Gene Test May Spot Which Kidney Transplants More Likely to Fail

Researchers suggest it could be way to spot trouble earlier.

TUESDAY, Nov. 11, 2014 (HealthDay News) -- A preliminary gene test may help identify kidney transplant patients at risk of organ rejection, researchers report.

 

Organ rejection occurs in 15 percent to 20 percent of kidney transplant patients, even when they are given drugs to suppress their immune system.

 

Typically, an increase in serum creatinine -- a sign of kidney function -- warns of impending kidney rejection. A kidney biopsy is then performed to confirm whether a new kidney is being rejected by the body, according to background information in the study.

 

The study was published Nov. 11 in the journal PLoS Medicine.

 

However, testing for elevated creatinine levels does not always provide early warning about rejection and is not specific enough to prevent some unnecessary kidney biopsies. So a noninvasive method of identifying rejection is needed, wrote the study authors, from the University of California, San Francisco, and the University of Cincinnati.

 

The researchers developed a blood test that analyzes 17 genes that identify which kidney transplant patients are at risk of rejection. They then developed and assessed the test using hundreds of patients in the United States, Mexico and Spain.

 

The test was highly accurate in pinpointing patients at risk of rejection, the study authors said in a journal news release.

 

This "is a simple, robust, and clinically applicable blood test," said the researchers.

 

They added, however, that they are conducting further studies of the test.

 

More information

 

The United Network for Organ Sharing has more about preventing rejection of transplanted organs.

SOURCE: PLoS Medicine, news release, Nov. 11, 2014

 

-- Robert Preidt

 

MicroRNAs in kidney physiology and disease

 Abstract

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression. They have important roles during kidney development, homeostasis and disease. In particular, miRNAs participate in the onset and progression of tubulointerstitial sclerosis and end-stage glomerular lesions that occur in various forms of chronic kidney disease (CKD). Therefore, miRNAs represent potential new therapeutic targets for a debilitating disease that continues to increase in prevalence worldwide and for which fully effective therapies are lacking. Several lines of research aimed at improving common CKD diagnostic tools and avoiding invasive kidney biopsies have also identified circulating miRNAs as possible diagnostic and even prognostic biomarkers of kidney disease. This Review discusses current understanding of the function of miRNAs in CKD, focusing on functions specifically involved in the transforming growth factor β1 pathway, which is activated in CKD. miRNAs that, according to available evidence, seem to be involved in diabetic nephropathy, IgA nephropathy, lupus nephritis, polycystic kidney disease and graft rejection, are also discussed.

 Key points

• MicroRNAs (miRNAs) are key players in kidney development and physiology
• Transforming growth factor β1 is a major regulator of kidney fibrosis; its signalling is finely regulated by miRNAs
• miRNAs contribute to both the induction and progression of chronic kidney disease (CKD)
• Current translational research on miRNAs in kidney disease is mainly focused on developing reliable biomarkers for diagnosis and prognosis of CKD and renal transplantation
• miRNAs represent novel therapeutic targets for CKD, but delivery and safety issues must be taken into account before translation into clinical practice

Introduction

Chronic kidney disease (CKD) is an important public health problem that is closely linked to major non-communicable diseases such as diabetes mellitus and hypertension.¹ Less common causes of CKD are hereditary diseases such as polycystic kidney disease (PKD), IgA nephropathy (IgAN) and lupus nephritis, or herbal and environmental toxins. Regardless of the disease aetiology, progression of CKD results in tubulointerstitial and glomerular fibrosis owing to excessive deposition of extracellular matrix (ECM). Other key features of CKD are inflammatory cell infiltration, tubular cell atrophy, mesangial cell hypertrophy and podocyte apoptosis.² All of these pathological events are mainly instigated by the cytokine transforming growth factor β1 (TGF-β1).³

 

Current therapies that target the renin–angiotensin–aldosterone system⁴ are not always effective in halting progression to end-stage renal disease, a condition that requires renal replacement therapy by dialysis or kidney transplantation. Blockade of TGF-β1 signalling using TGF-β1 neutralizing antibodies or specific inhibitors of TGF-β1 receptors might be a promising therapeutic approach for CKD as these strategies have been shown to attenuate renal fibrosis in various animal models.⁵ The success of these approaches in preclinical studies has prompted the search for therapies that interfere with the epigenetic regulation of TGF-β and genes involved in the TGF-β signalling pathway. In this context, non-coding RNA species, including microRNAs (miRNAs), have been widely implicated in the pathogenesis and progression of CKD, particularly in the regulation of TGF-β1-mediated fibrosis. Correction of miRNA expression by in vivo delivery of miRNA mimics or inhibitors has therefore emerged as a promising novel therapeutic strategy for the treatment of CKD. In addition, several lines of current research aimed at improving common CKD diagnostic tools and avoiding invasive kidney biopsies have identified circulating miRNAs as possible diagnostic and even prognostic biomarkers.^{6, 7} This Review focuses on current research investigating the roles of miRNAs in normal kidney physiology and diseases; particular attention is given to the TGF-β1 pathway and its regulation by miRNAs.

