Renal function of gene-targeted mice lacking both SGK1 and SGK3

Serum- and glucocorticoid-inducible kinase (SGK) 1 and SGK3 share the ability to upregulate several ion channels, including the epithelial Na(+) channel. Whereas SGK1 is under genomic control of mineralocorticoids and glucocorticoids, SGK3 is constitutively expressed. The SKG1-knockout (sgk1(-/-)) mouse is seemingly normal when it is fed a standard diet, but its ability to retain NaCl is impaired when it is fed a salt-deficient diet. In the SGK3-knockout (sgk3(-/-)) mouse fed standard and salt-deficient diets, hair growth is strikingly delayed but NaCl excretion is normal. Thus the possibility was considered that SGK1 and SGK3 could mutually replace each other, thus preventing severe NaCl loss in sgk1(-/-) and sgk3(-/-) mice. We crossed SGK1- and SGK3-knockout mice and compared renal electrolyte excretion of the double mutants (sgk1(-/-)/sgk3(-/-)) with that of their wild-type littermates (sgk1(+/+)/sgk3(+/+)). Similar to sgk3(-/-) mice, the sgk1(-/-)/sgk3(-/-) mice display delayed hair growth. Blood pressure was slightly, but significantly (P < 0.03), lower in sgk1(-/-)/sgk3(-/-) (102 +/- 4 mmHg) than in sgk1(+/+)/sgk3(+/+) (114 +/- 3 mmHg) mice, a difference that was maintained in mice fed low- and high-salt diets. Plasma aldosterone concentrations were significantly (P < 0.01) higher in sgk1(-/-)/sgk3(-/-) than in sgk1(+/+)sgk3(+/+) mice fed control (511 +/- 143 vs. 143 +/- 32 pg/ml) and low-salt (1,325 +/- 199 vs. 362 +/- 145 pg/ml) diets. During salt depletion, absolute and fractional excretions of Na(+) were significantly (P < 0.01) higher in sgk1(-/-)/sgk3(-/-) (1.2 +/- 0.2 micromol/24 h g body wt, 0.12 +/- 0.03%) than in sgk1(+/+)/sgk3(+/+) (0.4 +/- 0.1 micromol/24 h g body wt, 0.04 +/- 0.01%) mice. The sgk1(-/-)/sgk3(-/-) mice share the delayed hair growth with sgk3(-/-) mice and the modestly impaired renal salt retention with sgk1(-/-) mice. Additional lack of the isoform kinase does not substantially compound the phenotype for either property.     

Role of maternal glucocorticoid inducible kinase SGK1 in fetal programming of blood pressure in response to prenatal diet

Maternal stress and malnutrition modify intrauterine fetal development with impact on postnatal blood pressure, nutrient, water, and electrolyte metabolism. The present study explored the possible involvement of maternal serum- and glucocorticoid-inducible kinase (SGK)-1 in fetal programming of blood pressure. To this end, wild-type (sgk1(+/+)) male mice were mated with SGK1 knockout (sgk1(-/-)) female mice, and sgk1(-/-) males with sgk1(+/+) females, resulting in both cases in heterozygotic (sgk1(-/+)) offspring. Following prenatal protein restriction, the offspring of sgk1(+/+) mothers gained weight significantly slower and had significantly higher blood pressure after birth. Moreover, a sexual dimorphism was apparent in fasting blood glucose and plasma corticosterone concentrations, with higher levels in female offspring. In contrast, prenatal protein restriction of sgk1(-/-) mothers had no significant effect on postnatal weight gain, blood pressure, plasma glucose concentration, or corticosterone levels, irrespective of offspring sex. Plasma aldosterone concentration, urinary flow rates, and urinary excretions of Na(+) and K(+) were not significantly modified by either maternal genotype or nutritional manipulation. In conclusion, maternal signals mediated by SGK1 may play a decisive role in fetal programming of hypertension induced by prenatal protein restriction.     

