The ClC-K2 Chloride Channel Is Critical for Salt Handling in the Distal Nephron (original) (raw)
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Impairment in renal medulla development underlies salt wasting in Clc-k2 channel deficiency
JCI Insight
The prevailing view is that ClC-Ka chloride channel (mouse Clc-k1) functions in thin ascending limb for urine concentration, whereas ClC-Kb (mouse Clc-k2) in thick ascending limb (TAL) for salt reabsorption, respectively. Mutations of ClC-Kb cause classic Bartter syndrome with renal salt wasting with onset from perinatal to adolescent. We study the roles of Clc-k channels in perinatal mouse kidneys using constitutive or inducible kidney-specific gene ablation and 2-D and advanced 3-D imaging of optically cleared kidneys. We show that Clc-k1 and-k2 are broadly expressed and colocalized in perinatal kidneys. Deletion of Clc-k1 and-k2 reveals that both participate in NKCC2-and NCC-mediated NaCl reabsorption in neonatal kidneys. Embryonic deletion of Clc-k2 causes tubular injury and impairs renal medulla and TAL development. Inducible deletion of Clc-k2 begins after medulla maturation produces mild salt wasting resulting from reduced NCC activity. Thus, both Clc-k1 and-k2 contribute to salt reabsorption in TAL and DCT in neonates, potentially explaining less severe phenotypes in classic Bartter. As opposed to the current understanding that salt wasting in adult Bartter patients is due to Clc-k2 deficiency in adult TAL, our results suggest that it is mainly originated from medulla and TAL defects during development. Table 2. Plasma and urine biochemistries of 8-week-old Clc-k1-null and Clc-k2-null mice WT Clc-k1-/-WT Clc-k2-/-Plasma BUN (mg/dL) 29.7±1.6 26.6±2.0 30.4±1.7 63.6±3.0** Creatinine (mg/dL) 0.25±0.03 0.29±0.03 0.26±0.01 0.39±0.03** Table 3 Plasma and urine biochemistries in 10-week-old inducible Clc-k2 deficient mice.
Journal of Biological Chemistry, 2002
ROMK is an apical K ؉ channel expressed in the thick ascending limb of Henle (TALH) and throughout the distal nephron of the kidney. Null mutations in the ROMK gene cause type II Bartter's syndrome, in which abnormalities of electrolyte, acid-base, and fluid-volume homeostasis occur because of defective NaCl reabsorption in the TALH. To understand better the pathogenesis of type II Bartter's syndrome, we developed a mouse lacking ROMK and examined its phenotype. Young null mutants had hydronephrosis, were severely dehydrated, and ϳ95% died before 3 weeks of age. ROMKdeficient mice that survived beyond weaning grew to adulthood; however, they had metabolic acidosis, elevated blood concentrations of Na ؉ and Cl ؊ , reduced blood pressure, polydipsia, polyuria, and poor urinary concentrating ability. Whole kidney glomerular filtration rate was sharply reduced, apparently as a result of hydronephrosis, and fractional excretion of electrolytes was elevated. Micropuncture analysis revealed that the single nephron glomerular filtration rate was relatively normal, absorption of NaCl in the TALH was reduced but not eliminated, and tubuloglomerular feedback was severely impaired. These data show that the loss of ROMK in the mouse causes perturbations of electrolyte, acid-base, and fluid-volume homeostasis, reduced absorption of NaCl in the TALH, and impaired tubuloglomerular feedback.
AJP: Renal Physiology, 2008
We investigated which of the NaCl transporters are involved in the maintenance of salt sensitive hypertension. Milan hypertensive (MHS) rats were studied 3 months after birth. In MHS, as compared to normotensive strain (MNS), mRNA abundance, quantified by competitive PCR on isolated tubules, was unchanged both for Na + -H + isoform 3 (NHE3) and Na + -K + -2Cl -(NKCC2), but higher (119%, n=5, p<0.005) for Na + -Cl -(NCC) in distal convoluted tubules (DCT). These results were confirmed by Western blots which revealed: the renal tubule cells to reabsorb sodium (13). Therefore the importance of the Na + -K + -ATPase in the pathogenesis of hypertension is fully established (3).
