Proteinuria in mice expressing PKB/SGK-resistant GSK3

Am J Physiol Renal Physiol 296: F153–F159, 2009. First published November 5, 2008; doi:10.1152/ajprenal.90398.2008. Proteinuria in mice expressing PKB/SGK-resistant GSK3 Krishna M. Boini,1* Kerstin Amann,2* Daniela Kempe,1 Dario R. Alessi,3 and Florian Lang1 1 Department of Physiology, University of Tübingen, Tübingen; 2Department of Pathology, University of Erlangen, Erlangen, Germany; and 3MRC Protein Phosphorylation Unit, University of Dundee, Nethergate, Dundee, Scotland, United Kingdom Submitted 4 July 2008; accepted in final form 29 October 2008 Boini KM, Amann K, Kempe D, Alessi DR, Lang F. Proteinuria in mice expressing PKB/SGK-resistant GSK3. Am J Physiol Renal Physiol 296: F153–F159, 2009. First published November 5, 2008; doi:10.1152/ajprenal.90398.2008.—SGK1 is critically important for mineralocorticoid/salt-induced glomerular injury. SGK1 inactivates GSK3, which downregulates Snail, a DNA-binding molecule repressing the transcription of nephrin, a protein critically important for the integrity of the glomerular slit membrane. PKB/SGK-dependent GSK regulation is disrupted in mice carrying a mutation, in which the serine in the SGK/PKB-phosphorylation consensus sequence is replaced by alanine. The present study explored whether PKB/SGK-dependent GSK3 regulation influences glomerular proteinuria. Gene-targeted knockin mice with mutated and thus PKB/SGK-resistant GSK3␣,␤ (gsk3KI) were compared with their wild-type littermates (gsk3WT). gsk3KI and gsk3WT mice were implanted with DOCA release pellets and offered 1% saline as drinking water for 21 days. Under standard diet, tap water intake and absence of DOCA, urinary flow rate, glomerular filtration rate, and urinary albumin excretion were significantly larger and blood pressure was significantly higher in gsk3KI than in gsk3WT mice. Within 18 days, DOCA/salt treatment significantly increased fluid intake and urinary flow rate, urinary protein and albumin excretion, and blood pressure in both genotypes but the respective values were significantly higher in gsk3KI than in gsk3WT mice. Plasma albumin concentration was significantly lower in gsk3KI than in gsk3WT mice. Proteinuria was abrogated by lowering of blood pressure with ␣1-blocker prazosin (1 ␮g/g body wt) in 8-mo-old mice. According to immunofluorescence, nephrin at 3 and 8 mo and podocin expression at 3 mo were significantly lower in gsk3KI than in gsk3WT mice. After 18 days, DOCA/salt treatment renal glomerular sclerosis and tubulointerstitial damage were significantly more pronounced in gsk3KI than in gsk3WT mice. The observations reveal that disruption of PKB/SGK-dependent regulation of GSK3 leads to glomerular injury with proteinuria, which may at least partially be secondary to enhanced blood pressure. glomerular filtration rate; water; DOCA; albumin; nephrin and glucocorticoid-inducible kinase SGK1 is critically important for the development of proteinuria and glomerular fibrosis during mineralocorticoid and salt excess (1). Accordingly, gene-targeted mice lacking functional SGK1 are protected against the glomerular injury following treatment with DOCA/high salt (1). Similar to protein kinase B Akt/PKB (8, 33), SGK1 phosphorylates and thus inactivates glycogen synthase kinase GSK3␤ (32). GSK3␤, in turn, phosphorylates and thus inactivates the DNA-binding molecule Snail (3, 9) and inhibition of GSK3␤ is followed by upregulation of snail (9, 13). Snail represses the expression of nephrin (17, 18), which is critically important for the integrity of the glomerular THE SERUM * K. M. Boini and K. Amann contributed equally to this work and thus share first authorship. Address for reprint requests and other correspondence: F. Lang, Dept. of Physiology, Univ. of Tübingen, Gmelinstr. 