David B. Mount

Pathogenesis of Gout

Clinical Principles The overall disease burden of gout is substantial and may be increasing. As more scientific data on the modifiable risk factors and comorbidities of gout become available, integration of these data into gout care strategies may become essential. Hyperuricemia and gout are associated with the insulin resistance syndrome and related comorbid conditions. Lifestyle modifications that are recommended for gout generally align with those for major chronic disorders (such as the insulin resistance syndrome, hypertension, and cardiovascular disorders); thus, these measures may be doubly beneficial for many patients with gout and particularly for individuals with these comorbid conditions. Effective management of risk factors for gout and careful selection of certain therapies for comorbid conditions (such as hypertension or the insulin resistance syndrome) may also aid gout care. The urateanion exchanger URAT1 (urate transporter-1) is a specific target of action for both antiuricosuric and uricosuric agents. The long-term health effect of hyperuricemia (beyond the increased risk for gout) needs to be clarified, including any potential consequences associated with the chronic hyperuricemia that anti-inflammatory treatment does not correct. Pathophysiologic Principles A direct causal relationship exists between serum urate levels and the risk for gout. Lifestyle factors, including adiposity and dietary habits, appear to contribute to serum uric acid levels and the risk for gout. Urate is extensively reabsorbed from the glomerular ultrafiltrate in the proximal tubule via the brush-border urateanion exchanger URAT1. Sodium-dependent reabsorption of anions increases their concentration in proximal tubule cells, resulting in increased urate exchange via URAT1, increased urate reabsorption by the kidney, and hyperuricemia. Genetic variation in renal urate transporters or upstream regulatory factors may explain the hereditary susceptibility to conditions associated with high urate levels and a patients particular response to medications; these transporters may also serve as targets for future drug development. Urate crystals are able to directly initiate, to amplify, and to sustain an intense inflammatory attack because of their ability to stimulate the synthesis and release of humoral and cellular inflammatory mediators. Cytokines, chemokines, proteases, and oxidants involved in acute urate crystalinduced inflammation also contribute to the chronic inflammation that leads to chronic gouty synovitis, cartilage loss, and bone erosion. For definition of terms used, see Glossary. Gout is a type of inflammatory arthritis that is triggered by the crystallization of uric acid within the joints and is often associated with hyperuricemia (Figure 1). Acute gout is typically intermittent, constituting one of the most painful conditions experienced by humans. Chronic tophaceous gout usually develops after years of acute intermittent gout, although tophi occasionally can be part of the initial presentation. In addition to the morbidity that is attributable to gout itself, the disease is associated with such conditions as the insulin resistance syndrome, hypertension, nephropathy, and disorders associated with increased cell turnover (1, 2). Figure 1. Overview of the pathogenesis of gout. The overall disease burden of gout remains substantial and may be increasing. The prevalence of self-reported, physician-diagnosed gout in the Third National Health and Nutrition Examination Survey was found to be greater than 2% in men older than 30 years of age and in women older than 50 years of age (3). The prevalence increased with increasing age and reached 9% in men and 6% in women older than 80 years of age (4). Furthermore, the incidence of primary gout (that is, patients without diuretic exposure) doubled over the past 20 years, according to the Rochester Epidemiology Project (4). Dietary and lifestyle trends and the increasing prevalence of obesity and the metabolic syndrome may explain the increasing incidence of gout. Researchers have recently made great advances in defining the pathogenesis of gout, including elucidating its risk factors and tracing the molecular mechanisms of renal urate transport and crystal-induced inflammation. This article reviews key aspects of the pathogenesis of gout with a focus on the recent advances. Absence of Uricase in Humans Humans are the only mammals in whom gout is known to develop spontaneously, probably because hyperuricemia only commonly develops in humans (5). In most fish, amphibians, and nonprimate mammals, uric acid that has been generated from purine (see Glossary) metabolism undergoes oxidative degradation through the uricase enzyme, producing the more soluble compound allantoin. In humans, the uricase gene is crippled by 2 mutations that introduce premature stop codons (see Glossary) (6). The absence of uricase, combined with extensive reabsorption of