Potassium depletion stimulates Na-Cl cotransporter via phosphorylation and inactivation of the ubiquitin ligase Kelch-like 3.

Kelch-like 3 (KLHL3) is a component of an E3 ubiquitin ligase complex that regulates blood pressure by targeting With-No-Lysine (WNK) kinases for degradation. Mutations in KLHL3 cause constitutively increased renal salt reabsorption and impaired K+ secretion, resulting in hypertension and hyperkalemia. Although clinical studies have shown that dietary K+ intake affects blood pressure, the mechanisms have been obscure. In this study, we demonstrate that the KLHL3 ubiquitin ligase complex is involved in the low-K+-mediated activation of Na-Cl cotransporter (NCC) in the kidney. In the distal convoluted tubules of mice eating a low-K+ diet, we found increased KLHL3 phosphorylation at S433 (KLHL3S433-P), a modification that impairs WNK binding, and also reduced total KLHL3 levels. These changes are accompanied by the accumulation of the target substrate WNK4, and activation of the downstream kinases SPAK (STE20/SPS1-related proline-alanine-rich protein kinase) and OSR1 (oxidative stress-responsive 1), resulting in NCC phosphorylation and its accumulation at the plasma membrane. Increased phosphorylation of S433 was explained by increased levels of active, phosphorylated protein kinase C (but not protein kinase A), which directly phosphorylates S433. Moreover, in HEK cells expressing KLHL3 and WNK4, we showed that the activation of protein kinase C by phorbol 12-myristate 13-acetate induces KLHL3S433-P and increases WNK4 levels by abrogating its ubiquitination. These data demonstrate the role of KLHL3 in low-K+-mediated induction of NCC; this physiologic adaptation reduces distal electrogenic Na+ reabsorption, preventing further renal K+ loss but promoting increased blood pressure.

Acetazolamide prevents vacuolar myopathy in skeletal muscle of K(+) -depleted rats.

BACKGROUND AND PURPOSE: Acetazolamide and dichlorphenamide are carbonic anhydrase (CA) inhibitors effective in the clinical condition of hypokalemic periodic paralysis (hypoPP). Whether these drugs prevent vacuolar myopathy, which is a pathogenic factor in hypoPP, is unknown. The effects of these drugs on the efflux of lactate from skeletal muscle were also investigated. EXPERIMENTAL APPROACH: For 10 days, K(+)-depleted rats, a model of hypoPP, were administered 5.6 mg kg(-1) day(-1) of acetazolamide, dichlorphenamide or bendroflumethiazide (the last is not an inhibitor of CA). Histological analysis of vacuolar myopathy and in vitro lactate efflux measurements were performed in skeletal muscles from treated and untreated K(+)-depleted rats, and also from normokalemic rats. KEY RESULTS: About three times as many vacuoles were found in the type II fibres of tibialis anterioris muscle sections from K(+)-depleted rats as were found in the same muscle from normokalemic rats. In ex vivo experiments, a higher efflux of lactate on in vitro incubation was found in muscles of K(+)-depleted rats compared with that found in muscles from normokalemic rats. After treatment of K(+)-depleted rats with acetazolamide, the numbers of vacuoles in tibialis anterioris muscle decreased to near normal values. Incubation with acetazolamide in vitro inhibited efflux of lactate from muscles of K(+)-depleted rats. In contrast, bendroflumethiazide and dichlorphenamide failed to prevent vacuolar myopathy after treatment in vivo and failed to inhibit lactate efflux in vitro. CONCLUSIONS AND IMPLICATIONS: Acetazolamide prevents vacuolar myopathy in K(+)-depleted rats. This effect was associated with inhibition of lactate transport, rather than inhibition of CA.

On the mechanism of rhabdomyolysis in potassium depletion.

