Pathophysiology of cystic fibrosis: an overview

Cystic fibrosis (CF) is a monogenetic autosomal recessive disease that affects approximately 75,000 people worldwide.(1) Patients with CF show symptoms in the lungs and upper airways but also in the gastrointestinal (GI) tract and reproductive system.(2)

Fibrotic, scarred tissue and fluid-filled cysts in the exocrine pancreas were the pathological findings that eventually led scientists to name the disease ‘cystic fibrosis of the pancreas’.(3)  Another common feature found in all organs affected in CF are highly viscous mucus plugs that impair the function of these systems. The abnormal mucus rheology has prompted some investigators to coin the term ‘mucoviscidosis’.(4)  Most patients with CF also lose an increased amount of salt during perspiration, something that bears no immediate danger for the patients but which has, so far, been used as diagnostic test.(5) CF is still a lethal disease but the reports that not only the lifespan but also the quality of life for CF patients has increased dramatically over the past ten years are encouraging. Nowadays, many patients with CF reach adulthood and lead an otherwise seemingly normal life. The development of the median survival time of patients diagnosed with CF against time is shown in Figure 1.

It is surprising that this trend cannot be attributed to the introduction of a new drug with groundbreaking effects on the cause of the disease. Nevertheless, the outcome of CF still remains fatal and imposes a tremendous burden on patients and relatives. But why do mutations in a single protein have such an impact on the physiological functions of the body?

The defect underlying the disease results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The gene product, CFTR, belongs to the family of the ATP binding cassette (ABC) proteins.(6) Similar to other members of this family, CFTR consists of 12 transmembrane spanning domains that are separated by a nucleotide-binding fold and a regulatory subunit.(7) A second nucleotide-binding fold is located at the C-terminal end.(7)  The protein is heavily glycosylated and functions as a cAMP activated Cl– channel.(8)

CFTR not only conducts Cl– ions but also anions such as HCO3–, ATP  and glutathione.(9–11) CFTR is believed to interact with several other intracellular and transmembraneous proteins.(12) However it is not absolutely clear to what extent these interactions, or the lack thereof, impact on the functions of cells expressing CFTR. To date, almost 2000 different mutations in the CFTR gene have been reported and most of these mutations have been classified as five different types (Table 1).

A majority of CF patients carry, at least on one allele, a mutation that leads to deletion of a phenylalanine in position 508 of the protein (F508del). The mutated protein is improperly folded and degraded within the ubiquitin pathway.(13)  Mutations that prevent proper delivery of the protein are usually denoted as class II mutations. More than 70% of all CF patients are homozygous for the F508del mutation and more than 90% of all patients carry that mutation on at least one allele. Another class of mutations introduces a stop codon in the genetic sequence that results in the complete absence of any channel protein (class I mutations). Some studies have tried to find a genotype–phenotype correlation.(14,15) To this end, none of these studies have succeeded in demonstrating a clear correlation. This might, in part, be due to the strong impact that environmental and social factors have on the disease. Most recently a variety of ‘modifier genes’ are thought to influence the outcome of the disease.(14)

Despite the abundant analyses by geneticists, most clinicians prefer a somewhat simpler classification by considering patients with pancreatic insufficiency as severe cases, compared with those who retain pancreatic function as mild cases.

After the cloning of the gene encoding for CFTR in 1989,(16) the scientific community was full of optimism that CF as a fatal disease could be cured within a decade. In fact, the scientific efforts undertaken to understand the complex nature of CFTR and the consequences of CFTR mutations on the human physiology had been unprecedented. Nevertheless, while the molecular functions of the protein are somewhat understood, it is difficult to explain the pathophysiology of CF patients by a mere lack of electrolyte transport. The majority of scientists favour as explanation an imbalance between electrolyte absorption and secretion that results in an impaired hydration of the epithelial surface liquid. Cellular models have supported this hypothesis, while transgenic animal models in which the CF-causing mutations have been introduced did not reproduce the expected phenotype. A transgenic mouse in which the beta subunit of the epithelial Cl– channel is overexpressed is currently the best model for CF-like lung disease in rodents despite the fact that the CFTR protein is functioning properly.(17) Transgenic pig models bearing CFTR mutations that lead to problems in the GI tract and which have a higher likelihood of developing lung disease than a normal litter have also been developed.(18,19)

Airway epithelia can be divided in two different functional entities: primarily absorptive and secretory cells. The absorptive surface epithelia of the airways express high levels of the epithelial sodium channel, eNaC, whereas the CFTR expression pattern is rather scant (surface epithelium). Measurements of the transepithelial voltage difference across nasal mucosae revealed a substantially increased voltage in CF patients compared with normal.(20) The only explanation for this phenomenon was that the fractional Na+ conductance was elevated. Because no abnormality in the structure and subunit composition of eNaC in CF was reported, the apparent increase in Na+ transport remains puzzling. Some studies have proposed that activation of CFTR leads to an apparent downregulation of eNaC activity that is absent when CFTR is mutated.(21)

