Christie P. Thomas

Glucocorticoid Induction of Epithelial Sodium Channel Expression in Lung and Renal Epithelia Occurs via trans-Activation of a Hormone Response Element in the 5′-Flanking Region of the Human Epithelial Sodium Channel α Subunit Gene

In airway and renal epithelia, the glucocorticoid-mediated stimulation of amiloride-sensitive Na+ transport is associated with increased expression of the epithelial Na+ channel α subunit (αENaC). In H441 lung cells, 100 nm dexamethasone increases amiloride-sensitive short-circuit current (3.3 μA/cm2 to 7.5 μA/cm2), correlating with a 5-fold increase in αENaC mRNA expression that could be blocked by actinomycin D. To explore transcriptional regulation of αENaC, the human αENaC 5′-flanking region was cloned and tested in H441 cells. By deletion analysis, a ∼150-base pair region 5′ to the upstream promoter was identified that, when stimulated with 100 nm dexamethasone, increased luciferase expression 15-fold. This region, which contains two imperfect GREs, also functioned when coupled to a heterologous promoter. When individually tested, only the downstream GRE functioned in cis and bound GR in a gel mobility shift assay. In the M-1 collecting duct line Na+ transport, mαENaC expression and luciferase expression from αENaC genomic fragments were also increased by 100 nm dexamethasone. In a colonic cell line, HT29, trans-activation via a heterologously expressed glucocorticoid receptor restored glucocorticoid-stimulated αENaC gene transcription. We conclude that glucocorticoids stimulate αENaC expression in kidney and lung via activation of a hormone response element in the 5′-flanking region of hαENaC and this response, in part, is the likely basis for the up-regulation of Na+transport in these sites.

Comprehensive Genetic Analysis of Complement and Coagulation Genes in Atypical Hemolytic Uremic Syndrome

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy caused by uncontrolled activation of the alternative pathway of complement at the cell surface level that leads to microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney failure. In approximately one half of affected patients, pathogenic loss-of-function variants in regulators of complement or gain-of-function variants in effectors of complement are identified, clearly implicating complement in aHUS. However, there are strong lines of evidence supporting the presence of additional genetic contributions to this disease. To identify novel aHUS-associated genes, we completed a comprehensive screen of the complement and coagulation pathways in 36 patients with sporadic aHUS using targeted genomic enrichment and massively parallel sequencing. After variant calling, quality control, and hard filtering, we identified 84 reported or novel nonsynonymous variants, 22 of which have been previously associated with disease. Using computational prediction methods, 20 of the remaining 62 variants were predicted to be deleterious. Consistent with published data, nearly one half of these 42 variants (19; 45%) were found in genes implicated in the pathogenesis of aHUS. Several genes in the coagulation pathway were also identified as important in the pathogenesis of aHUS. PLG, in particular, carried more pathogenic variants than any other coagulation gene, including three known plasminogen deficiency mutations and a predicted pathogenic variant. These data suggest that mutation screening in patients with aHUS should be broadened to include genes in the coagulation pathway.

Pre-emptive Eculizumab and Plasmapheresis for Renal Transplant in Atypical Hemolytic Uremic Syndrome

The case of a 12-year-old with a hybrid CFH/CFHL1 gene and atypical hemolytic uremic syndrome (aHUS) that had previously developed native kidney and then renal allograft loss is reported. This case illustrates the relatively common occurrence of renal loss from the late presentation of aHUS. Also presented is a protocol for the pre-emptive use of eculizumab and plasmapheresis as part of a renal transplant plan for the treatment of aHUS in patients deemed at high risk for recurrent disease. This protocol was a result of a multidisciplinary approach including adult and pediatric nephrology, transplant surgery, transfusion medicine, and infectious disease specialists. This protocol and the justifications and components of it can function as a guideline for the treatment of a group of children that have waited in limbo for the first U.S. transplant to open the door to this type of definitive care for this devastating disease.

Intronic polyadenylation signal sequences and alternate splicing generate human soluble Flt1 variants and regulate the abundance of soluble Flt1 in the placenta