5, D-72076, Tübingen, Germany (e-mail: [email protected]). http://www.ajprenal.org slit membrane (2, 10, 14, 16, 23, 27, 29, 31, 35, 43). Defective nephrin leads to proteinuria with subsequent development of renal failure (15, 28). Moreover, deranged regulation of nephrin is considered to participate in the proteinuria of diabetic nephropathy (21, 34, 43), focal segmental glomerulosclerosis (26), experimental nephrotic syndrome (25), and preeclampsia (7). In theory, SGK1 could have at least partially been effective through phosphorylation and thus inhibition of GSK3␤ with subsequent snail upregulation, suppression of nephrin expression, and development of glomerular injury. The present study explored whether PKB/SGK-dependent inhibition of GSK participates in the pathophysiology of proteinuria and glomerular injury during salt and mineralocorticoid excess. The signaling of PKB/SGK to GSK3 could be disrupted by replacement of serine within the PKB phosphorylation site by alanine (GSK3␣21A/21A, GSK3␤9A/9A) thus yielding GSK3, which is resistant to inactivation by PKB/SGK (19). In knockin mice carrying these mutations (gsk3KI), the effect of insulin on muscle glycogen synthase is abrogated (19). To elucidate the role of SGK1-dependent regulation of GSK3 in glomerular function following salt/mineralocorticoid excess, kidneys from gsk3KI mice were compared with their wild-type littermates (gsk3WT) after treatment with DOCA/high salt. As shown previously (5), gsk3KI mice have significantly higher body temperature, blood pressure, food and water intake, fecal excretion, glomerular filtration rate, urinary flow rate, urine osmolarity, as well as urinary excretion of Na⫹, K⫹, and urea. Surprisingly, unlike SGK1 knockout mice (1), gsk3KI mice are not protected against DOCA/high salt-induced glomerular injury. Instead, nephrin and podocin expression are decreased in gsk3KI mice and those mice suffer from spontaneous proteinuria, which is at least partially due to enhanced blood pressure. METHODS All animal experiments were conducted according to the guidelines of the American Physiological Society as well as the German law for the welfare of animals and were approved by local authorities. Mice were generated, in which the codon encoding Ser9 of GSK3␤ gene was changed to encode nonphosphorylatable alanine (GSK3␤9A/9A), and simultaneously the codon encoding Ser21 of GSK3␣ was changed to encode the nonphosphorylatable GSK3␣21A/21A thus yielding the GSK3␣/␤21A/21A/9A/9A double knockin mouse (gsk3KI) as described previously (19). The mice were compared with their wild-type littermates (gsk3WT). The mice (6 –7 females, 6 males, age 8 mo) were fed a control diet (1314, Altromin, Heidenau, Germany). Where indicated, the mice The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 0363-6127/09 $8.00 Copyright © 2009 the American Physiological Society Downloaded from journals.physiology.org/journal/ajprenal (054.083.151.133) on January 26, 2022. F153 F154 GSK-SENSITIVE PROTEIN EXCRETION were offered saline. All other mice had free access to tap drinking water. For evaluation of renal excretion, both gsk3KI and gsk3WT mice were placed individually in metabolic cages (Techniplast, Hohenpeissenberg, Germany) for 24-h urine collection as described previously (36). They were allowed a 3-day habitation period when food and water intake, urinary flow rate, urinary excretion of salt, fecal excretion, and body weight were recorded every day to ascertain that the mice were adapted to the new environment. Subsequently, 24-h collection of urine was performed for 3 consecutive days to obtain the urinary parameters. To ensure quantitative urine collection, metabolic cages were siliconized and urine was collected under watersaturated oil. To induce mineralocorticoid excess, gsk3KI and gsk3WT mice (8 mo old, n ⫽ 12 each group) were implanted with 21-day-release 50-mg DOCA (2.4 mg/day) pellets (Innovative Research of America, Sara- Fig. 2. Urinary protein and albumin excretion in gsk3KI and gsk3WT mice before and following DOCA/salt treatment. Arithmetic means ⫾ SE (n ⫽ 11–12 each group) of urinary protein (top) and albumin (bottom) excretion