Rhabdomyolysis and myoglobinuria occur commonly in men who sustain environmental heat injury during intensive physical training in hot climates. These also occur in patients with potassium depletion. Since physical training in hot climates may be accompanied by serious losses of body potassium, the possibility was considered that performance of strenuous exercise when potassium deficient might enhance susceptibility to rhabdomyolysis. Potassium is released from contracting skeletal muscle fibers and its rising concentration in interstitial fluid is thought to dilate arterioles thereby mediating the normal rise of muscle blood flow during exercise. If potassium release from deficient muscle were subnormal, exercise would not be accompanied by sufficient muscle blood flow and rhabdomyolysis could occur by ischemia. This hypothesis was examined by comparing the effect of electrically stimulated exercise on muscle blood flow, potassium release, and histology of the intact gracilis muscle preparation in normal and potassium-depleted dogs. In normal dogs, muscle blood flow and potassium release rose sharply during exercise. In contrast, muscle blood flow and potassium release were markedly subnormal in depleted dogs despite brisk muscle contractions. Although minor histologic changes were sometimes observed in nonexercised potassium-depleted muscle, frank rhabdomyolysis occurred in each potassium-depleted animal after exercise. These findings support the hypothesis that ischemia may be the mechanism of rhabdomyolysis with exercise in potassium depletion.

L1 cell adhesion molecule is neuroprotective of alcohol induced cell death.

L1 cell adhesion molecule (L1), a protein critical for appropriate development of the central nervous system, is a target for ethanol teratogenicity. Ethanol inhibits both L1 mediated cell adhesion as well as L1 mediated neurite outgrowth. L1 has been shown to increase cell survival in cerebellar granule cells while ethanol has been shown to increase cell death. We sought to determine if L1 protected cells from ethanol induced cell death. Cerebellar granule cells from postnatal day 6 rat pups were cultured on either poly l-lysine with or without an L1 substratum. Alcohol was added at 2h post-plating and cell survival was measured at various times. L1 substratum significantly increased cell survival at 72 and 120 h. Ethanol significantly reduced cell survival at 48 h, with no effect at 72 or 120 h, both in the presence and absence of L1. At 48 h, L1 significantly increased cell survival in the presence of ethanol. We conclude that ethanol interferes with processes other than L1-L1 interactions in causing cell death, and that ethanol effects would be more severe in the absence of L1.

Prolonged K+ deficiency increases intracellular ATP, cell cycle arrest and cell death in renal tubular cells.

BACKGROUND: Chronic potassium (K+) deficiency can cause renal damage namely hypokalemic nephropathy with unclear pathogenic mechanisms. In the present study, we investigated expression and functional alterations in renal tubular cells induced by prolonged K+ deficiency. METHODS: MDCK cells were maintained in normal-K+ (CNK) (K+=5.3mmol/L), low-K+ (CLK) (K+=2.5mmol/L), or K+-depleted (CKD) (K+=0mmol/L) medium for 10days (n=5 independent cultures/condition). Differentially expressed proteins were identified by a proteomics approach followed by various functional assays. RESULTS: Proteomic analysis revealed 46 proteins whose levels significantly differed among groups. The proteomic data were confirmed by Western blotting. Gene Ontology (GO) classification and protein network analysis revealed that majority of the altered proteins participated in metabolic process, whereas the rest involved in cellular component organization/biogenesis, cellular process (e.g., cell cycle, regulation of cell death), response to stress, and signal transduction. Interestingly, ATP measurement revealed that intracellular ATP production was increased in CLK and maximum in CKD. Flow cytometry showed cell cycle arrest at S-phase and G2/M-phase in CLK and CKD, respectively, consistent with cell proliferation and growth assays, which showed modest and marked degrees of delayed growth and prolonged doubling time in CLK and CKD, respectively. Cell death quantification also revealed modest and marked degrees of increased cell death in CLK and CKD, respectively. CONCLUSIONS: In conclusion, prolonged K+ deficiency increased intracellular ATP, cell cycle arrest and cell death in renal tubular cells, which might be responsible for mechanisms underlying the development of hypokalemic nephropathy.

Alteration of skeletal muscle cellular structures by potassium depletion.

After rats had been fed a low-potassium diet for 4 to 8 weeks, skeletal muscle showed ultrastructural changes involving membranous organelles. Mitochondria were often swollen, condensed, or disintegrated. The transverse tubules were disoriented, focally dilated, and tortuous. The sarcoplasmic reticulum showed various degrees of dilatation. Vacuoles of different sizes occurred frequently. Whirls of membranes were closely associated with any of the membranous organelles, especially near an orifice of a transverse tubule. Supplementation of potassium reversed these changes. These findings are very similar to those in patients with hypokalemic periodic paralysis. The vacuolar myopathy in these patients may be secondary to the electrolyte alteration in skeletal muscles, and the chronic weakness of some patients may be due to excitation-contraction uncoupling as a result of the involvement of sarcotubular systems.