Hypertensinogenic factors and renal alpha-adrenoceptors in young SHR and WKY rats

The effects of high sodium intake (drinking 1% NaCl), DOCA and DOCA + 1%NaCl for 6 weeks on renal alpha 1- and alpha 2-adrenoceptors and on systolic blood pressure (SBP) were examined in young spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). On normal sodium intake, SHR rats had higher renal alpha 1 (p less than .001) and alpha 2-adrenoceptor densities (p less than .001) and SBP (p less than .001) than WKY rats. Although, WKY rats given either 1% NaCl, DOCA, and DOCA + 1% NaCl developed hypertension after 6 weeks of treatment, only 1% NaCl administration for the same period produced an increase in the alpha 1- and alpha 2-adrenoceptor densities when compared to the control (p less than .01 and p less than .001, respectively). In the SHR rats, to the contrary, ingestion of 1% NaCl and DOCA + 1% NaCl increased the already elevated alpha 2-adrenoceptor density (p less than .001) and SBP even more in this strain after 6 weeks of treatment. Equilibrium dissociation constants (KD), however, were similar for both classes of receptors in experimental and control rats. This study indicates that postweaning exposure of the WKY and SHR rats to a high salt treatment and DOCA can influence the renal alpha-adrenoceptor densities. Although the functional significance of the changes is unclear, it is reasonable to speculate that postweaning exposure to a hypertensinogenic stimuli such as a 1% NaCl and/or DOCA may ultimately interfere with the functional development of the kidney differently in rats genetically predisposed to hypertension (SHR) from normotensive (WKY) rats.     

Deranged Kv channel regulation in fibroblasts from mice lacking the serum and glucocorticoid inducible kinase SGK1

Coexpression of the serum and glucocorticoid inducible kinase 1 (SGK1) up-regulates Kv channel activity in Xenopus oocytes and human embryonic kidney cells. To investigate the physiological impact of SGK1 dependent Kv channel regulation, we recorded whole-cell currents in lung fibroblasts from SGK1 knockout mice (sgk1-/-) and wild-type littermates (sgk1+/+). Serum-grown mouse lung fibroblasts (MLF) from both genotypes exhibited voltage-gated outwardly rectifying K(+)-currents with time-dependent activation (tau(act) approximately 3 msec), slow inactivation (tau(inact) approximately 700 msec), use-dependent inactivation, and (partial) inhibition by K(+) channel blockers TEA, 4-AP, and margatoxin. In serum grown MLF peak Kv current density at +100 mV was significantly lower in sgk1-/- (14 +/- 2 pA/pF, n = 13) than in sgk1+/+ (31 +/- 4 pA/pF, n = 16). PCR amplification of different Kv1 and Kv3 subunits from mouse fibroblasts demonstrated the expression of Kv1.1-1.7, Kv3.1, and Kv3.3 mRNA in both sgk1+/+ and sgk1-/- cells. Upon serum deprivation Kv currents almost disappeared in sgk1+/+ (4 +/- 1 pA/pF, n = 11) but not in sgk1-/- (10 +/- 1 pA/pF, n = 6) MLF. Accordingly, following serum deprivation Kv current density was significantly lower in sgk1+/+ than in sgk1-/-. Stimulation of serum-depleted cells with dexamethasone (dex) (1 microM, 1 day), IGF-1 (6.7 microM, 4-6 h) or both, significantly activated Kv currents in sgk1+/+ but not in sgk1-/- MLF. In the presence of both, dex and IGF-1, the Kv current density was significantly larger in sgk1+/+ (27 +/- 3 pA/pF, n = 12) than in sgk1-/- (13 +/- 3 pA/pF, n = 10) cells. Similar to MLF, Kv currents were significantly higher in sgk1+/+ mouse tail fibroblasts (MTF). In sgk1+/+ but not sgk1-/- MTF the Kv currents were inhibited upon serum deprivation and reincreased after stimulation of serum deprived MTF with dex (1 microM, 1 day) and afterwards with IGF-1 (6.7 microM, 4-6 h). According to Fura-2-fluorescence capacitative Ca(2+) entry was lower in sgk1-/- MTF compared to sgk1+/+ MTF. Upon serum deprivation capacitative Ca(2+) entry decreased significantly in sgk1+/+ but not in sgk1-/- MTF. Stimulation of depleted cells with dex (1 microM, 1 day) and afterwards with IGF-1 (6.7 microM, 4-6 h) reincreased capacitative Ca(2+) entry in sgk1+/+ MTF, whereas in sgk1-/- cells it remained unchanged. In conclusion, lack of SGK1 does not abrogate Kv channel activity but abolishes regulation of those channels by serum, glucocorticoids and IGF-1, an effect influencing capacitative Ca(2+) entry.     

