Inner marginal strength of CAD/CAM materials is not affected by machining protocol.

PURPOSE: Here we aimed to compare two machining strategies regarding the marginal strength of CAD/CAM materials using a hoop-strength test in model sphero-cylindrical dental crowns, coupled with finite element analysis. MATERIALS AND METHODS: Five CAD/CAM materials indicated for single posterior crowns were selected, including a lithium disilicate (IPS e.max® CAD), a lithium (di)silicate (Suprinity® PC), a polymer-infiltrated ceramic scaffold (Enamic®), and two indirect resin composites (Grandio® Blocs and Lava™ Ultimate). A sphero-cylindrical model crown was built on CAD Software onto a geometrical abutment and machined using a Cerec MC XL system according to the two available protocols: rough-fast and fine-slow. Specimens were fractured using a novel hoop-strength test and analyzed using the finite element method to obtain the inner marginal strength. Data were evaluated using Weibull statistics. RESULTS: Machining strategy did not affect the marginal strength of any restorative material tested here. Ceramic materials showed a higher density of chippings in the outer margin, but this did not reduce inner marginal strength. IPS e.max® CAD showed the statistically highest marginal strength, and Enamic® and Lava™ Ultimate were the lowest. Grandio® Blocs showed higher performance than Suprinity® PC. CONCLUSIONS: The rough-fast machining strategy available in Cerec MC XL does not degrade the marginal strength of the evaluated CAD/CAD materials when compared to its fine-fast machining strategy. Depending on the material, resin composites have the potential to perform better than some glass-ceramic materials.

Inhibition of the heterotetrameric K+ channel KCNQ1/KCNE1 by the AMP-activated protein kinase.

The heterotetrameric K(+)-channel KCNQ1/KCNE1 is expressed in heart, skeletal muscle, liver and several epithelia including the renal proximal tubule. In the heart, it contributes to the repolarization of cardiomyocytes. The repolarization is impaired in ischemia. Ischemia stimulates the AMP-activated protein kinase (AMPK), a serine/threonine kinase, sensing energy depletion and stimulating several cellular mechanisms to enhance energy production and to limit energy utilization. AMPK has previously been shown to downregulate the epithelial Na(+) channel ENaC, an effect mediated by the ubiquitin ligase Nedd4-2. The present study explored whether AMPK regulates KCNQ1/KCNE1. To this end, cRNA encoding KCNQ1/KCNE1 was injected into Xenopus oocytes with and without additional injection of wild type AMPK (AMPKα1 + AMPKß1 + AMPKγ1), of the constitutively active (γR70Q)AMPK (α1ß1γ1(R70Q)), of the kinase dead mutant (αK45R)AMPK (α1(K45R)ß1γ1), or of the ubiquitin ligase Nedd4-2. KCNQ1/KCNE1 activity was determined in two electrode voltage clamp experiments. Moreover, KCNQ1 abundance in the cell membrane was determined by immunostaining and subsequent confocal imaging. As a result, wild type and constitutively active AMPK significantly reduced KCNQ1/KCNE1-mediated currents and reduced KCNQ1 abundance in the cell membrane. Similarly, Nedd4-2 decreased KCNQ1/KCNE1-mediated currents and KCNQ1 protein abundance in the cell membrane. Activation of AMPK in isolated perfused proximal renal tubules by AICAR (10 mM) was followed by significant depolarization. In conclusion, AMPK is a potent regulator of KCNQ1/KCNE1.

Regulation of renal tubular glucose reabsorption by Akt2/PKBß.

Akt/PKB is known to regulate the facilitative glucose carrier GLUT4. Nothing is known, however, of the role of Akt/PKB in the regulation of renal epithelial transport. To explore whether Akt2/PKBß influences the Na(+)-coupled glucose cotransporter SGLT1, human SGLT1 was expressed in Xenopus laevis oocytes with or without Akt/PKB, and electrogenic glucose transport was determined by dual-electrode voltage clamp. The coexpression of Akt/PKB in SGLT1-expressing oocytes was followed by an increase in glucose-induced currents. To study the functional significance of Akt/PKB-sensitive renal glucose transport, further experiments were performed in gene-targeted mice lacking functional Akt2/PKBß (akt2(-/-)) and in their wild-type littermates (akt2(+/+)). Plasma glucose concentration was significantly higher in akt2(-/-) mice than in akt2(+/+) mice but was virtually identical to the plasma glucose concentration in fructose-treated akt2(+/+) mice. Urinary glucose excretion was significantly higher in akt2(-/-) mice compared with akt2(+/+) mice with or without fructose treatment. Moreover, the glucose-induced depolarization of proximal tubular cells was significantly smaller in isolated, perfused renal tubules from akt2(-/-) mice than in those from akt2(+/+) mice. In conclusion, Akt2/PKBß plays a role in the regulation of renal glucose transport.