Biogenesis and function of miRNAs

miRNAs are epigenetic regulators of gene expression that are able to modulate several cellular processes, from development to disease conditions. The human miRNAome is composed of 1,881 precursors and 2,588 mature miRNAs, which regulate at least 60% of protein-coding genes.⁸ Since 2010, the number of miRNAs included in miRBase has grown by approximately two-thirds owing to the advent of small RNA deep-sequencing techniques.⁹

 

Here, we briefly mention the key points of miRNA biogenesis and function, as these processes are described thoroughly elsewhere.^{10, 11} Transcription of miRNAs occurs from individual or clustered genes (that is, the miR-194–192 cluster), although some miRNAs can be encoded from distinct genomic loci.¹² Genes encoding miRNAs are located in non-coding sequences or in introns of either protein-coding genes (miR-trons) or non-coding RNA.¹³ Intronic miRNAs are usually coordinately expressed with their host gene and most of the time they both affect the same signalling pathway.^{14, 15, 16}

 

RNA polymerase II transcribes miRNAs in the nucleus as long capped and polyadenylated hairpin transcripts, called primary miRNAs (pri-miRNAs). These are processed into smaller ~70 nucleotide stem–looped structures, called precursor miRNAs (pre-miRNAs) by the ribonuclease III-like enzyme Drosha together with the microprocessor complex subunit DGCR8. Pre-miRNAs are then exported to the cytoplasm by exportin-5/GTP-binding nuclear protein Ran, where the ribonuclease Dicer yields 22 nucleotide miRNA duplexes consisting of the guide and passenger strands (miRNA:miRNA*). The guide strands are finally assembled into the RNA-induced silencing complex (RISC) and bind through their 'seed sequence' (nucleotides 2–8) to fully or partially complementary sites within the 3′ untranslated region of target mRNAs. miRNAs rarely bind to the coding regions of mRNA or genomic DNA, including promoter regions.¹⁷ Target recognition by miRNAs leads to mRNA translational repression and/or mRNA deadenylation and decay,^{18, 19} although positive regulation has been described in a few cases (Figure 1).^{20, 21} Beyond the simplistic concept that miRNAs act as repressors of a single transcript, emerging evidence indicates that they are modulators of many hundreds of proteins often involved in related signalling pathways.^{22, 23}

Figure 1: Interplay between TGF-β1/Smad signalling and the miRNA machinery. 

TGF-β1/Smad signalling can modulate miRNA expression by regulating transcription and/or facilitating the processing of pri-miRNA into pre-miRNA in the nucleus. Pre-miRNA is exported to the cytoplasm where it is processed into the final mature miRNA, which is loaded into the RISC. Mature miRNA guides the RISC to silence target mRNAs through mRNA cleavage, translational repression or deadenylation. Rarely, transcriptional activation can occur. Abbreviations: Co-Smad, common mediator Smad; miRNA, microRNA; P, phosphorylation; pre-miRNA, precursor miRNA; pri-miRNA, primary miRNA; RISC, RNA-induced silencing complex; R-Smad, receptor-regulated Smad; SBS, Smad binding site; TGF-β1, transforming growth factor β1; TGFBR1, TGF-β receptor type-1; TGFBR2, TGF-β receptor type-2; UTR, untranslated region.

miRNAs are usually stable with a long half-life, but individual miRNAs can undergo rapid decay in specific cellular contexts in the presence of particular environmental stimuli or cellular factors. Of note, the presence of certain sequences in miRNAs that determine decay rate, together with several ribonucleases that degrade miRNAs, have been identified.²⁴

miRNAs also have a key role as regulators of cellular crosstalk.⁶ They can be actively secreted into the extracellular microenvironment or into body fluids and captured by other cells, thus altering their transcriptional programme. In addition, circulating miRNAs can be derived from apoptotic cells, with the likely purpose of carrying alarm signals from apoptotic cells to other cells.⁶ miRNAs circulate in blood, urine and other body fluids either packaged into microvesicles and/or exosomes, or transported by RNA-binding proteins (Argonaute2 complexes) and lipoproteins, which protect them from degradation by ribonucleases.⁶ The consequent stability of miRNAs in biological fluids, together with the fact that miRNAs can be repeatedly collected by non-invasive means and detected with high accuracy and specificity by amplification methods, renders miRNAs potentially better biomarkers than proteins and mRNAs. However, isolation and quantification of miRNAs is time-consuming and expensive, and further optimization of these procedures is required before they can be used in routine clinical practice.