Effect of saline infusion on kidney and collecting duct function in atrial natriuretic peptide (ANP) gene "knockout" mice.

Atrial natriuretic peptide (ANP) is thought to play a role in renal regulation of salt balance by reducing tubular reabsorption of sodium and chloride. Therefore, in the chronic absence of this hormone, a defect of salt excretion should be evident. We used an ANP gene deletion model to test this premise. F2 homozygous mutant mice (-/-) and their wild-type littermates (+/+) were fed an 8% NaCl diet prior to an acute infusion of isotonic saline. Arterial blood pressures, renal excretions of salt and water, as well as collecting duct transport of fluid and electrolytes were measured. Pressures were significantly higher in -/- compared with +/+ mice (139 +/- 4 vs. 101 +/- 2 mmHg; 1 mmHg = 133.3 Pa). There was no difference in glomerular filtration rate (-/- = 0.84 +/- 0.06; +/+ = 0.81 +/- 0.04 mL x min(-1) x g(-1) kidney weight). In the collecting duct, sodium and chloride reabsorptions were significantly higher in the -/- group than in the +/+ group. As a result, natriuresis and chloruresis were relatively reduced (U(Na)V: -/- = 8.6 +/- 1.1; +/+ = 14.0 +/- 1.1; U(Cl)V: -/- = 10.1 +/- 1.4; +/+ = 16.0 +/- 1.1 micromol x min(-1) x g(-1) kidney weight). We conclude that the absence of endogenous ANP activity in mice on a high-salt diet subjected to acute saline infusion causes inappropriately high reabsorption of sodium and chloride in the medullary collecting duct, resulting in a relative defect in renal excretory capacity for salt.     

Increased reactive oxygen species contributes to kidney injury in mineralocorticoid hypertensive rats.

Hypertension is associated with increased reactive oxygen species (ROS). Renal ROS production and their effects on renal function have never been investigated in mineralocorticoid hypertensive rats. In this study we hypothesized that increased ROS production in kidneys from deoxycorticosterone (DOCA)-salt rats contributes to adverse renal morphological changes and impaired renal function in DOCA-salt hypertensive rats. We also determined whether ROS-induced renal injury was dependent on blood pressure. DOCA-salt hypertensive rats exhibited a marked increase in blood pressure, renal ROS production, glomerular and tubular lesions, and microalbuminuria compared to sham rats. Treatment of DOCA-salt hypertensive rats with apocynin for 28 days resulted in attenuation of systolic blood pressure and improvement of renal morphology. Renal superoxide level in DOCA-salt rats was 215% of sham-operated rats and it was significantly decreased to 140% with apocynin treatment. Urinary protein level was decreased from 27 +/- 3 mg/day in DOCA-salt hypertensive rats to 9 +/- 2 mg/day. 28 days of Vitamin E treatment also reduced renal injury in regard to urinary protein level and renal morphology but had no effect on blood pressure in DOCA-salt rats. Increased urinary 8-isoprostane, a marker for oxidative stress, in DOCA-salt hypertensive rats (55 +/- 8 ng/day) was diminished by vitamin E treatment (24 +/- 6 ng/day). These data suggest that renal injury characteristic of mineralocorticoid hypertension is associated with oxidative stress and is partly independent of blood pressure.     

Implication of CD38 gene in podocyte epithelial-to-mesenchymal transition and glomerular sclerosis

CD38 is a multifunctional protein involving in a number of signalling pathways. Given that the lack of CD38 is considered as a dedifferentiation marker of lymphocytes and other cells, we hypothesized that CD38 and its signalling pathway may participate in the epithelial-to-mesenchymal transition (EMT) process of podocytes and thereby regulates the integrity of glomerular structure and function. Western blot analysis and RT-PCR demonstrated that renal tissue CD38 expression was lacking in CD38(-/-) mice or substantially reduced in renal CD38 shRNA-transfected WT (CD38-shRNA) mice compared to CD38(+/+) littermates. Confocal fluorescent microscopy demonstrated the reduced expression of epithelial markers (P-Cadherin, ZO-1 and podocin) and increased expression of mesenchymal markers (FSP-1, α-SMA and desmin) in the glomeruli of CD38(-/-) and CD38-shRNA mice compared to CD38(+/+) mice. Morphological examinations showed profound injury in the glomeruli of CD38(-/-) or CD38-shRNA mice compared to CD38(+/+) mice. This enhanced glomerular injury in CD38(-/-) or CD38-shRNA mice was accompanied by increased albuminuria and proteinuria. DOCA/high salt treatment further decreased the expression of epithelial markers and increased the abundance of mesenchymal markers, which were accompanied by more increased glomerular damage index and mean arterial pressure in CD38(-/-) and CD38-shRNA mice than CD38(+/+) mice. In vitro studies showed that inhibition of CD38 enhances the EMT in podocytes. In conclusion, our observations reveal that the normal expression of CD38 importantly contributes to the differentiation and function of podocytes and the defect of this gene expression may be a critical mechanism inducing EMT and consequently resulting in glomerular injury and sclerosis.     