Renal Potassium Handling and Associated Inherited Tubulopathies Leading to Hypokalemia
Basic Nephrology and Acute Kidney Injury, 2012
Basic Nephrology and Acute Kidney Injury 90 patients with chronic kidney disease. Generalized defects in proximal tubule handling of solutes result in Fanconi syndrome syndrome. Specific proximal tubular handling defects leading to hypokalemia include proximal renal tubular acidosis, and have been recently reviewed elsewhere (Fry &Karet, 2007). 2.2 Potassium movement in the thin loop of Henle Potassium may be secreted in the thin descending loops of Henle that penetrate the inner medulla, whilst the thin ascending loop is permeable to sodium and potassium and allows some uptake. The thin ascending limbs of the loops of Henle (among other nephron segments) express the CLC-KA chloride channel, with its subunit Barttin. Polymorphisms in CLCNKA have recently been associated with a hyperreninemic hyperaldosteronism, implicating a key role for this channel in regulating renal salt handling and determining a set point for renin and angiotensin levels (Cappola, et al., 2011). 2.3 Potassium movement in thick ascending loop of Henle In this part of nephron, ~25% of filtered sodium is reabsorbed together with ~15% of the filtered potassium. Transcellular sodium and potassium transport is achieved by the Na + K + 2Cl-cotransporter (NKCC2) in the apical membrane, driven by the basolateral Na + K + ATPase pump. Alternative names for NKCC2 include the bumetanide sensitive cotransporter, (BSC). NKCC2 is exclusively expressed in kidney tissue and is encoded by SLC12A1 gene (Simon, et al., 1996a). The NKCC2 transporter may also transport ammonium ions, which compete with potassium ions. Potassium entering the cell via NKCC2 is recycled back into tubule lumen via the apical membrane channel, ROMK1 (Simon, et al., 1996b), generating a lumen positive potential driving paracellular resorption of calcium and magnesium. Tight junction proteins, such as paracellin 1, mediate this divalent cation transport. ROMK1, also known as a KCNJ, is an ATPase sensitive potassium channel. Functional coupling of ROMK1 with NKCC2 is essential for NaCl reabsorption. Chloride exits the basolateral membrane of the TAL via the CLC chloride channel, CLC-KB, which is co-expressed with the subunit Barttin. 2.4 The distal convoluted tubule The distal convoluted tubule is responsible for ~8% of filtered sodium reabsorption. This is achieved via an apical Na + Clcotransporter (NCCT, alias the thiazide sensitive sodium chloride transporter). This transporter is regulated by a group of serine-threonine protein kinases, incluidng WNK4. In healthy individuals, WNK4 inhibits NCCT function by reducing its expression on the membrane. Recent data has suggested that potassium channels control DCT function. An apically expressed potassium channel Kv1.1 is postulated to stabilise the luminal membrane potential in this nephron segment (Glaudemans, et al., 2009). and facilitates effective magnesium transport via the apical TRPM6 magnesium channel. At the basolateral membrane of the DCT a potassium channel Kir4.1 is thought to allow potassium recycling, allowing maintenance of the basolateral Na + K + ATPase activity, the driving force for NaCl reabsorption via NCCT in this nephron segment (Bockenhauer, et al., 2009, Scholl, et al., 2009).
Pflügers Archiv - European Journal of Physiology, 2013
ClC-Kb, a member of the ClC family of Cl − channels/transporters, plays a major role in the absorption of NaCl in the distal nephron. CLCNKB mutations cause Bartter syndrome type 3, a hereditary renal salt-wasting tubulopathy. Here, we investigate the functional consequences of a Val to Met substitution at position 170 (V170M, α helix F), which was detected in eight patients displaying a mild phenotype. Conductance and surface expression were reduced by~40-50 %. The regulation of channel activity by external H + and Ca 2+ is a characteristic property of ClC-Kb. Inhibition by external H + was dramatically altered, with pK H shifting from 7.6 to 6.0. Stimulation by external Ca 2+ on the other hand was no longer detectable at pH 7.4, but was still present at acidic pH values. Functionally, these regulatory modifications partly counterbalance the reduced surface expression by rendering V170M hyperactive. Pathogenic Met170 seems to interact with another methionine on α helix H (Met227) since diverse mutations at this site partly removed pH sensitivity alterations of V170M ClC-Kb. Exploring other disease-associated mutations, we found that a Pro to Leu substitution at position 124 Olga Andrini and Mathilde Keck contributed equally to this work.