miRNAs in development and physiology

Analyses of miRNA expression profiles have identified a set of miRNAs expressed mainly in the adult human kidney (including miR-215, miR-146a and miR-886); other miRNAs, such as miR-192, miR-194, miR-21, miR-200a, miR-204 and let-7a–g, are enriched in the kidney as well as in other organs.^{14, 25, 26} However, some discordant findings regarding miRNA expression profiles have been reported, probably owing to use of different detection methods and to differences in the intrinsic nature of the analysed samples. The picture is further complicated by the fact that expression levels of selected miRNAs differ between fetal and adult kidneys, suggesting that besides being tissue-specific, miRNA expression is specific to developmental stage.²⁷

The generation of mouse models with conditional knockdown of miRNA-processing enzymes or knockdown of specific miRNAs in various cell lineages has enabled the roles of miRNAs during kidney development and homeostasis to be studied. In nephron progenitor cells, conditional deletion of Dicer results in their apoptosis—mediated by the proapoptotic protein Bim—and in the premature termination of nephrogenesis.^{28, 29} Ablation of the miR-17~92 cluster, composed of miRNAs normally expressed in the developing kidney with well-known roles in development, leads to defective proliferation of progenitor cells and reduced numbers of developing nephrons.³⁰ Mice deficient in miR-17~92 are characterized by renal hypodysplasia and develop glomerular dysfunction and proteinuria.³⁰

In ureteric buds, impaired miRNA processing leads to excessive cell proliferation and apoptosis accompanied by disrupted ciliogenesis within the epithelium of ureteric buds and consequent development of renal cysts.^{29, 31} In addition, one study suggested that miRNAs modulate later stages of renal tubule maturation as removal of Dicer from maturing renal tubules and collecting ducts leads to the formation of tubular and glomerular cysts in mice through a mechanism that probably involves modulation of Pkd1 gene dosage by miRNAs.³²

miRNAs also have a key role in podocyte homeostasis. Indeed, podocyte-specific deletion of Dicer or Drosha in the developing kidney causes disruption of the glomerular filtration barrier, leading to proteinuria and collapsing glomerulopathy with glomerular and tubulointerstitial fibrosis.^{33, 34, 35, 36, 37} Moreover, miRNA processing is important for maintenance of adult podocyte function and differentiation as selective deletion of Drosha in adult mice results in collapsing glomerulopathy,³⁷ whereas inactivation of Dicer in postnatal proximal tubules does not affect their histology or function and protects mice from renal ischaemia–reperfusion injury.³⁸

miRNAs are also involved in regulating renal physiology, from the control of blood pressure to the maintenance of whole-body fluid and electrolyte homeostasis. The role of miRNAs in blood pressure control was ascertained by generation of mice with specific inactivation of Dicer in renin-expressing juxtaglomerular cells, which leads to loss of these cells accompanied by reduced plasma renin concentration, hypotension and kidney fibrosis.^{39, 40} Several miRNAs are modulated by hypertonic conditions and have a key role in the control of osmolarity balance by regulating Na⁺ and K⁺ levels in the different portions of the nephron. miR-192 suppresses the Na⁺/K⁺-ATPase β1 subunit gene in human renal epithelial cells,⁴¹ and in the distal nephron it inhibits the serine/threonine kinase WNK1, a protein that is essential for the coordinated regulation of electrolyte transport in the kidney.⁴² Regulation of Na⁺ transport by miRNAs has been ascertained in the mouse cortical collecting duct where aldosterone reduces the expression of a subset of miRNAs that target ankyrin-3. In turn, overexpression of ankyrin-3 increases sodium transport mediated by the epithelial Na channel.⁴³ Moreover, renal medullary cortical collecting duct cells exposed to high Na⁺ concentration are characterized by reduced levels of miR-200 and miR-717 in association with increased levels of their common target NFAT5,⁴⁴ which controls the cellular response to osmotic stress. miRNAs are also able to regulate K⁺ secretion. Indeed, in the cortical collecting duct, high dietary K⁺ intake stimulates the transcription of miR-802 and miR-194, which regulate ROMK channel activity by targeting caveolin-1 (a negative regulator of the ROMK channel)⁴⁵ and intersectin-1 (involved in mediating the WNK-induced endocytosis of the ROMK channel),⁴⁶ respectively. Finally, in the thick ascending limb of Henle, miR-9 and miR-374 regulate the expression of claudin-14, which is critical for Ca²⁺ reabsorption in the kidney.⁴⁷ Interestingly, the transcriptional levels of both miRNAs are directly regulated by the Ca²⁺-sensing receptor CASR.⁴⁸

Figure 2: miRNA-regulatory networks in proximal tubular epithelial cells and mesangial cells in response to TGF-β1, and in podocytes in response to TGF-β1 and high glucose.