Renal denervation chronically lowers arterial pressure independent of dietary sodium intake in normal rats

The present study was designed to test the hypothesis that renal nerves chronically modulate arterial pressure (AP) under basal conditions and during changes in dietary salt intake. To test this hypothesis, continuous telemetric recording of AP in intact (sham) and renal denervated (RDNX) Sprague-Dawley rats was performed and the effect of increasing and decreasing dietary salt intake on AP was determined. In protocol 1, 24-h AP, sodium, and water balances were measured in RDNX (n = 11) and sham (n = 9) rats during 5 days of normal (0.4% NaCl) and 10 days of high (4.0% NaCl) salt intake, followed by a 3-day recovery period (0.4% NaCl). Protocol 2 was similar with the exception that salt intake was decreased to 0.04% NaCl for 10 days after the 5-day period of normal salt (0.04% NaCl) intake (RDNX; n = 6, sham; n = 5). In protocol 1, AP was lower in RDNX (91 +/- 1 mmHg) compared with sham (101 +/- 2 mmHg) rats during the 5-day 0.4% NaCl control period. During the 10 days of high salt intake, AP increased <5 mmHg in both groups so that the difference between sham and RDNX rats remained constant. In protocol 2, AP was also lower in RDNX (93 +/- 2 mmHg) compared with sham (105 +/- 4 mmHg) rats during the 5-day 0.4% NaCl control period, and AP did not change in response to 10 days of a low-salt diet in either group. Overall, there were no between-group differences in sodium or water balance in either protocol. We conclude that renal nerves support basal levels of AP, irrespective of dietary sodium intake in normal rats.     

Gastrointestinal osmoreceptors and renal sodium excretion in humans

The hypothesis that natriuresis can be induced by stimulation of gastrointestinal osmoreceptors was tested in eight supine subjects on constant sodium intake (150 mmol NaCl/day). A sodium load equivalent to the amount contained in 10% of measured extracellular volume was administered by a nasogastric tube as isotonic or hypertonic saline (850 mM). In additional experiments, salt loading was replaced by oral water loading (3.5% of total body water). Plasma sodium concentration increased after hypertonic saline (+3.1 +/- 0.7 mM), decreased after water loading (-3.8 +/- 0.8 mM), and remained unchanged after isotonic saline. Oncotic pressure decreased by 9.4 +/- 1.2, 3.7 +/- 1.2, and 10.7 +/- 1.3%, respectively. Isotonic saline induced an increase in renal sodium excretion (104 +/- 15 to 406 +/- 39 micromol/min) that was larger than seen with hypertonic saline (85 +/- 15 to 325 +/- 39 micromol/min) and water loading (88 +/- 11 to 304 +/- 28 micromol/min). Plasma ANG II decreased to 22 +/- 6, 35 +/- 6, and 47 +/- 5% of baseline after isotonic saline, hypertonic saline, and water loading, respectively. Plasma atrial natriuretic peptide (ANP) concentrations and urinary excretion rates of endothelin-1 were unchanged. In conclusion, stimulation of osmoreceptors by intragastric infusion of hypertonic saline is not an important natriuretic stimulus in sodium-replete subjects. The natriuresis after intragastric salt loading was independent of ANP but can be explained by inhibition of the renin-angiotensin system.     