SGK1-sensitive renal tubular glucose reabsorption in diabetes.

The hyperglycemia of diabetes mellitus increases the filtered glucose load beyond the maximal tubular transport rate and thus leads to glucosuria. Sustained hyperglycemia, however, may gradually increase the maximal renal tubular transport rate and thereby blunt the increase of urinary glucose excretion. The mechanisms accounting for the increase of renal tubular glucose transport have remained ill-defined. A candidate is the serum- and glucocorticoid-inducible kinase SGK1. The kinase has been shown to stimulate Na(+)-coupled glucose transport in vitro and mediate the stimulation of electrogenic intestinal glucose transport by glucocorticoids in vivo. SGK1 expression is confined to glomerula and distal nephron in intact kidneys but may extend to the proximal tubule in diabetic nephropathy. To explore whether SGK1 modifies glucose transport in diabetic kidneys, Akita mice (akita(+/-)), which develop spontaneous diabetes, have been crossbred with gene-targeted mice lacking SGK1 on one allele (sgk1(+/-)) to eventually generate either akita(+/-)/sgk1(-/-) or akita(+/-)/sgk1(+/+) mice. Both akita(+/-)/sgk1(-/-) and akita(+/-)/sgk1(+/+) mice developed profound hyperglycemia (>20 mM) within approximately 6 wk. Body weight and plasma glucose concentrations were not significantly different between these two genotypes. However, urinary excretion of glucose and urinary excretion of fluid, Na(+), and K(+), as well as plasma aldosterone concentrations, were significantly higher in akita(+/-)/sgk1(-/-) than in akita(+/-)/sgk1(+/+) mice. Studies in isolated perfused proximal tubules revealed that the electrogenic glucose transport was significantly lower in akita(+/-)/sgk1(-/-) than in akita(+/-)/sgk1(+/+) mice. The data provide the first evidence that SGK1 participates in the stimulation of renal tubular glucose transport in diabetic kidneys.

Reduced intestinal and renal amino acid transport in PDK1 hypomorphic mice.

The phosphoinositide-dependent kinase PDK1 activates the serum- and glucocorticoid-inducible kinase isoforms SGK1, SGK2, and SGK3 and protein kinase B, which in turn are known to up-regulate a variety of sodium-coupled transporters. The present study was performed to explore the role of PDK1 in amino acid transport. As mice completely lacking functional PDK1 are not viable, mice expressing 10-25% of PDK1 (pdk1(hm)) were compared with their wild-type (WT) littermates (pdk1(wt)). Body weight was significantly less in pdk1(hm) than in pdk1(wt) mice. Despite lower body weight of pdk1(hm) mice, food and water intake were similar in pdk1(hm) and pdk1(wt) mice. According to Ussing chamber experiments, electrogenic transport of phenylalanine, cysteine, glutamine, proline, leucine, and tryptophan was significantly smaller in jejunum of pdk1(hm) mice than in pdk1(wt) mice. Similarly, electrogenic transport of phenylalanine, glutamine, and proline was significantly decreased in isolated perfused proximal tubules of pdk1(hm) mice. The urinary excretion of proline, valine, guanidinoacetate, methionine, phenylalanine, citrulline, glutamine/glutamate, and tryptophan was significantly larger in pdk1(hm) than in pdk1(wt) mice. According to immunoblotting of brush border membrane proteins prepared from kidney, expression of the Na+-dependent neutral amino acid transporter B(0)AT1 (SLC6A19), the glutamate transporter EAAC1/EAAT3 (SLC1A1), and the transporter for cationic amino acids and cystine b(0,+)AT (SLC7A9) was decreased but the Na+/proline cotransporter SIT (SLC6A20) was increased in pdk1(hm) mice. In conclusion, reduction of functional PDK1 leads to impairment of intestinal absorption and renal reabsorption of amino acids. The combined intestinal and renal loss of amino acids may contribute to the growth defect of PDK1-deficient mice.

Impaired intestinal and renal glucose transport in PDK-1 hypomorphic mice.