Up and down black arrows represent upregulated and downregulated, respectively. miRNA-dependent signalling loops through which TGF-β1 amplifies its signal are shown in green. Abbreviations: ECM, extracellular matrix; HDAC, histone deacetylase; miRNA, microRNA; TGF-β1, transforming growth factor β1; TGFBR1, TGF-β receptor type-1.

miRNAs in immune diseases

IgA nephropathy (IgAN) is characterized by aberrant glycosylation of IgA1, a reaction catalysed in part by C1GALT1, which has been proposed to be a target of miR-148b. miR-148b is upregulated in peripheral blood mononuclear cells (PBMCs) isolated from patients with IgAN, and patients carrying the 1365G/G genotype in the miR-148b-binding site of C1GALT1 have increased C1GALT1 binding affinity for miR-148b and consequently lower enzyme expression (Table 1).¹⁰⁵

Global miRNA expression analysis in kidney biopsy samples from patients with IgAN found that dysregulated levels of miRNAs related to fibrosis (downregulation of miR-200c and upregulation of miR-192, miR-141 and miR-205)¹⁰⁶ and to the immune response (upregulation of miR-155 and miR-146a)¹⁰⁷ correlated with disease severity and progression. Moreover, glomerular endothelial cells of patients with IgAN are characterized by reduced expression of miR-223, which causes glomerular endothelial proliferation, a pathological hallmark of IgAN.¹⁰⁸ Low levels of miR-223 were also found in circulating endothelial cells, providing a possible non-invasive method for evaluating the severity of IgAN (Table 1). Other potential biomarkers of IgAN are miR-155,¹⁰⁹ miR-146a,¹⁰⁹ miR-17¹⁰⁴ and miR-93—which are found at increased levels—and miR-29b and miR-29c—which are found at decreased levels—in the urine of patients with IgAN.¹⁰⁹

miRNA dysregulation also has a key role in lupus nephritis, an autoimmune disorder with a complex pathophysiology. miRNA expression analysis in kidney biopsy samples of patients with class II lupus nephritis (characterized by pure mesangial involvement), identified 36 upregulated and 30 downregulated miRNAs compared with healthy controls.¹¹⁰ miR-150 has also been demonstrated to promote renal fibrogenesis in lupus nephritis (Table 1).^{111, 112} miR-150 targets SOCS1, a negative regulator of the JAK/STAT signalling pathway, which regulates a wide range of genes involved in cell proliferation, inflammation and fibrosis. In human mesangial cells and pTECs, TGF-β1 upregulates miR-150, resulting in decreased expression of SOCS1 and increased production of profibrotic proteins. SOCS1-knockout mice spontaneously develop a lupus-like disease; likewise, the kidneys and in particular the tubular cells of patients with fibrosing lupus nephritis are characterized by increased expression of miR-150 in association with lower levels of SOCS1.¹¹¹

In patients with lupus nephritis of differing severities, a correlation was identified between clinical disease severity and glomerular and tubulointerstitial expression of miR-638, miR-198 and miR-146.¹¹³ Analysis of miRNA expression profiles in plasma,¹¹⁴ urinary sediment¹¹⁵ and PBMCs¹¹⁶ revealed a subset of miRNAs associated with lupus nephritis (miR-342-3p, miR-223 and miR-20a in plasma, miR-221 and miR-222 in urinary sediment, and miR-371-5p, miR-1224-3p and miR-423-5p in PBMCs), which are potential promising disease biomarkers.¹¹⁷

miRNAs in kidney transplantation

miRNA profiling could be a promising tool for monitoring the status of transplanted kidneys, which despite advances in immunosuppressive therapy can undergo acute rejection (an event that is becoming increasingly less common) or chronic rejection. Two research groups have identified miRNAs that were differentially expressed in biopsy samples from the kidneys of patients with acute rejection, although no overlapping miRNAs were found.^{127, 128} In particular, Anglicheau and colleagues demonstrated that increased intragraft levels of miR-142-5p, miR-155 and miR-223, probably derived by graft-invading immune cells, and decreased levels of miR-10b, miR-30a-3p and let-7c, expressed by resident renal cells, were predictive of renal graft function.¹²⁸ One study identified miRNA signatures in kidney biopsy samples that were able to distinguish between acute cellular and humoral rejection and delayed graft function.¹²⁹ Of note, Lorenzen et al.¹³⁰ analysed miRNA expression in the urine of patients with acute rejection and proposed miR-210 as a novel biomarker of acute rejection and predictor of long-term graft function.