in gsk3KI (filled bars) and gsk3WT (open bars) mice before and following an 18-day DOCA/salt treatment. #P ⬍ 0.05 vs. respective value before DOCA/ salt treatment. *P ⬍ 0.05 vs. respective value of gsk3WT mice. Fig. 1. Fluid intake, urinary flow rate, and creatinine clearance in genetargeted knockin mice with mutated and thus PKB/SGK-resistant GSK3␣,␤ (gsk3KI) and wild-type littermates (gsk3WT) before and following DOCA/salt treatment. Arithmetic means ⫾ SE (n ⫽ 9 –13 each group) of fluid intake (top), urinary excretion (middle), and creatinine clearance (bottom) in gsk3KI (filled bars) and gsk3WT (open bars) mice before and following an 18-day DOCA/salt treatment. #P ⬍ 0.05 vs. respective value before DOCA/salt treatment. *P ⬍ 0.05 vs. respective value of gsk3WT mice. AJP-Renal Physiol • VOL sota, FL) in the neck area (12, 38) during superficial general anesthesia (5 mg/kg ip midazolam ⫹ 50 mg/kg ip ketamin), which was partially antagonized by flumazenil (0.5 mg/kg ip) afterwards (1). Before the pellet implantation (control period), the mice had free access to plain tap water. After the implantation, the tap water was replaced by 1% NaCl (high salt). Throughout the entire study, mice had free access to a standard mouse diet (C1314, Altromin). Renal excretion was determined before and after 18 days of DOCA ⫹ 1% NaCl treatment. To obtain blood specimen, animals were lightly anesthetized with diethylether (Roth, Karlsruhe, Germany) and ⬃200 ␮l of blood were withdrawn into heparinized capillaries by puncturing the retro-orbital plexus. Creatinine concentration in urine was determined using the Jaffe reaction (Sigma, St. Louis, MO), and creatinine concentration in serum was measured using an enzymatic kit (creatinine PAP, Lehmann, Berlin, Germany) according to the manufacturer’s instructions. Plasma proteins were separated by capillary electrophoresis in a Paragon CZE 2000 (Beckman Coulter). Electrophoresis was performed using the manufacturer’s instructions and reagents. Fraction limits were manually adjusted. Total plasma protein was measured using the Biuret method, and urinary total protein was measured quantitatively using Coomassie Brilliant Blue G-250 dye (Bio-Rad protein assay, Hercules, CA). A standard curve was generated with bovine albumin (Sigma). Urinary and plasma albumin was measured fluorometrically using the albumin-sensitive dye albumin blue 580 at 595-nm excitation and 642-nm emission on a multi-label counter (Victor 1420, PerkinElmer) according to the manufacturer’s instructions (microfluoral, Progen, Heidelberg, Germany). Standard curves were generated with mouse albumin (Sigma, Taufkirchen, Germany) and measurements were performed within the linear range (0 –156 mg/l). 296 • JANUARY 2009 • www.ajprenal.org Downloaded from journals.physiology.org/journal/ajprenal (054.083.151.133) on January 26, 2022. GSK-SENSITIVE PROTEIN EXCRETION F155 Fig. 3. Plasma protein concentration in gsk3KI and gsk3WT mice before and following DOCA/salt treatment. Arithmetic means ⫾ SE (n ⫽ 6 each group) of plasma protein concentration in gsk3KI (filled bars) and gsk3WT (open bars) mice before (left) and following (right) a 21-day DOCA/salt treatment. #P ⬍ 0.05 vs. respective value before DOCA/salt treatment. *P ⬍ 0.05 vs. respective value of gsk3WT mice. Systolic arterial blood pressure was determined by the tail-cuff method before and 21 days following the initiation of DOCA ⫹ 1% NaCl treatment. As reviewed recently (20), the tail-cuff approach to determine arterial blood pressure requires certain precautions to reduce the stress of the animals, including appropriate training of the mice over multiple days, prewarming to an ambient temperature of 29°C, measurement in a quiet and semidarkened and clean environment, and performance of the measurements by one person (KMB) and during a defined day time, when blood pressure is stable (between 1 and 3 PM). All these precautions were taken in the present study. The readings from 2 days were