A study published in 1996 proposed a new explanation for the pathophysiology in the airways of CF patients.(22) The investigators added bacteria to cultured airway epithelial cells collected either from CF patients or normal specimens and grown on an air–liquid interface. Their striking observation was that the growth rate in the presence of CF cells was relatively high compared with normal cells. When the culture media of the CF cells was diluted, the growth rate was identical in the two groups. Analysis of surface liquid samples obtained from CF patients and normals suggested that the NaCl content in CF lungs was increased. The authors proposed that the altered composition of the surface liquid produced by CF cells would result in a decreased first-strike capacity of antimicrobial peptides secreted on the surface of the cells. These peptides, beta-defensins,(23) were inactive when the ionic strength of the surrounding liquid exceeded plasma levels. The implications of these observations led to an ongoing discussion between competing labs worldwide but as yet, a general consensus has not been reached. What is clear is that exact knowledge of the composition of the airway surface liquid will have an enormous impact on future therapeutic regimens.

A less complicated situation is seen in the case of the secretory epithelia. The secretory serous cells of the submucosal glands and the goblet cells do not express eNaC and are believed to be the predominant site of CFTR expression in the airways. Other Cl– channels than CFTR might contribute to the overall transport capacity but their molecular identity and importance in the airways is still a matter of debate.(24)

Secretory epithelia, such as the submucosal glands, secrete mucins, protease inhibitors, antibiotic peptides and enzymes that must be flushed from the glands onto the airway surface epithelium. Recent data obtained from small airway specimens of CF patients have demonstrated no apparent difference in the electrolyte content of the gland secretions but an increased viscosity of the mucus compared control samples.(25)  At this stage, it is hard to reconcile these findings with a mere lack of CFTR expression in these glands.

It is generally accepted that defective CFTR leads to an imbalance between fluid absorption and secretion in the lungs of CF patients, resulting in a relatively dehydrated mucus layer on the airways. The depletion of liquid and the increased viscosity of the mucus impair the function of the cilia and thereby restrict mucociliary clearance. Small particles, bacteria and fungi remain stuck in the airways, which house perfect conditions for growth. The onset of clear symptoms of an impaired lung function remains highly variable. Carriers of CFTR mutations are often referred to clinical centres for the first time when lung problems occur. Persistent coughing, obstruction of the upper airways and recurrent lower respiratory tract infections are often mistaken for symptoms of a general bronchitis, and are therefore treated in a similar fashion. Because of the significant implications in making a diagnosis of CF, functional testing and genotype assessment are usually recommended. However, once the diagnosis CF is confirmed the therapeutic regimen might not necessarily need to be changed completely.

The progression of CF lung disease is in many ways similar to that of other chronic airway diseases such as COPD, emphysema and bronchiectasis.(2) In striking contrast, however, is that a variety of bacteria, such as Pseudomonas aeruginosa, Stenotropomonas maltophilia and Burgholderia cepacea, chronically colonise the airways of CF patients. The low susceptibility of these bacteria to antibiotics complicate the treatment and makes eradication challenging. During the recurrent inflammatory episodes that highlight the fight of the body against bacteria, lung tissue is lost and scarred. Clinical parameters such as vital capacity, FEV1 and O2 saturation decline as the disease takes its course. While the lung function deteriorates, CF patients often develop pulmonary hypertension resulting from loss of lung tissue and the hypoxic constriction of pulmonary blood vessels. Compensatory mechanisms lead to hypertrophy of the right ventricle, generally referred to as Cor pulmonale. During end-stage CF, patients are dependent on supplementary oxygen and mechanical ventilation before they eventually die from respiratory failure or cardiopulmonary complications. The only hope for end-stage patients is to receive a lung transplant, which cures the lung disease to an extent but which is also associated with additional problems, such as lifelong immunosuppression and possible graft rejection. The decision to put a patient on the waiting list for a lung transplant should be made before the progression of the disease renders the patient too weak to tolerate the consequences of this intervention.

The GI tract has an astonishing capacity for fluid transport. CFTR is present in almost all epithelia lining the GI tract, from the salivary glands to the distal colon; therefore, severe consequences of the CFTR mutations for the entire GI tract would be expected. However, the organ most affected in CF is the pancreas. Approximately 70–90% of all CF patients are born with pancreatic insufficiency, which means that more than 98% of the pancreatic capacity is already lost in the newborn.