 

Interestingly, analysis of a blood-derived miRNA signature of drug-free tolerant patients after kidney transplantation identified eight modulated miRNAs. Among these miRNAs, overexpression of miR-142-3p in PBMCs was correlated with drug-free tolerance, probably owing to the negative regulation of TGF-β signalling by miR-142-3p. Thus, miR-142-3p expression represents a promising predictor of patients who could reduce or even avoid immunosuppressive therapies.¹³¹

 

Three reports studied miRNA changes during chronic rejection characterized by interstitial fibrosis and tubular atrophy (IF/TA). miR-142-3p, miR-204 and miR-211 were differentially expressed in kidney biopsy samples and urine of patients with chronic rejection characterized by IF/TA compared with patients with normal histology and a functioning allograft.¹³² A subsequent study confirmed miR-142-3p overexpression in biopsy samples with IF/TA, and also reported dysregulation of miR-21, miR-142-5p, the cluster comprising miR-506 on chromosome X, miR-30b and miR-30c in these samples.¹³³ Investigation of miRNA expression in urinary cell pellets from patients diagnosed with chronic rejection with IF/TA compared with those with normal renal function identified 22 differentially expressed miRNAs, mainly associated with inflammation.¹³⁴ More importantly, these researchers identified a subset of miRNAs (miR-200b, miR-375, miR-423-5p, miR-193b and miR-345), which are promising biomarkers for monitoring graft function and anticipating progression to chronic rejection, as they were differentially expressed between the two groups soon after kidney transplantation but before histological injury was evident.¹³⁴

Potential uses of miRNAs for therapy

Mature miRNAs possess distinct features that make them potentially suitable as therapeutic agents, including their short sequence and their high homology across multiple vertebrate species. Manipulation of the activity of specific miRNAs in the kidney can be achieved by in vivo delivery of mimics to restore miRNA levels or inhibitors to block miRNA function. miRNA mimics are double-stranded synthetic oligonucleotides that accomplish the endogenous functions of the miRNA of interest, but following chemical modifications possess increased stability and are efficiently taken up by cells. The most widely adopted strategy so far to block miRNA function is with chemically modified oligonucleotides (anti-miRs) designed against the mature miRNA sequence that are stable in circulation and are cell permeable (2′-O-methyl-group-modified oligonucleotides or locked nucleic acid anti-miRs). In addition to anti-miRs, miRNA inhibition can be achieved by expression of miRNA-target sequences able to capture pathogenic miRNAs (miRNA sponge), short hairpin RNA plasmids to abrogate miRNA expression via RNA interference, or using oligonucleotides complementary either to the 3′ untranslated region of the target mRNA binding site sequence (masking approach) or to the sequence of the miRNA (erasers).

 

Many studies in experimental animal models have focused on the therapeutic potential of miRNAs in CKD and promising results in halting renal fibrosis have been obtained by knocking down miR-21,^{22, 65, 92} miR-29c,⁹⁹ miR-214,²³ miR-433,⁹⁴ and miR-192^{69, 76} or overexpressing miR-29b (Table 2).^{66, 80} Successful kidney transfection was achieved by intraperitoneal, intravenous or subcutaneous injection of either mimics or inhibitors or, more frequently, by intravenous injection of plasmids expressing miRNAs or short-hairpin RNAs.

 

However, many obstacles must be overcome before miRNA-based therapies for CKD can be translated into clinical practice, including delivery methods and safety concerns. Indeed, the target miRNA should be kidney-specific in order to avoid any potential adverse effects in other tissues and organs, and should affect only one target (or targets acting in the same pathway) to avoid effects on unintended templates, as in the case of miR-21, the knockdown of which induces cell death in addition to halting renal fibrosis.⁶⁵ So far, these limitations have been partially overcome either by local administration of miRNA-based drugs or by using vectors containing kidney-specific and inducible promoters.

Conclusions

 

The importance of miRNAs in the kidney field is increasingly recognized as they enable researchers to understand in-depth the pathways that have a role in kidney physiology and disease. They can also provide an explanation for divergent transcriptomic and proteomic data. Moreover, an miRNA-based therapy that either restores or blocks miRNA expression and activity is very attractive, especially now that the first miRNA-targeted drug (miravirsen for the treatment of hepatitis C) has entered a phase II clinical trial.¹³⁵ So far, the potential of miRNAs as an effective antifibrotic therapy has been demonstrated only in experimental models of CKD because many safety concerns must be resolved, from the delivery method to the adverse effects on alternative templates. Instead, a more impending application is the detection and quantification of circulating miRNAs as a novel non-invasive, repeatable method to identify and monitor the degree of disease. However, because most miRNAs are highly pleiotropic and act differently depending on the cell type, a single miRNA is unlikely to be able to diagnose and predict a form of CKD. Instead, a network of correlated miRNAs must be considered.