filtered urate, results in urate levels in human plasma that are approximately 10 times those of most other mammals (30 to 59 mol/L) (7). The evolutionary advantage of these findings is unclear, but urate may serve as a primary antioxidant in human blood because it can remove singlet oxygen and radicals as effectively as vitamin C (8). Of note, levels of plasma uric acid (about 300 M) are approximately 6 times those of vitamin C in humans (8, 9). Other potential advantages of the relative hyperuricemia in primate species have been speculated (8, 10, 11). However, hyperuricemia can be detrimental in humans, as demonstrated by its proven pathogenetic roles in gout and nephrolithiasis and by its putative roles in hypertension and other cardiovascular disorders (12). The Role of Urate Levels Uric acid is a weak acid (pKa, 5.8) that exists largely as urate, the ionized form, at physiologic pH. As urate concentration increases in physiologic fluids, the risk for supersaturation and crystal formation generally increases. Population studies indicate a direct positive association between serum urate levels and a future risk for gout (13, 14), as shown in Figure 2. Conversely, the use of antihyperuricemic medication is associated with an 80% reduced risk for recurrent gout, confirming the direct causal relationship between serum uric acid levels and risk for gouty arthritis (15). The solubility of urate in joint fluids, however, is influenced by other factors in the joint, as shown in Figure 3. Such factors include temperature, pH, concentration of cations, level of articular dehydration, and the presence of such nucleating agents as nonaggregated proteoglycans, insoluble collagens, and chondroitin sulfate (see Glossary) (16-18). Variation in these factors may account for some of the difference in the risk for gout associated with a given elevation in serum urate level (13, 14). Furthermore, these factors may explain the predilection of gout in the first metatarsal phalangeal joint (a peripheral joint with a lower temperature) and osteoarthritic joints (18) (degenerative joints with nucleating debris) and the nocturnal onset of pain (because of intra-articular dehydration) (19). Figure 2. The relationship between serum uric acid levels and the incidence of gout. Figure 3. Mechanisms of monosodium urate crystal formation and induction of crystal-induced inflammation. MSU Urate Balance The amount of urate in the body depends on the balance between dietary intake, synthesis, and the rate of excretion (20), as shown in Figure 1. Hyperuricemia results from urate overproduction (10%), underexcretion (90%), or often a combination of the two. The purine precursors come from exogenous (dietary) sources or endogenous metabolism (synthesis and cell turnover). The Relationship between Purine Intake and Urate Levels The dietary intake of purines contributes substantially to the blood uric acid. For example, the institution of an entirely purine-free diet over a period of days can reduce blood uric acid levels of healthy men from an average of 297 mol/L to 178 mol/L (21, 22). The bioavailable purine content of particular foods would depend on their relative cellularity and the transcriptional (see Glossary) and metabolic activity of the cellular content (20). Little is known, however, about the precise identity and quantity of individual purines in most foods, especially when cooked or processed (23). When a purine precursor is ingested, pancreatic nucleases break its nucleic acids into nucleotides (see Glossary), phosphodiesterases break oligonucleotides into simple nucleotides, and pancreatic and mucosal enzymes remove phosphates and sugars from nucleotides (20). The addition of dietary purines to purine-free dietary protocols has revealed a variable increase in blood uric acid levels, depending on the formulation and dose of purines administered (21). For example, RNA has a greater effect than an equivalent amount of DNA (24), ribomononucleotides have a greater effect than nucleic acid (21), and adenine has a greater effect than guanine (25, 26). A recent large prospective study showed that men in the highest quintile of meat intake had a 41% higher risk for gout compared with the lowest quintile, and men in the highest quintile of seafood intake had a 51% higher risk compared with the lowest quintile (27). Correspondingly, in a nationally representative sample of men and women in the United States, higher levels of meat and seafood consumption were associated with higher serum uric acid levels (28). However, consumption of oatmeal and purine-rich vegetables (for example, peas, beans, lentils, spinach, mushrooms, and cauliflower) was not associated with an increased risk for gout (27). The variation in the risk for gout associated with different purine-rich foods may be explained by varying amounts and type of purine content and their bioavailability for metabolizing purine to uric acid (28). At the practical level, these data suggest that dietary p

