Hypokalaemic metabolic alkalosis, hypertension and diabetes: what is the link.

Two years after diagnosis of a metastatic neuroendocrine gastrin-secreting tumour and after several cycles of chemotherapy and peptide receptor radionuclide therapy, a 56-year-old woman presented with hypokalaemic metabolic alkalosis, hypertension, leg oedema and new-onset diabetes mellitus. Further investigations revealed renal potassium loss confirmed by a transtubular potassium gradient of 16, fully suppressed serum aldosterone, but instead highly elevated blood levels of morning cortisol and adrenocorticotropic hormone as well as increased urinary excretion of glucocorticoid and mineralocorticoid metabolites. Ruling out other causes, paraneoplastic hypercortisolism was diagnosed. Pharmacological inhibition of the steroid 11ß-hydroxylase with metyrapone resulted in complete resolution of metabolic alkalosis, hypokalaemia, hypertension, hyperglycaemia and leg oedema within 1 week.

Patients with hypokalemia develop WNK bodies in the distal convoluted tubule of the kidney.

Hypokalemia contributes to the progression of chronic kidney disease, although a definitive pathophysiological theory to explain this remains to be established. K+ deficiency results in profound alterations in renal epithelial transport. These include an increase in salt reabsorption via the Na+-Cl- cotransporter (NCC) of the distal convoluted tubule (DCT), which minimizes electroneutral K+ loss in downstream nephron segments. In experimental conditions of dietary K+ depletion, punctate structures in the DCT containing crucial NCC-regulating kinases have been discovered in the murine DCT and termed "WNK bodies," referring to their component, with no K (lysine) kinases (WNKs). We hypothesized that in humans, WNK bodies occur in hypokalemia as well. Renal needle biopsies of patients with chronic hypokalemic nephropathy and appropriate controls were examined by histological stains and immunofluorescence. Segment- and organelle-specific marker proteins were used to characterize the intrarenal and subcellular distribution of established WNK body constituents, namely, WNKs and Ste20-related proline-alanine-rich kinase (SPAK). In both patients with hypokalemia, WNKs and SPAK concentrated in non-membrane-bound cytoplasmic regions in the DCT, consistent with prior descriptions of WNK bodies. The putative WNK bodies were located in the perinuclear region close to, but not within, the endoplasmic reticulum. They were closely adjacent to microtubules but not clustered in aggresomes. Notably, we provide the first report of WNK bodies, which are functionally challenging structures associated with K+ deficiency, in human patients.

Role of WNK4 and kidney-specific WNK1 in mediating the effect of high dietary K+ intake on ROMK channel in the distal convoluted tubule.

With-no-lysine kinase 4 (WNK4) and kidney-specific (KS)-WNK1 regulate ROMK (Kir1.1) channels in a variety of cell models. We now explore the role of WNK4 and KS-WNK1 in regulating ROMK in the native distal convoluted tubule (DCT)/connecting tubule (CNT) by measuring tertiapin-Q (TPNQ; ROMK inhibitor)-sensitive K+ currents with whole cell recording. TPNQ-sensitive K+ currents in DCT2/CNT of KS- WNK1-/- and WNK4-/- mice were significantly smaller than that of WT mice. In contrast, the basolateral K+ channels (a Kir4.1/5.1 heterotetramer) in the DCT were not inhibited. Moreover, WNK4-/- mice were hypokalemic, while KS- WNK1-/- mice had normal plasma K+ levels. High K+ (HK) intake significantly increased TPNQ-sensitive K+ currents in DCT2/CNT of WT and WNK4-/- mice but not in KS- WNK1-/- mice. However, TPNQ-sensitive K+ currents in the cortical collecting duct (CCD) were normal not only under control conditions but also significantly increased in response to HK in KS- WNK1-/- mice. This suggests that the deletion of KS-WNK1-induced inhibition of ROMK occurs only in the DCT2/CNT. Renal clearance study further demonstrated that the deletion of KS-WNK1 did not affect the renal ability of K+ excretion under control conditions and during increasing K+ intake. Also, HK intake did not cause hyperkalemia in KS- WNK1-/- mice. We conclude that KS-WNK1 but not WNK4 is required for HK intake-induced stimulation of ROMK activity in DCT2/CNT. However, KS-WNK1 is not essential for HK-induced stimulation of ROMK in the CCD, and the lack of KS-WNK1 does not affect net renal K+ excretion.