 

Nature Reviews Nephrology | Review

Piera Trionfini^1, Ariela Benigni^1, Giuseppe Remuzzi^1,

Published online 11 November 2014

                                                                                                                                                                                                                                                                                                                                                                                                                             

Mother’s Microbes Protect Baby’s Brain

Bacteria in the gut of a pregnant mouse strengthen the blood-brain barrier of her developing fetus.

V. ALTOUNIAN/SCIENCE TRANSLATIONAL MEDICINEThe brain—the most exalted and enigmatic of organs, which is closed off from the rest of the body by a largely impermeable barrier—could not seem more disconnected from the intestine. Yet, according to a paper published today (November 19) in Science Translational Medicine, it’s thanks to the contents of the gut—specifically, the resident bacteria—that the mouse brain’s impermeable barrier develops properly, both before and after birth.

 

“It’s absolutely fascinating to think that gut bacteria can control permeability of the blood-brain barrier,” said Caltech microbiologist Sarkis Mazmanian who was not involved in the study. “Many neuroscientists staunchly believe that the blood-brain barrier is an incredibly impermeable membrane to many molecules and . . . would hardly believe that gut bacteria would control such an integral part of our neurobiology.”

 

The blood-brain barrier (BBB), which shields the organ from blood-borne infections, toxins, and more, is created by steadfast connections called tight junctions between the endothelial cells that line its blood vessels. So effective is the barrier that most proteins and molecules cannot pass through; those that do generally require selective transport via specific receptors.

 

A similar barrier—made up of epithelial cells and tight junctions—lines the intestine and stops the trillions of microbes present in the gut from escaping into the body. It’s known that the gut bacteria themselves control integrity of this intestinal barrier, said Sven Pettersson of the Karolinska Institute in Sweden who led the new study.

 

It’s also known, he said, “that [gut] microbes could modulate brain function and development.” Pettersson and his colleagues therefore wondered whether the effect of gut microbes on the brain might be manifested in part by control of the BBB.

 

The team compared development of the BBB between germ-free fetal mice and those with normal microbiomes. As expected, the mice with normal microbiomes exhibited normal closure of the BBB toward the late stages of fetal development—a traceable antibody that could be detected readily entering the brain in the early fetus became restricted to blood vessels later on. In the fetuses whose mothers were germ-free, however, the antibody continued to enter brain tissue even late in pregnancy.

 

This increased barrier permeability was associated with low expression and disorganization of tight junction proteins and was shown by additional methods to persist into adult life. That is, pups that were born to germ-free mothers and that remained germ-free throughout life had leakier BBBs as adults.

 

“The interesting thing here is that it is [controlled by] the mother’s microbiome,” said John Cryan, chair of anatomy and neuroscience at University College Cork in Ireland. “We largely think of the influence of the microbiome as having a postnatal effect, but here they show clearly that even before the animals get exposed to microbes, the fact that their mothers are germ-free is already impacting on their development,” he said.

 

In the adult mice, transplantation of fecal matter from animals with normal microbiomes into the germ-free animals, not only corrected the expression of BBB tight junction proteins but reduced barrier permeability, the team showed. A similar effect was obtained by giving the germ-free mice bacteria-derived short chain fatty acids, suggesting that the production of metabolites may be part of the mechanism by which the bacteria control barrier integrity.

 

Although the precise mechanisms remain to be determined, said Cryan, “we know that the microbiota is critical for the developing brain and now we know it is also critical for the blood-brain barrier.”

“Therefore,” he added, “anything that threatens the microbiota homeostasis could potentially threaten the integrity of the blood-brain barrier.”

 

Indeed, “anything that happens to the mother during pregnancy that can have a negative effect on her intestinal bacteria could adversely affect the development of the fetal brain,” speculated Stephen Collins, director of the Farncombe Family Digestive Health Research Institute at McMaster University in Ontario. “For example, antibiotic usage during pregnancy theoretically—if these results can be extrapolated to humans—could actually have some impact on brain development.”

 

And that’s not all. “The development and final closure of the blood-brain barrier isn’t quite finished at birth,” added Collins, explaining that the process continues during the early days of postnatal life. “So, again, anything that interferes with intestinal bacteria . . . after the child has been born”—such as antibiotic use or delivery by cesarean section, which is thought to prevent the offspring receiving a dose of microbes from the mother’s birth canal—“could also prevent the proper closure of that barrier and therefore the proper development of the brain,” he said.