Role of PKC in the Regulation of the Human Kidney Chloride Channel ClC-Ka
Scientific Reports, 2020
The physiological role of the renal ClC-Ka/ClC-K1 channels is to confer a high Clpermeability to the thin Ascending Limb of Henle (tAL), which in turn is essential for establishing the high osmolarity of the renal medulla that drives water reabsorption from collecting ducts. Here, we investigated by whole-cell patch-clamp measurements on HEK293 cells co-expressing ClC-Ka (tagged with GFP) and the accessory subunit barttin (tagged with m-Cherry) the effect of a natural diuretic extract from roots of Dandelion (DRE), and other compounds activating PKC, such as ATP, on ClC-Ka activity and its membrane localization. Treatment with 400 µg/ml DRE significantly inhibited Clcurrents time-dependently within several minutes. Of note, the same effect on Clcurrents was obtained upon treatment with 100 µM ATP. Pretreatment of cells with either the intracellular Ca 2+ chelator BAPTA-AM (30 μM) or the PKC inhibitor Calphostin C (100 nM) reduced the inhibitory effect of DRE. Conversely, 1 µM of phorbol meristate acetate (PMA), a specific PKC activator, mimicked the inhibitory effect of DRE on ClC-Ka. Finally, we found that pretreatment with 30 µM Heclin, an E3 ubiquitin ligase inhibitor, did not revert DRE-induced clcurrent inhibition. In agreement with this, live-cell confocal analysis showed that DRE treatment did not induce ClC-Ka internalization. In conclusion, we demonstrate for the first time that the activity of ClC-Ka in renal cells could be significantly inhibited by the activation of PKC elicited by classical maneuvers, such as activation of purinergic receptors, or by exposure to herbal extracts that activates a PKC-dependent pathway. Overall, we provide both new information regarding the regulation of ClC-Ka and a proof-of-concept study for the use of DRE as new diuretic.
Physiology and pathophysiology of the renal Na-K-2Cl cotransporter (NKCC2
is located in the apical membrane of the epithelial cells of the thick ascending limb of the loop of Henle (TAL). NKCC2 facilitates 20-25% of the reuptake of the total filtered NaCl load. NKCC2 is therefore one of the transport proteins with the highest overall reabsorptive capacity in the kidney. Consequently, even subtle changes in NKCC2 transport activity considerably alter the renal reabsorptive capacity for NaCl and eventually lead to perturbations of the salt and water homoeostasis. In addition to facilitating the bulk reabsorption of NaCl in the TAL, NKCC2 transport activity in the macula densa cells of the TAL constitutes the initial step of the tubular-vascular communication within the juxtaglomerular apparatus (JGA); this communications allows the TAL to modulate the preglomerular resistance of the afferent arteriole and the renin secretion from the granular cells of the JGA. This review provides an overview of our current knowledge with respect to the general functions of NKCC2, the modulation of its transport activity by different regulatory mechanisms, and new developments in the pathophysiology of NKCC2-dependent renal NaCl transport. differential splicing; macula densa; NKCC2; Slc12a1; thick ascending limb HUMAN KIDNEYS FILTER APPROXIMATELY 1.5 kg of NaCl and 180 liters of water each day. The bulk of the filtered load is reabsorbed along the tubular system and the collecting ducts, which results in the formation of 1.5 liters of urine/day. In total, 20-25% of the filtered NaCl is reabsorbed along the thick ascending limb of the loop of Henle (TAL), whereas virtually no water is reabsorbed in this portion of the nephron due to the lack of paracellular and transcellular water permeability. The major apical entry pathway for NaCl is provided by the Na-K-2Cl cotransporter, NKCC2 (BSC1, bumetanide-sensitive cotrans-porter 1), which accounts for 80% of the total salt reabsorption of the TAL (52, 64). The driving force for NKCC2-dependent salt transport is provided by the activity of the basolateral Na-K-ATPase; basolateral chloride conductivity (clcnkb channels) and apical K recycling via ROMK channels complete the net trans-cellular transport of NaCl in the TAL (53). NKCC2 is encoded by a single gene, but differential splicing of its pre-mRNA gives rise to several splice isoforms, which differ markedly in their transport characteristics and in their localization along the TAL (46, 63). Three different