Chronic potassium depletion increases adrenal progesterone production that is necessary for efficient renal retention of potassium.

Modern dietary habits are characterized by high-sodium and low-potassium intakes, each of which was correlated with a higher risk for hypertension. In this study, we examined whether long-term variations in the intake of sodium and potassium induce lasting changes in the plasma concentration of circulating steroids by developing a mathematical model of steroidogenesis in mice. One finding of this model was that mice increase their plasma progesterone levels specifically in response to potassium depletion. This prediction was confirmed by measurements in both male mice and men. Further investigation showed that progesterone regulates renal potassium handling both in males and females under potassium restriction, independent of its role in reproduction. The increase in progesterone production by male mice was time dependent and correlated with decreased urinary potassium content. The progesterone-dependent ability to efficiently retain potassium was because of an RU486 (a progesterone receptor antagonist)-sensitive stimulation of the colonic hydrogen, potassium-ATPase (known as the non-gastric or hydrogen, potassium-ATPase type 2) in the kidney. Thus, in males, a specific progesterone concentration profile induced by chronic potassium restriction regulates potassium balance.

Mechanism underlying hypokalemia induced by trimethyltin chloride: Inhibition of H+/K+-ATPase in renal intercalated cells.

Trimethyltin chloride (TMT), a byproduct of plastic stabilizers, has caused 67 poisoning accidents in the world; more than 98% (1814/1849) of the affected patients since 1998 have been in China. As a long-established toxic chemical, TMT severely affects the limbic system and the cerebellum; however, its relationship with hypokalemia, a condition observed in the majority of the cases in the last decade, remains elusive. To understand the mechanism underlying hypokalemia induced by TMT, Sprague-Dawley (SD) rats were administered TMT to determine the relationship between H(+)/K(+)-ATPase activity and the blood and urine K(+) concentration and pH, respectively. H(+)/K(+)-ATPase protein and mRNA were observed too. In vitro changes to intracellular pH, K(+) channels in renal cells were measured. The results showed that TMT increased potassium leakage from the kidney, raised urine pH, and inhibited H(+)/K(+)-ATPase activity both in vitro and in vivo. In the tested animals, H(+)/K(+)-ATPase activity was positively correlated with the decrease of plasma K(+) and blood pH but was negatively correlated with the increase of urine K(+) and urine pH (P<0.01), while TMT did not change the expression of H(+)/K(+)-ATPase protein and mRNA. TMT decreased intracellular pH and opened K(+) channels in renal intercalated cells. Our findings suggest TMT can directly inhibit the activity of H(+)/K(+)-ATPases in renal intercalated cells, reducing urine K(+) reabsorption and inducing hypokalemia.

The phenomenon of seasonal pseudohypokalemia: effects of ambient temperature, plasma glucose and role for sodium-potassium-exchanging-ATPase.

OBJECTIVES: There is limited data regarding the phenomenon of seasonal pseudohypokalemia. We aimed to demonstrate the incidence of spurious hypokalemia during the summer months and to investigate the mechanism of cause. DESIGN AND METHODS: Potassium and glucose results from primary care and hospital patients were collected retrospectively for a period of 1 year to assess the incidence of pseudohypokalemia. Experiments were undertaken to confirm that this was a reversible in vitro phenomenon due to increased temperature mediated by sodium-potassium-exchanging-ATPase. RESULTS: Our data show an increased incidence of hypokalemia associated with increasing ambient temperature during June-August in samples from primary care but not in hospital samples. In a subset of patients, we showed that the repeat results were within or at the lower limit of the reference range. Experiments showed that this phenomenon was mediated by the sodium-potassium-exchanging-ATPase. CONCLUSIONS: There is an increased incidence of pseudohypokalemia during the summer (seasonal pseudohypokalemia) in samples from primary care and this is an in vitro pseudo-phenomenon mediated by sodium-potassium-exchanging-ATPase.