Role of renal nerves in development of hypertension in DOCA-salt model in rats: a telemetric approach

Centrally mediated hyperactivity of the autonomic nervous system contributes to DOCA hypertension; however, the targeted peripheral vascular bed(s) remain unclear. We propose that if renal sympathetic activity is a factor in the development of DOCA-salt hypertension, then renal denervation (RDNX) should attenuate the hypertensive response. In protocol 1, uninephrectomized RDNX (n = 9) and sham-denervated (n = 6) Sprague-Dawley rats were allowed free access to 0.9% NaCl solution and 0.1% NaCl diet. Mean arterial pressure (MAP) and heart rate were telemetrically recorded for 4 days before and 36 days after DOCA (100 mg/rat) implantation; sodium and water balances were recorded daily. Protocol 2 was similar except that saline intake in sham rats (n = 7) was matched to that observed in RDNX rats of protocol 1 for 30 days; for the last 10 days, the rats were allowed free access to saline. Before DOCA in protocol 1, MAP was lower (P < 0.05) in RDNX rats (99 +/- 1 mmHg) compared with sham rats (111 +/- 3 mmHg); however, heart rate and sodium and water balances were similar between groups. RDNX attenuated the MAP response to DOCA by approximately 50% (DeltaMAP = 22 +/- 3 mmHg, where Delta is change in MAP) when compared with sham rats (DeltaMAP = 38 +/- 6). RDNX rats consumed significantly less saline than sham rats, and cumulative sodium and water balances were reduced by 33% and 23%, respectively. In protocol 2, a similar pattern in MAP elevation was observed in RDNX and saline-restricted, sham-denervated rats even when saline restriction was removed. These results indicate that the renal sympathetic nerves are important in hypertension development but that other factors are also involved.     

NAD(P)H oxidase/nitric oxide interactions in peroxisome proliferator activated receptor (PPAR)alpha-mediated cardiovascular effects

Activation of peroxisome proliferator activated receptor (PPAR)alpha and its protective role in cardiovascular function has been reported but the exact mechanism(s) involved is not clear. As we have shown that PPARalpha ligands increased nitric oxide (NO) production and cardiovascular function is controlled by a balance between NO and free radicals, we hypothesize that PPARalpha activation tilts the balance between NO and free radicals and that this mechanism defines the protective effects of PPARalpha ligands on cardiovascular system. Systolic blood pressure (SBP) was greater in PPARalpha knockout (KO) mice compared with its wild type (WT) litter mates (130+/-10 mmHg versus 107+/-4 mmHg). L-NAME (100mg/L p.o.), the inhibitor of NO production abolished the difference between PPARalpha KO and WT mice. In kidney homogenates, tissue lipid hydroperoxide generation was greater in KO mice (11.8+/-1.4 pM/mg versus 8.3+/-0.6 pM/mg protein). This was accompanied by a higher total NOS activity (46+/-6%, p<0.05) and a approximately 3 fold greater Ca2+-dependent NOS activity in kidney homogenates of untreated PPARalpha WT compared with the KO mice. Clofibrate, a PPARalpha ligand, increased NOS activity in WT but not KO mice. Bezafibrate (30 mg/kg) reduced SBP in conscious rats (19+/-4%, p<0.05), increased urinary NO excretion (4.06+/-0.53-7.07+/-1.59 microM/24 h; p<0.05) and reduced plasma 8-isoprostane level (45.8+/-15 microM versus 31.4+/-8 microM), and NADP(H) oxidase activity (16+/-5%). Implantation of DOCA pellet (20mg s.c.) in uninephrectomized mice placed on 1% NaCl drinking water increased SBP by a margin that was markedly greater in KO mice (193+/-13 mmHg versus 130+/-12 mmHg). In the rat, DOCA increased SBP and NAD(P)H oxidase activity and both effects were diminished by clofibrate. In addition, clofibrate reduced ET-1 production in DOCA/salt hypertensive rats. Thus, apart from inhibition of ET-1 production, PPARalpha activation exerts protective actions in hypertension via a mechanism that involves NO production and/or inhibition of NAD(P)H oxidase activity.     

sgk is an aldosterone-induced kinase in the renal collecting duct. Effects on epithelial na+ channels.