CLONING AND CHARACTERIZATION OF KCC3 AND KCC4, NEW MEMBERS OF THE CATION-CHLORIDE COTRANSPORTER GENE FAMILY

The K+-Cl−cotransporters (KCCs) belong to the gene family of electroneutral cation-chloride cotransporters, which also includes two bumetanide-sensitive Na+-K+-2Cl−cotransporters and a thiazide-sensitive Na+-Cl− cotransporter. We have cloned cDNAs encoding mouse KCC3, human KCC3, and human KCC4, three new members of this gene family. The KCC3 and KCC4 cDNAs predict proteins of 1083 and 1150 amino acids, respectively. The KCC3 and KCC4 proteins are 65–71% identical to the previously characterized transporters KCC1 and KCC2, with which they share a predicted membrane topology. The four KCC proteins differ at amino acid residues within key transmembrane domains and in the distribution of putative phosphorylation sites within the amino- and carboxyl-terminal cytoplasmic domains. The expression of mouse KCC3 in Xenopus laevis oocytes reveals the expected functional characteristics of a K+Cl− cotransporter: Cl−-dependent uptake of86Rb+ which is strongly activated by cell swelling and weakly sensitive to furosemide. A direct functional comparison of mouse KCC3 to rabbit KCC1 indicates that KCC3 has a much greater volume sensitivity. The human KCC3 and KCC4 genes are located on chromosomes 5p15 and 15q14, respectively. Although widely expressed, KCC3 transcripts are the most abundant in heart and kidney, and KCC4 is expressed in muscle, brain, lung, heart, and kidney. The unexpected molecular heterogeneity of K+-Cl− cotransport has implications for the physiology and pathophysiology of a number of tissues.

Roles of the cation-chloride cotransporters in neurological disease.

In the nervous system, the intracellular chloride concentration ([Cl−]i) determines the strength and polarity of γ-aminobutyric acid (GABA)-mediated neurotransmission. [Cl−]i is determined, in part, by the activities of the SLC12 cation–chloride cotransporters (CCCs). These transporters include the Na–K–2Cl cotransporter NKCC1, which mediates chloride influx, and various K–Cl cotransporters—such as KCC2 and KCC3—that extrude chloride. A precise balance between NKCC1 and KCC2 activity is necessary for inhibitory GABAergic signaling in the adult CNS, and for excitatory GABAergic signaling in the developing CNS and the adult PNS. Altered chloride homeostasis, resulting from mutation or dysfunction of NKCC1 and/or KCC2, causes neuronal hypoexcitability or hyperexcitability; such derangements have been implicated in the pathogenesis of seizures and neuropathic pain. [Cl−]i is also regulated to maintain normal cell volume. Dysfunction of NKCC1 or of swelling-activated K–Cl cotransporters has been implicated in the damaging secondary effects of cerebral edema after ischemic and traumatic brain injury, as well as in swelling-related neurodegeneration. CCCs represent attractive therapeutic targets in neurological disorders the pathogenesis of which involves deranged cellular chloride homoestasis.

Penetration of the stigma and style elicits a novel transcriptome in pollen tubes, pointing to genes critical for growth in a pistil.

Pollen tubes extend through pistil tissues and are guided to ovules where they release sperm for fertilization. Although pollen tubes can germinate and elongate in a synthetic medium, their trajectory is random and their growth rates are slower compared to growth in pistil tissues. Furthermore, interaction with the pistil renders pollen tubes competent to respond to guidance cues secreted by specialized cells within the ovule. The molecular basis for this potentiation of the pollen tube by the pistil remains uncharacterized. Using microarray analysis in Arabidopsis, we show that pollen tubes that have grown through stigma and style tissues of a pistil have a distinct gene expression profile and express a substantially larger fraction of the Arabidopsis genome than pollen grains or pollen tubes grown in vitro. Genes involved in signal transduction, transcription, and pollen tube growth are overrepresented in the subset of the Arabidopsis genome that is enriched in pistil-interacted pollen tubes, suggesting the possibility of a regulatory network that orchestrates gene expression as pollen tubes migrate through the pistil. Reverse genetic analysis of genes induced during pollen tube growth identified seven that had not previously been implicated in pollen tube growth. Two genes are required for pollen tube navigation through the pistil, and five genes are required for optimal pollen tube elongation in vitro. Our studies form the foundation for functional genomic analysis of the interactions between the pollen tube and the pistil, which is an excellent system for elucidation of novel modes of cell–cell interaction.

Molecular physiology of cation-coupled Cl- cotransport: the SLC12 family.