Downregulation of renal TonEBP in hypokalemic rats.

Hypokalemia causes a significant decrease in the tonicity of the renal medullary interstitium in association with reduced expression of sodium transporters in the distal tubule. We asked whether hypokalemia caused downregulation of the tonicity-responsive enhancer binding protein (TonEBP) transcriptional activator in the renal medulla due to the reduced tonicity. We found that the abundance of TonEBP decreased significantly in the outer and inner medullas of hypokalemic rats. Underlying mechanisms appeared different in the two regions because the abundance of TonEBP mRNA was lower in the outer medulla but unchanged in the inner medulla. Immunohistochemical examination of TonEBP revealed cell type-specific differences. TonEBP expression decreased dramatically in the outer and inner medullary collecting ducts, thick ascending limbs, and interstitial cells. In the descending and ascending thin limbs, TonEBP abundance decreased modestly. In the outer medulla, TonEBP shifted to the cytoplasm in the descending thin limbs. As expected, transcription of aldose reductase, a target of TonEBP, was decreased since the abundance of mRNA and protein was reduced. Downregulation of TonEBP appeared to have also contributed to reduced expression of aquaporin-2 and UT-A urea transporters in the renal medulla. In cultured cells, expression and activity of TonEBP were not affected by reduced potassium concentrations in the medium. These data support the view that medullary tonicity regulates expression and nuclear distribution of TonEBP in the renal medulla in cell type-specific manners.

Proteomic identification of alterations in metabolic enzymes and signaling proteins in hypokalemic nephropathy.

Hypokalemic nephropathy caused by prolonged K(+) deficiency is associated with metabolic alkalosis, polydipsia, polyuria, growth retardation, hypertension, and progressive tubulointerstitial injury. Its pathophysiology, however, remains unclear. We performed gel-based, differential proteomics analysis of kidneys from BALB/c mice fed with high-normal-K(+) (HNK), low-normal-K(+) (LNK), or K(+)-depleted diet for 8 wk (n = 6 in each group). Plasma K(+) levels were 4.62 +/- 0.35, 4.46 +/- 0.23, and 1.51 +/- 0.21 mmol/L for HNK, LNK, and KD mice, respectively (p < 0.0001; KD vs. others). With comparable amounts of food intake, the KD mice drank significantly more water than the other two groups and had polyuria. Additionally, the KD mice had growth retardation, metabolic alkalosis, markedly enlarged kidneys, renal tubular dilation, intratubular deposition of amorphous and laminated hyaline materials, and tubular atrophy. A total of 33 renal proteins were differentially expressed between the KD mice and others, whereas only eight proteins were differentially expressed between the HNK and LNK groups, as determined by quantitative intensity analysis and ANOVA with Tukey's post hoc multiple comparisons. Using MALDI-MS and/or quadrupole-TOF MS/MS, 30 altered proteins induced by K(+)-depletion were identified as metabolic enzymes (e.g., carbonic anhydrase II, aldose reductase, glutathione S-transferase GT41A, etc.), signaling proteins (14-3-3 epsilon, 14-3-3 zeta, and cofilin 1), and cytoskeletal proteins (gamma-actin and tropomyosin). Some of these altered proteins, particularly metabolic enzymes and signaling proteins, have been demonstrated to be involved in metabolic alkalosis, polyuria, and renal tubular injury. Our findings may lead to a new road map for research on hypokalemic nephropathy and to better understanding of the pathophysiology of this medical disease when the functional and physiological significances of these altered proteins are defined.

Liquorice and hypertension.

Glycyrrhetinic acid, the active constituent of liquorice, inhibits renal IIbeta-hydroxysteroid dehydrogenase. This allows cortisol to stimulate mineralocorticoid receptors, which can result in hypertension and hypokalaemia. Treatment options are based on pathophysiological understanding.

Correlation between decrease of 11beta-hydroxysteroid dehydrogenase activity and hypokalemia induced by furosemide in rats.