The phosphoinositide-dependent kinase-1 (PDK-1) activates the serum- and glucocorticoid-inducible kinase and protein kinase B isoforms, which, in turn, are known to stimulate the renal and intestinal Na+-dependent glucose transporter 1. The present study has been performed to explore the role of PDK-1 in electrogenic glucose transport in small intestine and proximal renal tubules. To this end, mice expressing approximately 20% of PDK-1 (pdk1hm) were compared with their wild-type littermates (pdk1wt). According to Ussing chamber experiments, electrogenic glucose transport was significantly smaller in the jejunum of pdk1hm than of pdk1wt mice. Similarly, proximal tubular electrogenic glucose transport in isolated, perfused renal tubule segments was decreased in pdk1hm compared with pdk1wt mice. Intraperitoneal injection of 3 g/kg body wt glucose resulted in a similar increase of plasma glucose concentration in pdk1hm and in pdk1wt mice but led to a higher increase of urinary glucose excretion in pdk1hm mice. In conclusion, reduction of functional PDK-1 leads to impairment of electrogenic intestinal glucose absorption and renal glucose reabsorption. The experiments disclose a novel element of glucose transport regulation in kidney and small intestine.

KCNQ1-dependent transport in renal and gastrointestinal epithelia.

Mutations in the gene encoding for the K+ channel alpha-subunit KCNQ1 have been associated with long QT syndrome and deafness. Besides heart and inner ear epithelial cells, KCNQ1 is expressed in a variety of epithelial cells including renal proximal tubule and gastrointestinal tract epithelial cells. At these sites, cellular K+ ions exit through KCNQ1 channel complexes, which may serve to recycle K+ or to maintain cell membrane potential and thus the driving force for electrogenic transepithelial transport, e.g., Na+/glucose cotransport. Employing pharmacologic inhibition and gene knockout, the present study demonstrates the importance of KCNQ1 K+ channel complexes for the maintenance of the driving force for proximal tubular and intestinal Na+ absorption, gastric acid secretion, and cAMP-induced jejunal Cl- secretion. In the kidney, KCNQ1 appears dispensable under basal conditions because of limited substrate delivery for electrogenic Na+ reabsorption to KCNQ1-expressing mid to late proximal tubule. During conditions of increased substrate load, however, luminal KCNQ1 serves to repolarize the proximal tubule and stabilize the driving force for Na+ reabsorption. In mice lacking functional KCNQ1, impaired intestinal absorption is associated with reduced serum vitamin B12 concentrations, mild macrocytic anemia, and fecal loss of Na+ and K+, the latter affecting K+ homeostasis.

Role of Sgk1 in salt and potassium homeostasis.

Aldosterone plays a pivotal role in NaCl and K(+) homeostasis by stimulation of Na(+) reabsorption and K(+) secretion in the aldosterone-sensitive distal nephron (ASDN). Recent studies demonstrated that the serum- and glucocorticoid-regulated kinase 1 (Sgk1) is induced by aldosterone in the ASDN and that polymorphisms of the kinase associate with arterial blood pressure in normotensive subjects. This review discusses the role of Sgk1 in NaCl and K(+) homeostasis as evidenced by in vivo studies, including those in Sgk1-deficient mice. The studies indicate that Sgk1 is not absolutely required for Na(+) reabsorption and K(+) secretion in the ASDN. On a standard NaCl and K(+) diet, modestly enhanced plasma aldosterone concentrations appear sufficient to establish a compensated phenotype in the absence of Sgk1. The kinase is necessary, however, for upregulation of transcellular Na(+) reabsorption in the ASDN. This may involve Sgk1-mediated stimulation of basolateral Na(+)-K(+)-ATPase as well as retention of epithelial Na(+) channel, ENaC, in the apical membrane. Such an upregulation is a prerequisite for adequate adaptation of 1) renal NaCl reabsorption during restricted dietary NaCl intake, as well as 2) K(+) secretion in response to enhanced K(+) intake. Thus gain-of-function mutations of Sgk1 are expected to result in renal NaCl retention and enhanced K(+) secretion. Further studies are required to elucidate renal and nonrenal aldosterone-induced effects of Sgk1, the role of other Sgk1 activators, as well as the link of Sgk1 polymorphisms to arterial hypertension in humans.

Impaired regulation of renal K+ elimination in the sgk1-knockout mouse.