isoforms, NKCC2B, NKCC2A, and NKCC2F, differ in the variable exon 4, which encodes the amino acids of the second transmembrane domain and parts of the adjacent intracellular loop of the cotransporter (96, 114). This specific portion of NKCC2 has been show to be crucial for chloride binding. In addition to these three full-length isoforms, truncated variants of NKCC2 have been reported; these variants differ in the C-terminal portion of the protein and increase the total number of NKCC2 isoforms to at least six (96, 114). In addition, isoforms with tandem repeats of exon 4, such as exon 4A/4F, 4B/4F, and 4B/4A, have been described (20, 43, 67, 161). Because of the high overall salt transport capacity of NKCC2 and its crucial role in the urinary concentrating mechanism , even subtle modulations in NKCC2 transport activity result in considerable changes in renal salt reabsorptive capacity (103). Thus inhibitors of NKCC2, known as loop diuretics, constitute the most potent class of diuretics. The profound impact of NKCC2 inhibition on renal salt reabsorption is further enhanced by the limited transport capacity of the portions of the nephron downstream of the TAL, such as the distal convoluted tubule or the collecting duct. The large amount of salt reabsorption mediated by NKCC2 is most likely responsible for the evolution of a complex regulatory network in the TAL; this network modulates NKCC2 expression, differential splicing of its pre-mRNA, surface trafficking, specific transport activity, and, ultimately, the TAL salt-reabsorptive capacity. The regulatory network that controls NKCC2 expression and activity comprises systemic hormones (such as angiotensin II, catecholamines, and vasopressin) and local paracrine/autocrine factors (such as nitric oxide), all of which modulate the complex intracellular signaling network of TAL epithelial cells (61). The NKCC2-dependent salt retrieval in the TAL substantially contributes to the overall salt-reabsorptive capacity of the kidney; in addition, NKCC2 transport activity initiates tubular-vascular cross talk within the kidney (81, 126). Thus NKCC2-dependent salt transport is the initial step that links the tubular chloride concentration at the macula densa (MD) to the control of the tone of the afferent arteriole and eventually the glomer-ular filtration rate (GFR) of the respective nephron (129). This mechanism is known as tubuloglomerular feedback (TGF)
Chloride Transport in the Kidney: Lessons from Human Disease and Knockout Mice
Journal of the American Society of Nephrology, 2005
Knockout mouse models and human inherited diseases have provided important new insights into the physiologic role of chloride transport by CLC Cl ؊ channels and KCC K-Cl co-transporters. ClC-K/barrtin Cl ؊ channels are important for renal salt reabsorption and possibly for acid secretion by intercalated cells. The endosomal ClC-5 protein is crucial for proximal tubular endocytosis. Its disruption in mice and patients with Dent's disease leads to hypercalciuria and kidney stones through a pathologic cascade that may be entirely explained by an impairment of endocytosis. KCC4 is important for recycling Cl ؊ for the basolateral anion exchanger in intercalated cells, as is evident from the renal tubular acidosis resulting from its knockout. Finally, both KCC3 and KCC4 are crucial for proximal tubular cell volume regulation.
AJP: Renal Physiology, 2010
The bumetanide-sensitive Na+-K+-2Cl− cotransporter NKCC2, located in the thick ascending limb of Henle's loop, plays a critical role in the kidney's ability to concentrate urine. In humans, loss-of-function mutations of the solute carrier family 12 member 1 gene ( SLC12A1), coding for NKCC2, cause type I Bartter syndrome, which is characterized by prenatal onset of a severe polyuria, salt-wasting tubulopathy, and hyperreninemia. In this study, we describe a novel chemically induced, recessive mutant mouse line termed Slc12a1I299F exhibiting late-onset manifestation of type I Bartter syndrome. Homozygous mutant mice are viable and exhibit severe polyuria, metabolic alkalosis, marked increase in plasma urea but close to normal creatininemia, hypermagnesemia, hyperprostaglandinuria, hypotension,, and osteopenia. Fractional excretion of urea is markedly decreased. In addition, calcium and magnesium excretions are more than doubled compared with wild-type mice, while uric acid ex...