Even in seemingly pancreatic-sufficient patients, the ratio between alkaline fluid and secreted digestive enzymes is decreased significantly. Fluid secretion in the exocrine pancreas is very similar to the mechanisms outlined for the submucosal glands of the airways. Under physiological conditions pancreatic ducts secrete a HCO3– – and electrolyte-rich fluid that serves to flush the digestive enzymes from the acini into the lumen of the small intestine. A lack of fluid secretion leads to an accumulation of lipo- and proteolytic enzymes in the pancreatic ducts, which eventually damage the pancreatic tissue. The tissue damage occurs in utero in pancreatic-insufficient patients; however, in some cases, the process may develop over a period of many years. Pancreatic insufficiency leads to maldigestion and severe steatorrhoea with concominant loss of lipid soluble vitamins and essential fatty acids. The resulting malnutrition renders CF patients more susceptible to infections, and thereby aggravates the lung disease.

In contrast to the lung, the symptoms in the GI tract can often be observed shortly after birth. Babies suffering from CF often have bulky, greasy stools that can be initially mistaken for symptoms of coeliac disease. The resulting failure to thrive is often reason enough for the first hospitalisation. Sometimes stool sticks in the gut, causing a so-called meconium ileus that might require surgical intervention. It is not clear whether this symptom is a result of maldigestion in the small intestine or is caused by hyperabsorption in the distal parts of the gut. As more and more patients reach adulthood, clinicians also observe hepatobiliary problems that result from obstructions in the bile duct, presumably as a result of the higher viscosity of the bile in CF. The subsequent destruction of liver tissue sometimes leads to jaundice, and even liver failure, that can only be overcome by transplanting a new organ.

Most of the recent drug developments are aimed to cure CF of the lung. One should not forget that a rational drug treatment should also be directed against the multiple manifestations in the GI tract.

Most CF patients carry a mutation that leads to early degradation of the premature protein that would otherwise be functional if inserted into the plasma membrane. The most prevailing causative mutation, F508del, leads to an improper folding of the protein. The quality control mechanisms located in the trans-Golgi network detect the misfolded protein and submit it to the ubiquitin-proteasome pathway.(13)  Tampering with the quality control system would eventually rescue enough protein to restore its activity. Currently it is estimated that reaching 10% of the wild-type CFTR activity would be sufficient to yield a normal phenotype in patients. It is unsurprising that most pharmacological attempts were aimed to bypass the quality control system. None of these approaches was nearly as successful as trying to activate dysfunctional CFTR that was already inserted into the membrane. The glycine-to-aspartate mutation, G551D, is the most promising candidate for such an approach. G551D is the third most common mutation, with a higher prevalence among people of Celtic origin. The mutation leads to a defect in the gating of the CFTR, which means that the channel is in a closed state most of the time and therefore unable to conduct anions correctly. A drug targeted to alter the conductive state of such a protein would show that it is the lack of anion transport that is responsible for the pathophysiology in CF.

Cystic fibrosis is the most frequent inherited disease among Caucasians. The genetic defect underlying CF results from mutations in the gene encoding CFTR. Most mutations in the CFTR gene result in a lack of functional CFTR expression in the membrane. It therefore seemed prudent to assume that impaired epithelial electrolyte transport is the primary cause for the pathophysiology of CF; however, patients suffering from this disease are affected by numerous other symptoms that cannot always be related to dysfunctional Cl– transport. CF is still a lethal disease, but the lifespan of CF patients has increased markedly over the past years. This can certainly be attributed to an improved knowledge about the disease, resulting in new and intensified therapeutic schemes. Novel drugs, however, are still rare and in high demand. Current strategies are aimed either to correct the genetic defect or to influence the pathophysiological consequences of the CFTR mutations. Because of the impact defective CFTR has on the disease, most developments are directed to rescue the defective protein and restore its activity in the cell membrane. For the first time, a drug designed to target a specific mutation in the CFTR gene, thereby correcting the transport defect, has shown clinical benefit for a subset of patients. In a series of clinical trials, the small molecule, ivacaftor (VX-770), demonstrated safety and effectiveness in a panel of outcome parameters. The drug, already marketed in the USA as Kalydeco™ has meanwhile been launched in a number of EU countries during the second half of 2012. Ivacaftor is most effective on a mutation present only in a small proportion of CF patients, and other compounds are currently under investigation in clinical trials. At this stage a real cure for all CF patients is still wishful thinking, but the combined use of novel drugs opens up the hope of greatly increased quality of life for CF patients.

• Cystic fibrosis (CF) is the most frequent inherited disease among Caucasians. The defect underlying the disease results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.
• Symptoms present in the lungs, upper airways, gastrointestinal tract and reproductive system.
• CF is still a lethal disease, but the lifespan of patients has markedly increased over the
• past years. Current strategies are aimed at either correcting the genetic defect or influencing the pathophysiological consequences of the CFTR mutations.
• Novel drugs are still rare and in high demand.
• In a series of clinical trials, the small molecule, ivacaftor (VX-770; Kalydeco™), demonstrated safety and effectiveness in a panel of outcome parameters. The drug is already marketed in the US and in some countries of the EU.

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