 

Collins pointed out, however, that “so far, this remains an important observation that has been made in a mouse system and hasn’t yet been proven to be the case in man.” Moreover, “there are situations in pregnancy and around birth where antibiotics are required to save the life of the mother or the fetus,” he added.

 

V. Braniste et al., “The gut microbiota influences blood-brain barrier permeability in mice,” Science Translational Medicine, 6:263ra158, 2014.

Tags

neuroscience, microbiome, disease/medicine, developmental biology and blood-brain barrier

 

By Ruth Williams | November 19, 2014

 

The Scientist » News & Opinion » Daily News

 

 

Cystic fibrosis genetics: from molecular understanding to clinical application

Abstract

The availability of the human genome sequence and tools for interrogating individual genomes provide an unprecedented opportunity to apply genetics to medicine. Mendelian conditions, which are caused by dysfunction of a single gene, offer powerful examples that illustrate how genetics can provide insights into disease. Cystic fibrosis, one of the more common lethal autosomal recessive Mendelian disorders, is presented here as an example. Recent progress in elucidating disease mechanism and causes of phenotypic variation, as well as in the development of treatments, demonstrates that genetics continues to play an important part in cystic fibrosis research 25 years after the discovery of the disease-causing gene.

Key points

• Investigation of disease-causing variants such as F508del is resolving the mechanisms underlying cystic fibrosis transmembrane conductance regulator (CFTR) folding and will inform rational design of compounds to correct the folding of mutant CFTR.
• New tissue culture methods will facilitate the evaluation of molecular targeted therapy for a wide array of CFTR genotypes, and new animal models should enable assessment of treatment at the earliest stages of the disease.
• Analyses of affected twin and sibling pairs have quantified the contribution of genetic and non-genetic modifiers to variation in key features of cystic fibrosis.
• Candidate and genome-wide approaches have identified biologically plausible gene modifiers of lung disease severity, neonatal intestinal obstruction and diabetes in cystic fibrosis.
• Annotation of variants in CFTR will increase the utility of genetic testing in newborn screening, carrier testing and diagnostic settings. Assignment of variants as disease-causing will validate efforts to target variants for molecular therapies.
• Small-molecule therapy for cystic fibrosis has been successful for patients carrying a subset of CFTR variants. Grouping of variants according to responses in cell-based assays (that is, theratypes) could expedite treatment of affected individuals with rare CFTR genotypes.

Introduction

Cystic fibrosis (OMIM 219700) is a life-limiting autosomal recessive disorder that affects ~70,000 individuals worldwide. The condition affects primarily those of European descent, although cystic fibrosis has been reported in all races and ethnicities. Abnormally viscous secretions in the airways of the lungs and in the ducts of the pancreas in individuals with cystic fibrosis cause obstructions that lead to inflammation, tissue damage and destruction of both organ systems (Fig. 1). Other organ systems containing epithelia — such as the sweat gland, biliary duct of the liver, the male reproductive tract and the intestine — are also affected. Loss of pancreatic exocrine function results in malnutrition and poor growth, which leads to death in the first decade of life for most untreated individuals. Replacement of pancreatic enzymes and intensive therapy guided by multidisciplinary teams have revolutionized the treatment of cystic fibrosis, resulting in progressive improvements in survival to a median predicted age of ~37 years for children born with cystic fibrosis today¹. Obstructive lung disease is currently the primary cause of morbidity and is responsible for ~80% of mortality².

 

Figure 1: Cardinal features of cystic fibrosis and relative contribution of genetic modifiers to variation in select cystic fibrosis traits.

       

 

A diagnosis of cystic fibrosis is based on the presence of clinical findings shown on the left, along with an elevated sweat chloride concentration (>60 mM). The degree of organ system dysfunction varies considerably among affected individuals. Genetic modifiers and non-genetic factors both contribute to airway obstruction and infection with Pseudomonas aeruginosa — two traits that define lung disease in cystic fibrosis. Cystic fibrosis transmembrane conductance regulator (CFTR) genotype is the primary determinant of the degree of pancreatic exocrine dysfunction. The presence of CFTR variants associated with severe pancreatic exocrine dysfunction is essentially a pre-requisite for the development of diabetes and intestinal obstruction. In the setting of severe endocrine dysfunction, genetic modifiers determine when, and if, diabetes occurs and whether neonatal intestinal obstruction occurs. Genetic variation plays the predominant part in nutritional status as assessed by body mass index (BMI)70.