The electroneutral cation-chloride-coupled cotransporter gene family (SLC12) was identified initially at the molecular level in fish and then in mammals. This nine-member gene family encompasses two major branches, one including two bumetanide-sensitive Na+-K+-2Cl− cotransporters and the thiazide-sensitive Na+:Cl− cotransporter. Two of the genes in this branch (SLC12A1 and SLC12A3), exhibit kidney-specific expression and function in renal salt reabsorption, whereas the third gene (SLC12A2) is expressed ubiquitously and plays a key role in epithelial salt secretion and cell volume regulation. The functional characterization of both alternatively-spliced mammalian Na+-K+-2Cl− cotransporter isoforms and orthologs from distantly related species has generated important structure-function data. The second branch includes four genes (SLC12A4–7) encoding electroneutral K+-Cl− cotransporters. The relative expression level of the neuron-specific SLC12A5 and the Na+-K+-2Cl− cotransporter SLC12A2 appears to determine whether neurons respond to GABA with a depolarizing, excitatory response or with a hyperpolarizing, inhibitory response. The four K+-Cl− cotransporter genes are co-expressed to varying degrees in most tissues, with further roles in cell volume regulation, transepithelial salt transport, hearing, and function of the peripheral nervous system. The transported substrates of the remaining two SLC12 family members, SLC12A8 and SLC12A9, are as yet unknown. Inactivating mutations in three members of the SLC12 gene family result in Mendelian disease; Bartter syndrome type I in the case of SLC12A1, Gitelman syndrome for SLC12A3, and peripheral neuropathy in the case of SLC12A6. In addition, knockout mice for many members of this family have generated important new information regarding their respective physiological roles.

The K-Cl cotransporter KCC3 is mutant in a severe peripheral neuropathy associated with agenesis of the corpus callosum

Peripheral neuropathy associated with agenesis of the corpus callosum (ACCPN) is a severe sensorimotor neuropathy associated with mental retardation, dysmorphic features and complete or partial agenesis of the corpus callosum. ACCPN is transmitted in an autosomal recessive fashion and is found at a high frequency in the province of Quebec, Canada. ACCPN has been previously mapped to chromosome 15q. The gene SLC12A6 (solute carrier family 12, member 6), which encodes the K+–Cl− transporter KCC3 and maps within the ACCPN candidate region, was screened for mutations in individuals with ACCPN. Four distinct protein-truncating mutations were found: two in the French Canadian population and two in non–French Canadian families. The functional consequence of the predominant French Canadian mutation (2436delG, Thr813fsX813) was examined by heterologous expression of wildtype and mutant KCC3 in Xenopus laevis oocytes; the truncated mutant is appropriately glycosylated and expressed at the cellular membrane, where it is non-functional. Mice generated with a targeted deletion of Slc12a6 have a locomotor deficit, peripheral neuropathy and a sensorimotor gating deficit, similar to the human disease. Our findings identify mutations in SLC12A6 as the genetic lesion underlying ACCPN and suggest a critical role for SLC12A6 in the development and maintenance of the nervous system.

Functional Comparison of the K+-Cl−Cotransporters KCC1 and KCC4

The K+-Cl−cotransporters (KCCs) are members of the cation-chloride cotransporter gene family and fall into two phylogenetic subgroups: KCC2 paired with KCC4 and KCC1 paired with KCC3. We report a functional comparison inXenopus oocytes of KCC1 and KCC4, widely expressed representatives of these two subgroups. KCC1 and KCC4 exhibit differential sensitivity to transport inhibitors, such that KCC4 is much less sensitive to bumetanide and furosemide. The efficacy of these anion inhibitors is critically dependent on the concentration of extracellular K+, with much higher inhibition in 50 mm K+ versus 2 mmK+. KCC4 is also uniquely sensitive to 10 mmbarium and to 2 mm trichlormethiazide. Kinetic characterization reveals divergent affinities for K+(K m values of ∼25.5 and 17.5 mm for KCC1 and KCC4, respectively), probably due to variation within the second transmembrane segment. Although the two isoforms have equivalent affinities for Cl−, they differ in the anion selectivity of K+ transport (Cl− > SCN− = Br− > PO4 −3 > I−for KCC1 and Cl− > Br− > PO4 −3 = I− > SCN−for KCC4). Both KCCs express minimal K+-Cl−cotransport under isotonic conditions, with significant activation by cell swelling under hypotonic conditions. The cysteine-alkylating agentN-ethylmaleimide activates K+-Cl− cotransport in isotonic conditions but abrogates hypotonic activation, an unexpected dissociation of N-ethylmaleimide sensitivity and volume sensitivity. Although KCC4 is consistently more volume-sensitive, the hypotonic activation of both isoforms is critically dependent on protein phosphatase 1. Overall, the functional comparison of these cloned K+-Cl− cotransporters reveals important functional, pharmacological, and kinetic differences with both physiological and mechanistic implications.