Serum- and glucocorticoid-regulated kinase 1 (Sgk1) contributes to Na+ reabsorption in the aldosterone-sensitive distal nephron. Sgk1-knockout (sgk1-/-) and littermate wild-type mice (sgk1+/+) were used to test the importance of Sgk1 in renal elimination of K+ . Intravenous application of K+ load under anesthesia increased plasma K+ concentration by 1.3 to 1.4 mM in both sgk1-/- (n = 6) and sgkl+/+ (n = 7) mice. However, the increase of absolute and fractional renal K+ excretion observed in sgk1+/+ was significantly blunted in sgk1-/- animals. Both groups of mice decreased or increased renal K+ excretion to a similar extent after a low (<0.03%) or high (5%) K+ diet for 6 d, respectively. In sgk1+/+, plasma K+ concentration was not significantly modified by either high or low K+ diet. In sgk1-/-, however, high K+ diet enhanced plasma K+ concentration by about 1.6 mM, despite an excessive increase of plasma aldosterone concentration reaching values about sixfold higher than in sgk1+/+. Electrophysiological and immunohistochemical studies under high K+ diet indicated that reduced epithelial Na+ channel ENaC and/or Na+/K+-ATPase activity in the aldosterone-sensitive distal nephron accounted for the impaired response in sgk1-/- and that an enhanced apical abundance of renal outer medullary K+ channel ROMK partly compensated for the defect. The acute and chronic regulation of renal K+ elimination involves Sgk1.

Loss of K-Cl co-transporter KCC3 causes deafness, neurodegeneration and reduced seizure threshold.

K-Cl co-transporters are encoded by four homologous genes and may have roles in transepithelial transport and in the regulation of cell volume and cytoplasmic chloride. KCC3, an isoform mutated in the human Anderman syndrome, is expressed in brain, epithelia and other tissues. To investigate the physiological functions of KCC3, we disrupted its gene in mice. This severely impaired cell volume regulation as assessed in renal tubules and neurons, and moderately raised intraneuronal Cl(-) concentration. Kcc3(-/-) mice showed severe motor abnormalities correlating with a progressive neurodegeneration in the peripheral and CNS. Although no spontaneous seizures were observed, Kcc3(-/-) mice displayed reduced seizure threshold and spike-wave complexes on electrocorticograms. These resembled EEG abnormalities in patients with Anderman syndrome. Kcc3(-/-) mice also displayed arterial hypertension and a slowly progressive deafness. KCC3 was expressed in many, but not all cells of the inner ear K(+) recycling pathway. These cells slowly degenerated, as did sensory hair cells. The present mouse model has revealed important cellular and systemic functions of KCC3 and is highly relevant for Anderman syndrome.

Impaired renal Na(+) retention in the sgk1-knockout mouse.

The serum- and glucocorticoid-regulated kinase (sgk1) is induced by mineralocorticoids and, in turn, upregulates heterologously expressed renal epithelial Na(+) channel (ENaC) activity in Xenopus oocytes. Accordingly, Sgk1 is considered to mediate the mineralocorticoid stimulation of renal ENaC activity and antinatriuresis. Here we show that at standard NaCl intake, renal water and electrolyte excretion is indistinguishable in sgk1-knockout (sgk1(-/-)) mice and wild-type (sgk1(+/+)) mice. In contrast, dietary NaCl restriction reveals an impaired ability of sgk1(-/-) mice to adequately decrease Na(+) excretion despite increases in plasma aldosterone levels and proximal-tubular Na(+) and fluid reabsorption, as well as decreases in blood pressure and glomerular filtration rate.

Role of KCNE1-dependent K+ fluxes in mouse proximal tubule.

The electrochemical gradient for K+ across the luminal membrane of the proximal tubule favors K+ fluxes to the lumen. Here it was demonstrated by immunohistochemistry that KCNE1 and KCNQ1, which form together the slowly activated component of the delayed rectifying K+ current in the heart, also colocalize in the luminal membrane of proximal tubule in mouse kidney. Micropuncture experiments revealed a reduced K+ concentration in late proximal and early distal tubular fluid as well as a reduced K+ delivery to these sites in KCNE1 knockout (-/-), compared with wild-type (+/+) mice. These observations would be consistent with KCNE1-dependent K+ fluxes to the lumen in proximal tubule. Electrophysiological studies in isolated perfused proximal tubules indicated that this K+ flux is essential to counteract membrane depolarization due to electrogenic Na+-coupled transport of glucose or amino acids. Clearance studies revealed an enhanced fractional urinary excretion of fluid, Na+, Cl-, and glucose in KCNE1 -/- compared with KCNE1 +/+ mice that may relate to an attenuated transport in proximal tubule and contribute to volume depletion in these mice, as indicated by higher hematocrit values.