 

Twenty-five years ago, a variant (p.Phe508del; also known as F508del in legacy nomenclature) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene was found to be the most common cause of cystic fibrosis^{3, 4, 5}. Demonstration that CFTR functions as a chloride channel regulated by cyclic AMP (cAMP)-dependent phosphorylation⁶ was consistent with the ion transport disturbances documented in cystic fibrosis tissues^{7, 8}. Key insights into cystic fibrosis pathophysiology were derived from the study of CFTR mutants⁹, correlation of CFTR dysfunction with the cellular manifestations of cystic fibrosis¹⁰, and elucidation of protein partners involved in biogenesis and membrane function¹¹. Identification of disease-causing variants in CFTR contributed a tool for both the diagnosis of cystic fibrosis and the identification of cystic fibrosis carriers¹², demonstrated the degree to which CFTR dysfunction correlates with clinical features¹³, and revealed that CFTR dysfunction can create phenotypes other than cystic fibrosis¹⁴. Over the past 5 years, there has been remarkable progress in the development of small-molecule therapy targeting CFTR bearing select disease-causing variants^{15, 16}.

 

The purpose of this Review is to highlight advances over the past decade in our understanding and treatment of cystic fibrosis that were informed by genetics. Given the breadth of the cystic fibrosis field, not all of the important contributions and publications relevant to the topic can be included. Examples have been chosen to illustrate that genetics continues to have a role in the research of Mendelian disorders long after the causative variants and the responsible gene have been discovered. This Review covers new insights into the processing defect caused by the F508del variant, advances in stem cell technology that can enable testing of therapeutics for a wide range of CFTR genotypes and the development of new animal models that are informing our understanding of organ pathology in cystic fibrosis. I also summarize progress in parsing genetic and non-genetic contributions to variability in cystic fibrosis and in the identification of modifier loci. The final section describes efforts to determine the molecular and phenotypic consequences of the majority of cystic fibrosis-causing variants and to develop molecular treatments for every defect in CFTR.

 

Figure 2: Molecular consequences of variants in CFTR.

       

 

The degree to which epithelial ion transport is altered in an individual with cystic fibrosis is determined by the effect of each disease-causing variant on the quantity and the function of cystic fibrosis transmembrane conductance regulator (CFTR). The key steps of CFTR biogenesis in an epithelial cell are depicted. The membrane-spanning domains of CFTR are shown as red boxes, the two nucleotide-binding domains as yellow circles, and the regulatory domain as a blue circle. The quantity of CFTR protein in the apical cell membrane is a product of the amount of RNA transcribed, the efficiency of RNA splicing, the fraction of protein correctly folded and the stability of the protein in the membrane. The level and/or content of CFTR transcripts can be affected by disease-causing variants in the promoter (for example, c.−234TA (also known as −102TA in legacy nomenclature))¹⁴⁰ and splice sites (for example, c.3717 + 12191 CT (legacy 3849 + 10 kb CT))¹⁴¹, or by variants that introduce a premature termination codon (PTC) and that lead to RNA decay (for example, p.Gly542X; (legacy G542X)¹⁴². The processing of CFTR can be altered by variants that cause aberrant folding of the protein, leading to degradation (for example, p.Phe508del (legacy F508del))¹⁸, or by variants that cause reduced membrane stability as a result of increased rates of endocytosis (for example, p.Asn287Tyr (legacy N287Y))¹⁴³. The function of CFTR is dependent on activity of the ion channel and on the efficiency of conductance of ions through the channel. Disease-causing variants cause reduction in activity (for example, p.Gly551Asp (legacy G551D)¹⁴⁴ or changes in the conduction properties of the chloride channel (for example, p.Arg334Trp (legacy R334W))¹⁴⁴. cAMP, cyclic AMP; ER, endoplasmic reticulum.

 

Figure 3: Molecular treatments for cystic fibrosis.

       

 

Conclusions

The discovery of CFTR 25 years ago was a triumph for genetics and a potent demonstration of its ability to deliver the molecular culprit in a Mendelian disorder. Cystic fibrosis is now positioned to reap the dividends of personalized medicine as variant-specific therapy is deployed, and a growing understanding of the genetic and environmental modifiers of cystic fibrosis enables targeting of individual risk factors. The development of new genetic models of cystic fibrosis in pigs, ferrets, rats and zebrafish provides opportunities to investigate pathophysiology and to explore therapies at the earliest stages of disease. Newborn and population screening enables prospective management of affected individuals from birth, and genomic variation will provide information on the trajectories that individual patients are likely to follow. Genetics has played and will continue to play a key part in achieving a normal lifespan for individuals with cystic fibrosis.

 

Nature Reviews Genetics | Review

Article series: Disease mechanisms

 

Garry R. Cutting^1, 

 

Published online 18 November 2014

Corrected online 20 November 2014