation and management may decrease the risk of high morbidity and mortality associated with aHUS.

Clinical Characteristics and Treatment Patterns of Children and Adults With IgA Nephropathy or IgA Vasculitis: Findings From the CureGN Study.

INTRODUCTION: The Cure Glomerulonephropathy Network (CureGN) is a 66-center longitudinal observational study of patients with biopsy-confirmed minimal change disease, focal segmental glomerulosclerosis, membranous nephropathy, or IgA nephropathy (IgAN), including IgA vasculitis (IgAV). This study describes the clinical characteristics and treatment patterns in the IgA cohort, including comparisons between IgAN versus IgAV and adult versus pediatric patients. METHODS: Patients with a diagnostic kidney biopsy within 5 years of screening were eligible to join CureGN. This is a descriptive analysis of clinical and treatment data collected at the time of enrollment. RESULTS: A total of 667 patients (506 IgAN, 161 IgAV) constitute the IgAN/IgAV cohort (382 adults, 285 children). At biopsy, those with IgAV were younger (13.0 years vs. 29.6 years, P < 0.001), more frequently white (89.7% vs. 78.9%, P = 0.003), had a higher estimated glomerular filtration rate (103.5 vs. 70.6 ml/min per 1.73 m2, P < 0.001), and lower serum albumin (3.4 vs. 3.8 g/dl, P < 0.001) than those with IgAN. Adult and pediatric individuals with IgAV were more likely than those with IgAN to have been treated with immunosuppressive therapy at or prior to enrollment (79.5% vs. 54.0%, P < 0.001). CONCLUSION: This report highlights clinical differences between IgAV and IgAN and between children and adults with these diagnoses. We identified differences in treatment with immunosuppressive therapies by disease type. This description of baseline characteristics will serve as a foundation for future CureGN studies.

Identification of a novel functional miR-143-5p recognition element in the Cystic Fibrosis Transmembrane Conductance Regulator 3'UTR.

MicroRNAs (miRNAs) are small non-coding RNAs involved in regulation of gene expression. They bind in a sequence-specific manner to miRNA recognition elements (MREs) located in the 3' untranslated region (UTR) of target mRNAs and prevent mRNA translation. MiRNA expression is dysregulated in cystic fibrosis (CF), affecting several biological processes including ion conductance in the epithelial cells of the lung. We previously reported that miR-143 is up-regulated in CF bronchial brushings compared to non-CF. Here we identified two predicted binding sites for miR-143-5p (starting at residues 558 and 644) on the CFTR mRNA, and aimed to assess whether CFTR is a true molecular target of miR-143-5p. Expression of miR-143-5p was found to be up-regulated in a panel of CF vs non-CF cell lines (1.7-fold, P = 0.0165), and its levels were increased in vitro after 20 hours treatment with bronchoalveolar lavage fluid from CF patients compared to vehicle-treated cells (3.3-fold, P = 0.0319). Luciferase assays were performed to elucidate direct miRNA::target interactions and showed that miR-143-5p significantly decreased the reporter activity when carrying the wild-type full length sequence of CFTR 3'UTR (minus 15%, P = 0.005). This repression was rescued by the disruption of the first, but not the second, predicted MRE, suggesting that the residue starting at 558 was the actual active binding site. In conclusion, we here showed that miR-143-5p modestly but significantly inhibits CFTR, improving the knowledge on functional MREs within the CFTR 3'UTR. This could lead to the development of novel therapeutic strategies where miRNA-mediated CFTR repression is blocked thereby possibly increasing the efficacy of the currently available CFTR modulators.