Experimental potassium depletion myopathy.

Myopathic morphological changes were observed in the calf muscles of rats maintained on a potassium-depleted diet for 30-50 days. Light-microscopic changes were more severe in the superficial gastrocnemius muscle fibres and included pallor, swelling and vacuolation, progressing to necrosis, mononuclear cell infiltration and phagocytosis. Mitochondrial disruption, transverse tubular dilatation, membranous body formation and vacuolation were seen on ultrastructural examination. Both vacuoles and subsarcolemmal membranous bodies were shown to be in continuity with the transverse tubular system. More advanced myopathic changes were those of sarcomere destruction and muscle fibre necrosis. Morphological changes resemble those of human potassium depletion myopathy. Ineffective energy metabolism and focal ischaemia due to impaired vasodilatation are proposed as mechanisms for the myopathy of potassium depletion.

A differential display protocol to identify differentially expressed mRNAs in potassium-deprived cerebellar granule cells.

Differential display (DD) has become a popular technique for the identification of differentially expressed genes. Here we present a DD protocol for studying mRNA expression changes during neuronal apoptosis. Neuronal apoptosis is typically dependent on macromolecular synthesis, thus suggesting that regulation of gene expression is involved in control of the activation of the cell-death machinery. In order to identify some of the genes involved, we employed the widely used cell culture model in which apoptosis is induced in rat cerebellar granule cells (CGCs) using potassium deprivation. Although DD has been applied productively in the study of various biological phenomena, the method has its drawbacks. In particular, the cloning and verification of cDNA fragments is frequently described as problematic or laborious, and often produces many "false positives". Here we report the successful use of DD including an efficient protocol for cDNA clone screening and verification. This protocol avoids some of the problems presented by heterogeneous DD bands, which may be a major cause of false-positive results. To identify the desired clones, we apply single-stranded conformational polymorphism (SSCP) and slot blot techniques.

[Potassium deficiency in rat cardiomyocytes after whole-body hyperthermia]. / Issledovanie kalievogo defitsita v kardiomiotsite krysy posle gipertermii vsego organizma.

Potassium distribution and content were studied in different compartments of rat heart papillary muscle by X-ray microanalysis. A higher concentration of potassium was measured in normal rat as compared to that in animals treated with high physiological temperature (45 degrees C), to be 120 and 80 mM, respectively.

[Morphological and functional alterations in the intercalated cells of outer medullary collecting duct in K-depleted rats--with special reference to ultrastructural change on electron microscopy].

It has been known that morphological changes are predominant at the site of intercalated cells (IC-cells) of the rat outer medullary collecting duct (OMCD) under K depletion. The changes are characterized by an increase in microplica and the stud of the apical plasma membrane in association with a decrease in the tubulovesicular membrane compartment of the apical region of the cells. It has also been shown that significant K reabsorption and acceleration of H secretion are taking place in these cells of OMCD under the K depletion. In order to clarify whether the changes are directly related to K reabsorption or H secretion, the animal experiment was carried out under any condition. In the control group, G-I, rat was fed with standard diet of normal K content (serum K; 4.5 +/- 0.2 mEq/l), whereas K content was decreased in the K depletion group, G-II, (serum K; 2.7 +/- 0.6 mEq/l). In the acid loaded group, G-III, 4 mEq/100 g BW of 2N ammonium chloride was given every day (serum K; 4.0 +/- 0.2 mEq/l). Animals were sacrificed after 14 days and kidneys were removed for morphological and biochemical examinations. As a result, a significant cell proliferation and interstitial PAS positive granules are observed in K-depleted rats under the light microscopic study. Under the electron microscope, the changes of the intracellular ultrastructure are predominant in the IC-cells of OMCD, such as 'membrane recycling'. On the contrary, no particular changes are seen in acid loaded rats under the light microscope.(ABSTRACT TRUNCATED AT 250 WORDS)

[The effect of elevated environmental temperature on the development of a potassium deficiency in the body]. / Vliianie povyshennoi temperatury okruzhaiushchei sredy na razvitie defitsita kaliia v organizme.