The gene FLT1 produces at least two transcripts from a common transcription start site: full‐length Flt1 contains 30 exons encoding a membrane‐bound VEGF receptor;soluble Flt1 (sFlt1) shares the first 13 exons but utilizes poly(A) signal sequences within intron 13 to create a transcript that lacks downstream exons. To address the mechanisms that regulate human sFlt1, we mapped the 3′ end of sFlt1 mRNA and defined the full extent of its 3′ untranslated region (UTR). We identified a 3.2 Kb sFlt1 transcript that is cleaved within an alternatively spliced exon downstream of exon 14 and is predicted to encode a C‐terminal variant of sFlt1 with an unusual polyserine tail. sFlt1 mRNA cleavage sites within intron 13 were identified in human placenta and in vascular endothe‐lium by ribonuclease protection assay (RPA). A proximal and two distal mRNA cleavage sites were identified by RPA downstream of consensus polyadenylation signals that create variant transcripts with a 3′ UTR ranging from 30 bases to ~4 Kb. Northern blot analysis and 3′ rapid amplification of cDNA ends (RACE) in placenta confirmed the existence of distal intronic sFlt1 cleavage sites that give rise to a sFlt1 transcript of ~7 Kb. The identity of the distal signal sequences were then confirmed by mutagenesis of putative signal elements in a polyadenylation reporter assay. We demonstrate the heterogeneity of human sFlt1 that arises from alternate splicing and from alternative polyadenylation directed by strong intronic poly(A) signal sequences leading to C‐terminal variants and to an sFlt1 transcript with a large 3′ UTR containing several AU rich elements and poly(U) regions that may regulate mRNA stability.— Thomas, C. P., Andrews, J. I., Liu, K. Z. Intronic polyadenylation signal sequences and alternate splicing generate human soluble Flt1 variants and regulate the abundance of soluble Flt1 in the placenta. FASEB J. 21, 3885–3895 (2007)

A recently evolved novel trophoblast-enriched secreted form of fms-like tyrosine kinase-1 variant is up-regulated in hypoxia and preeclampsia.

CONTEXT Recent published studies indicate a possible role for sFlt1 in the development of preeclampsia. OBJECTIVE The objective of the study was to investigate the expression and regulation of sFlt1-e15a, a recently described novel C-terminal variant isoform of sFlt1. DESIGN The studies included a computational comparative analysis of the genomic locus of sFlt1 across vertebrate species; an assessment of sFlt1 variants in human and rhesus cells and tissues; an analysis of sFlt1 variants transiently expressed in HeLa and COS-7 cells; an evaluation of the effect of hypoxia on sFlt1 expression in trophoblasts; and a comparison of placental sFlt1 expression between pregnancies complicated by preeclampsia and control pregnancies. RESULT AND CONCLUSIONS sFlt1-e15a emerged as an alternate transcript of Flt1 late in evolution with the insertion of an AluSq sequence into the primate genome after the emergence of the simian infraorder about 40 million years ago. sFlt1-e15a is particularly abundant in human placenta and trophoblasts and is also highly expressed in nonhuman primate placenta. The expressed protein has a C-terminal polyserine tail and, like reference sequence sFlt1 (sFlt1-i13), is glycosylated and secreted. Consistent with a role in placental pathophysiology, hypoxia stimulates sFlt1-e15a expression in isolated cytotrophoblasts and a trophoblast cell line, and differentiation into syncytiotrophoblasts further enhances the effect of hypoxia. Placental levels of sFlt1-e15a and sFlt1-i13 transcripts are significantly elevated in patients with preeclampsia compared with normal pregnancies. We speculate that sFlt1-e15a may contribute to the pathophysiology of preeclampsia.

Atypical hemolytic uremic syndrome: what is it, how is it diagnosed, and how is it treated?

Atypical hemolytic uremic syndrome (aHUS) is a rare syndrome of hemolysis, thrombocytopenia, and renal insufficiency. Genetic mutations in the alternate pathway of complement are well recognized as the cause in more than 60% of patients affected by this thrombotic microangiopathy. The identification of aHUS as a disease of the alternate pathway of complement enables directed therapeutic intervention both in the acute and chronic setting and may include one or all of the following: plasma therapy, complement blockade, and liver transplantation. Because aHUS shares many of the presenting characteristics of the other thrombotic microangiopathies, and confirmatory genetic results are not available at the time of presentation, the diagnosis relies heavily on the recognition of a clinical syndrome consistent with the diagnosis in the absence of signs of an alternate cause of thrombotic microangiopathy. Limited understanding of the epidemiology, genetics, and clinical features of aHUS has the potential to delay diagnosis and treatment. To advance our understanding, a more complete characterization of the unique phenotypical features of aHUS is needed. Further studies to identify additional genetic loci for aHUS and more robust biomarkers of both active and quiescent disease are required. Advances in these areas will undoubtedly improve the care of patients with aHUS.

New insights into epithelial sodium channel function in the kidney: site of action, regulation by ubiquitin ligases, serum- and glucocorticoid-inducible kinase and proteolysis.