AIM: To investigate the correlation between decrease of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) activity and hypokalemia induced by furosemide (Fur) in rats. METHODS: SD rats were given single dose or successive doses of Fur by gavage. The activity of 11beta-HSD was evaluated by measuring the ratio of 11-dehydrocorticosterone (A) and corticosterone (B) in urine and conversion rate of B to A in renal cortex microsome preparation was determined with HPLC. RESULTS: After giving single dose of Fur (40, 100, and 250 mg/kg) or multiple doses of Fur (10, 20, and 100 mg/kg, bid x 20 d), the ratio of A/B was reduced by 29.0 %, 58.6 %, and 60.9 % at 0 - 2 h; 14.4 %, 36.0 %, and 44.9 %, respectively; the conversion rate of B to A was decreased by 29 %, 33 %, and 37 %; 6 %, 17 %, and 23 %, respectively. The serum potassium was significantly reduced by multiple doses of Fur (20 and 100 mg/kg, bid x 20 d) (P < 0.01). The reduction in serum potassium was positively correlated with the lowering of A/B ratio and the conversion of B to A (P < 0.01). CONCLUSION: The inhibition of renal 11beta-HSD activity may be another new biochemical mechanism for hypokalemia induced by Fur.

Temporal responses of oxidative vs. glycolytic skeletal muscles to K+ deprivation: Na+ pumps and cell cations.

When K+ output exceeds input, skeletal muscle releases intracellular fluid K+ to buffer the fall in extracellular fluid (ECF) K+. To investigate the mechanisms and muscle specificity of the K+ shift, rats were fed K+-deficient chow for 2-10 days, and two muscles at phenotypic extremes were studied: slow-twitch oxidative soleus and fast-twitch glycolytic white gastrocnemius (WG). After 2 days of low-K+ chow, plasma K+ concentration ([K+]) fell from 4.6 to 3.7 mM, and Na+-K+-ATPase alpha2 (not alpha1) protein levels in both muscles, measured by immunoblotting, decreased 36%. Cell [K+] decreased from 116 to 106 mM in soleus and insignificantly in WG, indicating that alpha2 can decrease before cell [K+]. After 5 days, there were further decreases in alpha2 (70%) and beta2 (22%) in WG, not in soleus, whereas cell [K+] decreased and cell [Na+] increased by 10 mM in both muscles. By 10 days, plasma [K+] fell to 2.9 mM, with further decreases in WG alpha2 (94%) and beta2 (70%); cell [K+] fell 19 mM in soleus and 24 mM in WG compared with the control, and cell [Na+] increased 9 mM in soleus and 15 mM in WG; total homogenate Na+-K+-ATPase activity decreased 19% in WG and insignificantly in soleus. Levels of alpha2, beta1, and beta2 mRNA were unchanged over 10 days. The ratios of alpha2 to alpha1 protein levels in both control muscles were found to be nearly 1 by using the relative changes in alpha-isoforms vs. beta1- (soleus) or beta2-isoforms (WG). We conclude that the patterns of regulation of Na+ pump isoforms in oxidative and glycolytic muscles during K+ deprivation mediated by posttranscriptional regulation of alpha2beta1 and alpha2beta2 are distinct and that decreases in alpha2-isoform pools can occur early enough in both muscles to account for the shift of K+ to the ECF.

Molecular basis of hypokalemic myopathy caused by 17alpha-hydroxylase/17,20-lyase deficiency.

We describe a 28-year-old woman presenting with hypokalemic myopathy caused by 17alpha-hydroxylase/17,20-lyase deficiency caused by a homozygous mutation consisting of a G-to-C transition in the initiation codon in exon 1 of the CYP17 gene resulting in expression of an enzymatically inactive truncated P450c17 protein.

Expression of HKalpha2 protein is increased selectively in renal medulla by chronic hypokalemia.