Electron microscopy and roentgen microanalysis were used to study the ultrastructure and electrolytic balance of K+, Na+ and Cl- of the animal myocardial tissue during general body hyperthermia. It was established that long-term effect of heating microclimate causes a distinct reduction of K+ in the cardiomyocytes accompanied by essential changes of the ultrastructure of ischemic character.

Proposed mechanism in the change of cellular composition in the outer medullary collecting duct during potassium homeostasis.

Potassium depletion (K⁺-D) induces hypertrophy and hyperplasia of collecting duct cells, and potassium repletion (K⁺-R) induces regression of these changes. The purpose of this study was to examine the time courses of the changes in cellular composition, the origin of intercalated cells (ICs) and the mechanism responsible for these changes. SD rats received K⁺-depleted diets for 1, 7, or 14 days. After K⁺-D for 14 days some of the rats received normal diets for 1, 3, 5, or 7 days. In the inner stripe of the outer medulla, K⁺-D increased significantly the number and proportion of H⁺-ATPase-positive ICs, but decreased the proportion of H⁺-ATPase-negative principal cells (PCs). However, proliferation was limited to H⁺-ATPase-negative PCs. During K⁺-R, the cellular composition was recovered to control level. Apoptosis increased during K⁺-R and exclusively limited in H⁺-ATPase-negative PCs. Double immunolabeling with antibodies to PC and IC markers identified both cells negative or positive for all markers during both K⁺-D and K⁺-R. Electron microscopic observation showed that ultrastructure of AE1-positive some cells were similar to AE1-negative some cells during K⁺-R. LC3 protein expression increased significantly and autophagic vacuoles appeared particularly in PCs on days 14 of K⁺-D and in ICs on days 3 of K⁺-R. These results suggest that PCs and ICs may interconvert in response to changes in dietary K+ availability and that autophagic pathways may be involved in the interconversion.

WNK kinases and essential hypertension.

PURPOSE OF REVIEW: The present review summarizes recent literature and discusses the potential roles of WNKs in the pathogenesis of essential hypertension. RECENT FINDINGS: WNKs (with-no-lysine [K]) are a recently discovered family of serine-threonine protein kinases with unusual protein kinase domains. The role of WNK kinases in the control of blood pressure was first revealed by the findings that mutations of two members, WNK1 and WNK4, cause Gordon's syndrome. Laboratory studies have revealed that WNK kinases play important roles in the regulation of sodium and potassium transport. Animal models have been created to unravel the pathophysiology of sodium transport disorders caused by mutations of the WNK4 gene. Potassium deficiency causes sodium retention and increases hypertension prevalence. The expression of WNK1 is upregulated by potassium deficiency, raising the possibility that WNK1 may contribute to salt-sensitive essential hypertension associated with potassium deficiency. Associations of polymorphisms of WNK genes with essential hypertension in the general population have been reported. SUMMARY: Mutations of WNK1 and WNK4 cause hypertension at least partly by increasing renal sodium retention. The role of WNK kinases in salt-sensitive hypertension within general hypertension is suggested, but future work is required to firmly establish the connection.

K+, Na+, Mg2+, Ca2+, and water contents in human skeletal muscle: correlations among these monovalent and divalent cations and their alterations in K+ -depleted subjects.