Purpose of reviewThe epithelial sodium channel (ENaC) sets the rate of Na+ reabsorption in the collecting duct. This review describes recent advances in our understanding of ENaC function. Recent findingsFirst, collecting duct-specific deletion of αENaC does not cause Na+ wasting in mice, suggesting that other regions can compensate. Second, Nedd4 and Nedd4-2 are ubiquitin ligases that reduce surface expression of ENaC and inhibit Na+ transport. Nedd4-2, but not Nedd4, is negatively regulated by serum- and glucocorticoid-inducible kinase 1, an aldosterone-induced kinase, providing an attractive mechanism for the stimulatory effect of aldosterone on Na+ transport. However, mice with germline ablation of serum- and glucocorticoid-inducible kinase 1 show only modest hypotension and are able to decrease Na+ excretion rates substantially. Third, maturation of ENaC is associated with processing at consensus furin cleavage sites and this cleavage is critical for channel activity. A separate class of serine proteases, the channel-activating proteases, also stimulates ENaC activity. SummaryThe connecting tubule of the kidney has abundant ENaC and Na+- and K+-transport capacity and may provide much of ENaC-mediated Na+ transport in the kidney. Aldosterone may increase Na+ transport, in part, by serum- and glucocorticoid-inducible kinase 1-mediated inhibition of Nedd4-2 but this has not been demonstrated in the native collecting duct or connecting tubule. The mild phenotype of the serum- and glucocorticoid-inducible kinase 1-knockout mouse points to serum- and glucocorticoid-inducible kinase 1-independent mechanisms that regulate Na+ transport. Two separate classes of protease appear to regulate Na+ transport: one is furin or furin-like and cleaves ENaC subunits to stimulate transport; the other, the channel-activating proteases, may act on ENaC or a regulatory molecule.

Case Report: Eculizumab Rescue of Severe Accelerated Antibody-Mediated Rejection After ABO-Incompatible Kidney Transplant

ABO-incompatible (ABOI) living donor kidney transplantation has become a well-accepted practice with standard protocols using perioperative antibody-depleting therapies to lower blood group titers to an acceptable threshold for transplantation. However, a subset of patients will experience accelerated antibody-mediated rejection (AMR) after ABOI kidney transplantation and require aggressive intervention to prevent allograft loss. Here in we report the successful use of terminal complement inhibition with eculizumab to rescue an ABOI kidney allograft with accelerated AMR refractory to salvage splenectomy and daily plasmapheresis. This case emphasizes the fact that, despite close postoperative surveillance and aggressive intervention, graft loss from accelerated AMR after ABOI kidney transplantation remains a very real risk. Eculizumab may offer a graft-saving therapeutic option for isolated cases of severe AMR after ABOI kidney transplantation refractory to standard treatment.

High-Throughput Genetic Testing for Thrombotic Microangiopathies and C3 Glomerulopathies

The thrombotic microangiopathies (TMAs) and C3 glomerulopathies (C3Gs) include a spectrum of rare diseases such as atypical hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, C3GN, and dense deposit disease, which share phenotypic similarities and underlying genetic commonalities. Variants in several genes contribute to the pathogenesis of these diseases, and identification of these variants may inform the diagnosis and treatment of affected patients. We have developed and validated a comprehensive genetic panel that screens all exons of all genes implicated in TMA and C3G. The closely integrated pipeline implemented includes targeted genomic enrichment, massively parallel sequencing, bioinformatic analysis, and a multidisciplinary conference to analyze identified variants in the context of each patients specific phenotype. Herein, we present our 1-year experience with this panel, during which time we studied 193 patients. We identified 17 novel and 74 rare variants, which we classified as pathogenic (11), likely pathogenic (12), and of uncertain significance (68). Compared with controls, patients with C3G had a higher frequency of rare and novel variants in C3 convertase (C3 and CFB) and complement regulator (CFH, CFI, CFHR5, and CD46) genes (P<0.05). In contrast, patients with TMA had an increase in rare and novel variants only in complement regulator genes (P<0.01), a distinction consistent with differing sites of complement dysregulation in these two diseases. In summary, we were able to provide a positive genetic diagnosis in 43% and 41% of patients carrying the clinical diagnosis of C3G and TMA, respectively.

Recurrent Atypical Hemolytic Uremic Syndrome Associated With Factor I Mutation in a Living Related Renal Transplant Recipient

Atypical hemolytic uremic syndrome, or the nondiarrheal form of hemolytic uremic syndrome, is a rare disorder typically classified as familial or sporadic. Recent literature has suggested that approximately 50% of patients have mutations in factor H (CFH), factor I (CFI), or membrane cofactor protein (encoded by CD46). Importantly, results of renal transplantation in patients with mutations in either CFH or CFI are dismal, with recurrent disease leading to graft loss in the majority of cases. We describe an adult renal transplant recipient who developed recurrent hemolytic uremic syndrome 1 month after transplantation. Bidirectional sequencing of CFH, CFI, and CD46 confirmed that the patient was heterozygous for a novel missense mutation, a substitution of a serine reside for a tyrosine residue at amino acid 369, in CFI. This report reemphasizes the importance of screening patients with atypical hemolytic uremic syndrome for mutations in these genes before renal transplantation and shows the challenges in the management of these patients.