Our laboratory has demonstrated by Northern analysis that chronic hypokalemia increases HKalpha2 (i.e., alpha-subunit of the colonic H+-K+-ATPase) mRNA abundance in the rat. To determine whether the increase in mRNA correlated with an increase in HKalpha2 protein, an antibody was raised against a synthetic peptide derived from amino acids 686-698 of the HKalpha2 sequence. The anti-HKalpha2 antibody hybridized to rat distal colon membranes which migrated at approximately 100 kDa (expected mobility of HKalpha2). HKalpha2 protein was not detected in plasma membranes from rat whole kidney or stomach (100 microg) derived from control animals. The antibody was then used to investigate changes in expression of HKalpha2 in renal cortex, renal medulla, and distal colon in two pathophysiological conditions: 1) chronic hypokalemia (LK) and 2) chronic metabolic acidosis (CMA). In LK rats there was a marked, but selective, increase in the abundance of HKalpha2 protein in membranes prepared from renal medulla. Nevertheless, a corresponding increase in HKalpha2 protein abundance was not observed in membranes prepared from the distal colon of LK rats. HKalpha2 protein abundance in CMA was indistinguishable from controls. Moreover, chronic hypokalemia had no effect on expression of alpha1-Na+-K+-ATPase or HKalpha1 in kidney or distal colon under any experimental condition. Therefore, HKalpha2 protein is tissue- and site-specifically upregulated in response to chronic hypokalemia but not by CMA. Furthermore, this regulatory response is localized to the renal medulla.

Gossypol-induced hypokalemia and role of exogenous potassium salt supplementation when used as an antispermatogenic agent in male langur monkey.

In our earlier study, we have observed that hypokalemia in langur monkeys, following gossypol acetic acid (GAA) treatment (5 mg dose level) when used as an antispermatogenic agent, and potassium salt supplementation partially maintained body potassium level of the animals. The aims of the present investigation was to confirm further occurrence of hypokalemia in the monkey (comparatively at two higher dose levels) and the role of potassium salt in preventing occurrence of gossypol-induced hypokalemia. Highly purified gossypol acetic acid alone at two dose levels (7.5 and 10 mg/animal/day; oral) and in combination with potassium chloride (0.50 and 0.75 mg/animal/ day; oral) was given for 180 days. Treatment with gossypol alone as well as with the supplementation of potassium salt resulted in severe oligospermia and azoospermia. Animals receiving gossypol alone showed significant potassium deficiency with signs of fatigue at both dose levels. Enhanced potassium loss through urine was found in potassium-deficient animals, whereas animals receiving gossypol acetic acid plus potassium salt showed normal serum potassium with a less significant increase in urine potassium level during treatment phases. Other parameters of the body remained within normal range except gradual and significant elevation in serum transaminases activity. The animals gradually returned to normalcy following 150 and 180 days of termination of the treatment.

PIP: An earlier study conducted by the authors indicated that body potassium levels were partially maintained in male langur monkeys treated with gossypol acetic acid (5 mg) and potassium salt supplementation. The present study sought to confirm the persistence of hypokalemia at two higher dosage levels (7.5 and 10 mg/animal/day) and assess the role of exogenous potassium salt (0.50 and 0.75 mg/animal/day) in preventing gossypol-induced hypokalemia. The two dosages of highly purified gossypol acetic acid were administered alone and in combination with potassium chloride for 180 days. All regimens produced severe oligospermia and azoospermia. However, monkeys who received gossypol alone showed significant potassium deficiency with signs of fatigue at both doses. On the other hand, animals receiving gossypol acetic acid and potassium salt supplementation showed normal serum potassium with a less significant increase in urine potassium level during treatment. Also noted was a gradual but significant elevation in the activity of serum transaminases. All parameters returned to normal 150-180 days after treatment termination. The hypokalemic effect documented in this study with gossypol alone may be due to renal leakage and gastrointestinal disturbances.

Effect of chronic hypokalemia on H(+)-K(+)-ATPase expression in rat colon.