Localization of the K+–Cl− cotransporter, KCC3, in the central and peripheral nervous systems: expression in the choroid plexus, large neurons and white matter tracts

Na(+)-independent K(+)-Cl(-) cotransporters function in the regulation of cell volume, control of CNS excitability and epithelial ion transport. Several K(+)-Cl(-) cotransporter isoforms are expressed in the nervous system, and KCC3 in particular is expressed at significant levels in both the brain and spinal cord. The cellular localization of this transporter has, however, not been determined. In this study, we generated a polyclonal antibody against the KCC3 cotransporter in order to characterize and localize this protein in the brain. Western blot analysis of mouse kidney and brain demonstrated KCC3 proteins of different size, 150 and 170kDa, respectively; this disparity remained after deglycosylation. Northern blot confirmed the presence of two distinct forms of KCC3, KCC3a and KCC3b, generated by the inclusion of different first coding exons. KCC3a predominates in the brain, whereas KCC3b is more abundant in the kidney. Western blots with membrane protein from dissected mouse brain revealed abundant expression in all brain regions examined: the cerebral cortex, hippocampus, diencephalon, brainstem and cerebellum. The spinal cord showed the highest levels of KCC3 expression, whereas peripheral nerves did not contain immunoreactive KCC3 protein. Western blot analysis of whole brain from rats of various ages indicated increasing expression in the postnatal period, concurrent with CNS maturation and myelination. Immunofluorescence studies demonstrated strong signal in myelinated tracts of the spinal cord, consistent with individual myelin sheaths. Brain sections also showed white matter enhancement, but also cellular signal consistent with pyramidal neurons and Purkinje cells. The base of the choroid plexus epithelium was also strongly labeled. These data demonstrate the specificity and diversity of KCC3 expression in the mouse CNS.

Molecular, functional, and genomic characterization of human KCC2, the neuronal K-Cl cotransporter.

The expression level of the neuronal-specific K-Cl cotransporter KCC2 (SLC12A5) is a major determinant of whether neurons will respond to GABA with a depolarizing, excitatory response or a hyperpolarizing, inhibitory response. In view of the potential role in human neuronal excitability we have characterized the hKCC2 cDNA and gene. The 5.9 kb hKCC2 transcript is specific to brain, and is induced during in vitro differentiation of NT2 teratocarcinoma cells into neuronal NT2-N cells. The 24-exon SLC12A5 gene is on human chromosome 20q13, and contains a polymorphic dinucleotide repeat within intron 1 near a potential binding site for neuron-restrictive silencing factor. Expression of hKCC2 cRNA in Xenopus laevis oocytes results in significant Cl(-)-dependent (86)Rb(+) uptake under isotonic conditions; cell swelling under hypotonic conditions causes a 20-fold activation, which is blocked by the protein phosphatase inhibitor calyculin-A. In contrast, oocytes expressing mouse KCC4 do not mediate isotonic K-Cl cotransport but express much higher absolute transport activity than KCC2 oocytes under hypotonic conditions. Initial and steady state kinetics of hKCC2-injected oocytes were performed in both isotonic and hypotonic conditions, revealing K(m)s for K(+) and Cl(-) of 9.3+/-1.8 mM and 6.8+/-0.9 mM, respectively; both affinities are significantly higher than KCC1 and KCC4. The K(m) for Cl(-) is close to the intracellular Cl(-) activity of mature neurons, as befits a neuronal efflux mechanism.

The genetics of hyperuricaemia and gout

Gout is a common and very painful inflammatory arthritis caused by hyperuricaemia. This Review provides an update on the genetics of hyperuricaemia and gout, including findings from genome-wide association studies. Most of the genes that associated with serum uric acid levels or gout are involved in the renal urate-transport system. For example, the urate transporter genes SLC2A9, ABCG2 and SLC22A12 modulate serum uric acid levels and gout risk. The net balance between renal urate absorption and secretion is a major determinant of serum uric acid concentration and loss-of-function mutations in SLC2A9 and SLC22A12 cause hereditary hypouricaemia due to reduced urate absorption and unopposed urate secretion. However, the variance in serum uric acid explained by genetic variants is small and their clinical utility for gout risk prediction seems limited because serum uric acid levels effectively predict gout risk. Urate-associated genes and genetically determined serum uric acid levels were largely unassociated with cardiovascular–metabolic outcomes, challenging the hypothesis of a causal role of serum uric acid in the development of cardiovascular disease. Strong pharmacogenetic associations between HLA-B*5801 alleles and severe allopurinol-hypersensitivity reactions were shown in Asian and European populations. Genetic testing for HLA-B*5801 alleles could be used to predict these potentially fatal adverse effects.