None of previous studies had simultaneously analyzed the K(+), Na(+), Mg(2+), and Ca(2+) contents in human skeletal muscle. We examined extensively and simultaneously the levels of all these cations and examined water content in vastus lateralis and pectoralis major muscles in 30 northeastern Thai men who were apparently healthy but died from an accident. Specimen collection was performed within 6 h of death. We used atomic absorption or flame photometry to measure the level of muscle cation. Histopathology of muscle and kidney was also evaluated. K(+), Na(+), Mg(2+), and Ca(2+) contents in vastus lateralis were 84.74 +/- 1.50, 38.64 +/- 0.77, 7.58 +/- 0.17, and 0.94 +/- 0.06 micromol/g wet weight, respectively, whereas K(+), Na(+), and Mg(2+) contents in pectoralis major were 82.83 +/- 1.54, 37.57 +/- 0.72, and 7.30 +/- 0.17 micromol/g wet weight, respectively. The water component was comparable in vastus lateralis and pectoralis major (78.66 +/- 0.41 and 78.09 +/- 0.56 %, respectively). Based on muscle K(+) levels, we divided the subjects into 2 main groups: K(+)-depleted (KD) group (K(+) < 80 micromol/g wet weight; n = 7) and non-K(+)-depleted (NKD) group (K(+) > or = 80 micromol/g wet weight; n = 23). In the KD muscle, Na(+) and Ca(2+) levels were significantly higher, whereas the level of Mg(2+) was significantly lower. Linear regression analysis showed significant correlations of K(+) and Mg(2+) levels and between Na(+) and Ca(2+). However, K(+) and Mg(2+) had the negative correlation with Na(+) and Ca(2+). Histopathologic examination showed no change in the KD muscles, whereas 29% (2 of 7) of the KD kidneys had vacuolization in proximal renal tubular cells. Our study not only provided the descriptive data but also implied the balance or homeostasis of these monovalent and divalent cations in their muscle pools.

A primary Sjögren's syndrome patient with distal renal tubular acidosis, who presented with symptoms of hypokalemic periodic paralysis: Report of a case study and review of the literature.

Although renal tubular acidosis (RTA), secondary to autoimmune interstitial nephritis, develops in a large proportion of patients with Sjögren's syndrome (SS), most of the subjects are asymptomatic. Here, we shall present a 39-year-old female patient who came to us with hypokalemic periodic paralysis (HPP), and who was later diagnosed with distal RTA. The patient, who had xerostomia and xerophthalmia for a long period of time, was diagnosed with primary SS from serologic and histologic findings. The patient recovered by being prescribed potassium replacement therapy. Although renal biopsy was not performed, corticosteroids were administered because HPP indicated severe interstitial nephritis. HPP did not reoccur during a 2-year follow-up period. We also review cases with SS-related distal RTA and HPP.

The effect of a low potassium diet on the glomerular zone of the adrenal cortex of rats.

Rats were fed on low potassium diets in order to observe the effect of dietary low potassium on the adrenal cortex. The authors clarified morphological changes of the hypofunctional glomerular zone and compared these changes with those of the hyperfunctional glomerular zone. Three weeks after or 2 months after the start of a low potassium diet, slight narrowing of the glomerular zone of the adrenal cortex was observed followed by miniaturization of cells, presence of binuclear cells and an increase of lipid with enlarged lipid drops. Electron microscope mainly disclosed changes of mitochondrial cristae consisting of markedly reduced, enlarged and irregularly dilated cristae with shortening or elongation. Granules appeared in mitochondria. Lysosomes or dense bodies were enlarged. The Golgi's apparatus was atrophied but endoplasmic reticulum did not show remarkable changes. These changes were directly opposite to those of the hyperfunctional glomerular zone noted after a pottasium load or seen in sodium deficiency. Consequently, these changes were considered to be the changes of the hypofunctional glomerular zone associated with decrease of aldosterone production.

Ultrastructural changes in the renal papillary cells of rats during maintenance and repair of profound potassium depletion.

In rats with long-term diet-induced potassium depletion, the cytoplasm of markedly enlarged papillary cells was mainly occupied by membrane-bound droplets, many of which acquired massive proportions. Both free and attached ribosomes were decreased, the Golgi apparatus was inconspicuous and there was a paucity of mitochondria. Despite the overwhelming accumulation of droplets with concomitant loss of normal metabolic organelles, cell death did not occur. With potassium repletion, the organelles readily proliferated regardless of the progression of droplet dissolution. The shrinkage of the droplets was accompanied either by disintegration of the limiting membrane or by disappearance of the constituents within an intact membrane. Microtubules were conspicuous in many of the cells undergoing gradual reduction in size. These cytoplasmic changes in renal medullary cells of rats during long-term potassium depletion and immediate post-repletion periods essentially represented the consequences of maintenance and repair of a storage process.