Although the kidney plays the major role in the regulation of systemic K+ homeostasis, the colon also participates substantively in K+ balance. The colon is capable of both K+ absorption and secretion, the magnitude of which can be modulated in response to dietary K+ intake. The H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) has been proposed as a possible mediator of K+ absorption in distal colon, but inhibitor profiles obtained in recent studies suggest that two, and perhaps more, distinct H(+)-K(+)-ATPase activities may be present in mammalian distal colon. We have developed highly specific probes for the catalytic alpha-subunits of colonic and gastric H(+)-K(+)-ATPase, alpha 1-Na(+)-K(+)-ATPase, and beta-actin, which were used in Northern analysis of total RNA from whole distal colon and stomach obtained from one of three experimental groups of rats: 1) controls, 2) chronic dietary K+ depletion, and 3) chronic metabolic acidosis. The probe for the colonic but not the gastric H(+)-K(+)-ATPase alpha-isoform hybridized to distal colon total RNA in all groups. A significant increase in colonic H(+)-K(+)-ATPase mRNA abundance was observed in response to chronic dietary K+ depletion but not to chronic metabolic acidosis. The alpha 1-isoform of Na(+)-K(+)-ATPase, which is also expressed in distal colon, did not respond consistently to either chronic dietary K+ depletion or chronic metabolic acidosis. The gastric probe did not hybridize to total RNA from distal colon but, as expected, hybridized to total stomach RNA. However, the abundance of gastric H(+)-K(+)-ATPase or Na(+)-K(+)-ATPase in stomach was not altered consistently by either chronic dietary K+ depletion or metabolic acidosis. Under the conditions of this study, it appears that the mRNA encoding the colonic alpha-isoform is upregulated by chronic dietary K+ restriction, a condition shown previously to increase K+ absorption in the distal colon.

Skeletal muscle Na,K-ATPase alpha and beta subunit protein levels respond to hypokalemic challenge with isoform and muscle type specificity.

During potassium deprivation, skeletal muscle loses K+ to buffer the fall in extracellular K+. Decreased active K+ uptake via the sodium pump, Na,K-ATPase, contributes to the adjustment. Skeletal muscle expresses alpha1, alpha2, beta1, and beta2 isoforms of the Na, K-ATPase alphabeta heterodimer. This study was directed at testing the hypothesis that K+ loss from muscle during K+ deprivation is a function of decreased expression of specific isoforms expressed in a muscle type-specific pattern. Isoform abundance was measured in soleus, red and white gastrocnemius, extensor digitorum longus, and diaphragm by immunoblot. alpha2 expression was uniform across control muscles, whereas alpha1 and beta1 were twice as high in oxidative (soleus and diaphragm) as in fast glycolytic (white gastrocnemius) muscles, and beta2 expression was reciprocal: highest in white gastrocnemius and barely detectable in soleus and diaphragm. Following 10 days of potassium deprivation plasma K+ fell from 4.0 to 2.3 mM, and there were distinct responses in glycolytic versus oxidative muscles. In glycolytic white gastrocnemius alpha2 and beta2 fell 94 and 70%, respectively; in mixed red gastrocnemius and extensor digitorum longus both fell 60%, and beta1 fell 25%. In oxidative soleus and diaphragm alpha2 fell 55 and 30%, respectively, with only minor changes in beta1. Although decreases in alpha2 and beta2 expression are much greater in glycolytic than oxidative muscles during K+ deprivation, both types of muscle lose tissue K+ to the same extent, a 20% decrease, suggesting that multiple mechanisms are in place to regulate the release of skeletal muscle cell K+.

Chronic hypokalemia enhances expression of the H(+)-K(+)-ATPase alpha 2-subunit gene in renal medulla.

Recent molecular and physiological studies suggested that at least two H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) isozymes are expressed in the rat kidney and that these ion pumps respond to changes in dietary potassium balance. We used Northern analysis and in situ hybridization to analyze the expression of mRNA encoding the "colonic" isoform of the H(+)-K(+)-ATPase alpha-subunit (HK alpha 2) in normal and potassium-deprived (2 wk) rats. Control rats exhibited low levels of HK alpha 2 mRNA in the cortical and medullary thick ascending limb, distal convoluted tubule, connecting segment, and the entire collecting duct. The potassium-deprived rats expressed approximately fivefold higher levels of HK alpha 2 mRNA in the outer and inner medulla compared with controls, as well as hypertrophy and increased in situ hybridization signal in the intercalated cells of the inner stripe of the outer medullary collecting duct and the proximal inner medullary collecting duct. In contrast, renal cortical expression of HK alpha 2 mRNA was low and comparable in the two groups. Our results suggest that enhanced expression of the HK alpha 2 subunit gene in the renal medulla contributes to potassium conservation during chronic hypokalemia.