The early phase of the stimulatory effect of aldosterone on sodium reabsorption in renal epithelia is thought to involve activation of apical sodium channels. However, the genes initiating this effect are unknown. We used a combination of polymerase chain reaction-based subtractive hybridization and differential display techniques to identify aldosterone-regulated immediate early genes in renal mineralocorticoid target cells. We report here that aldosterone rapidly increases mRNA levels of a putative Ser/Thr kinase, sgk (or serum- and glucocorticoid-regulated kinase), in its native target cells, i.e. in cortical collecting duct cells. The effect occurs within 30 min of the addition of aldosterone, is mediated through mineralocorticoid receptors, and does not require de novo protein synthesis. The full-length sequences of rabbit and mouse sgk cDNAs were determined. Both cDNAs show significant homology to rat and human sgk (88-94% at the nucleotide level, and 96-99% at the amino acid level). Coexpression of the mouse sgk in Xenopus oocytes with the three subunits of the epithelial Na+ channel results in a significantly enhanced Na+ current. These results suggest that sgk is an immediate early aldosterone-induced gene, and this protein kinase plays an important role in the early phase of aldosterone-stimulated Na+ transport.     

Immune suppression blocks sodium-sensitive hypertension following recovery from ischemic acute renal failure

The present study determined the effect of immune suppression with mycophenolate mofetil (MMF) on sodium-sensitive hypertension following recovery from ischemia reperfusion (I/R)-induced acute renal failure. Male Sprague-Dawley rats fed 0.4% NaCl chow were subjected to 40 min bilateral I/R or control sham surgery. After 35 days of recovery, when plasma creatinine levels had returned to normal, the rats were switched to 4.0% NaCl chow for 28 days and administered vehicle or MMF (20 mg.kg(-1).day(-1) ip). High-salt mean arterial pressure was significantly higher in I/R rats (144 +/- 16 mmHg) compared with vehicle-treated sham rats (122 +/- 2 mmHg). Treatment of I/R rats with MMF during the period of high salt intake prevented the salt-induced increase in arterial pressure (114 +/- 3 mmHg). Conscious creatinine clearance was lower in I/R rats (0.27 +/- 0.07 ml.min(-1).100 g body wt(-1)) compared with vehicle-treated sham rats (0.58 +/- 0.04 ml.min(-1).100 g body wt(-1)); MMF treatment prevented the decrease in creatinine clearance in I/R rats (0.64 +/- 0.07 ml.min(-1).100 g body wt(-1)). I/R injury also significantly increased glomerular tissue damage and increased the presence of ED-1 positive (macrophages) and S100A4 positive cells (fibroblasts) in the renal interstitium. The I/R rats treated with MMF exhibited a significant reduction in infiltrating macrophages and fibroblasts and decreased histological damage. The present data indicate that infiltrating immune cells mediate or participate in the development of sodium-sensitive hypertension and renal damage in rats apparently recovered from renal I/R injury.     

Serum- and glucocorticoid-inducible kinase 1 (SGK1) increases neurite formation through microtubule depolymerization by SGK1 and by SGK1 phosphorylation of tau

Serum- and glucocorticoid-inducible kinase 1 (SGK1) is a member of the Ser/Thr protein kinase family that regulates a variety of cell functions. Recently, SGK1 was shown to increase dendritic growth but the mechanism underlying the increase is unknown. Here we demonstrated that SGK1 increased the neurite formation of cultured hippocampal neurons through microtubule (MT) depolymerization via two distinct mechanisms. First, SGK1 directly depolymerized MTs. In vitro MT depolymerization experiments revealed that SGK1, especially N-truncated SGK1, directly disassembled self-polymerized MTs and taxol-stabilized MTs in a dose-dependent and ATP-independent manner. The transfection of sgk1 to HeLa cells also inhibited MT assembly in vivo. Second, SGK1 indirectly depolymerized MTs through the phosphorylation of tau at Ser214. An in vitro kinase assay revealed that active SGK1 phosphorylated tau Ser214 specifically. In vivo transfection of sgk1 also phosphorylated tau Ser214 in HEK293T cells and hippocampal neurons. Further, sgk1 transfection significantly increased the number of primary neurites and shortened the length of the total process in cultured hippocampal neurons. These effects were antagonized by the cotransfection of the tauS214A mutant plasmid. Dexamethasone, a synthetic glucocorticoid, mimics the effect of sgk1 overexpression. Together, these results suggest that SGK1 enhances neurite formation through MT depolymerization by a direct action of SGK1 and by the SGK1 phosphorylation of tau.