then averaged to obtain a mean blood pressure. All recordings and data analysis were done using a computerized data-acquisition system and software (Power Lab 400 and Chart 4; AdInstruments). In a further series of experiments, gsk3KI and gsk3WT mice (male 8 mo old, n ⫽ 6 each) were treated after a control period with daily injections of prazosin (1 ␮g/g body wt ip) for 5 days. Systolic blood pressure was measured before and after 3 days of prazosin treatment. Renal protein excretion was determined before and after 5 days of prazosin treatment. Fig. 5. Urinary flow rate and urinary protein excretion in gsk3KI and gsk3WT mice before and following a prazosin treatment. A: arithmetic means ⫾ SE (n ⫽ 6 each group) of urinary flow rate (top) and urinary protein excretion (bottom) in gsk3KI (filled bars) and gsk3WT (open bars) mice before and following a prazosin treatment. #P ⬍ 0.05 vs. respective value before prazosin treatment. *P ⬍ 0.05 vs. respective value of gsk3WT mice. B: correlation between blood pressure and proteinuria before and following a prazosin treatment. Fig. 4. Systolic blood pressure in gsk3KI and gsk3WT mice before and following DOCA/salt treatment. Arithmetic means ⫾ SE (n ⫽ 10 –12 each group) of systolic blood pressure in gsk3KI (filled bars) and gsk3WT (open bars) mice before (left) and following (right) a 21-day DOCA/salt treatment. #P ⬍ 0.05 vs. respective value before DOCA/salt treatment. *P ⬍ 0.05 vs. respective value of gsk3WT mice. AJP-Renal Physiol • VOL For histology, fixed kidneys and samples were stored in 4% paraformaldehyde/0.1 M sodium phosphate buffer. Kidneys were dissected into 1-mm-thick slices perpendicular to the longitudinal axis. Using area weighted sampling 10 small pieces of the kidney cortex were selected for later embedding in epon araldite. All remaining kidney slices were embedded in paraffin yielding one representative section of each slice for qualitative morphological investigations. Four-micrometer paraffin sections were cut and stained with hematoxylin/eosin (HE), periodic acidSchiff stain (PAS), and a fibrous tissue stain (Sirius red). Sections were evaluated using well-established semiquantitative scoring indexes (score: 0 – 4) for glomerular, tubulointerstitial, and vascular damage (1). Criteria of glomerular damage were mesangial cell and matrix expansion, focal segmental sclerosis, and podocyte damage. Tubulointerstitial damage 296 • JANUARY 2009 • www.ajprenal.org Downloaded from journals.physiology.org/journal/ajprenal (054.083.151.133) on January 26, 2022. F156 GSK-SENSITIVE PROTEIN EXCRETION Fig. 6. Nephrin and podocin expression in gsk3KI and gsk3WT mice. Nephrin (top) and podocin (bottom) immunofluorescence of kidney sections from 3-mo-old gsk3KI (right) and gsk3WT (left) mice. Antibodies are directed against nephrin and podocin (green fluorescence). Nuclei are counterstained in blue with DAPI. consisted of tubular atrophy, tubular dilatation, interstitial inflammation, and interstitial fibrosis and vascular damage was assessed as wall thickening, vascular inflammation, or fibrinoid necrosis, respectively. All semiquantitative, morphometric, and stereological measurements were performed in a blinded manner by an observer who was unaware of the study protocol. Nephrin and podocin expression in the kidney were analyzed by immunofluorescence in 3-mo-old and nephrin expression in 8-mo-old mice. To this end, the mice were killed by CO2 and cervical dislocation and the kidneys were rapidly frozen on liquid nitrogen. Frozen sections (3 ␮m) were fixed in aceton (10 min at ⫺20°C), air-dried, and stored in Tris 䡠 CSA buffer for 20 min. Then, blocking was performed with 20% goat serum (blotto, 1:5, 30 min). Afterwards, the primary antibodies against nephrin (polyclonal anti-nephrin, guinea pig, Acris, 1:100 in Tris 䡠 CSA) and podocin (polyclonal anti-podocin, rabbit, Sigma, 1:100 in Tris 䡠 CSA) were applied (1 h; room temperature) and the sections were washed in Tris 䡠 CSA buffer (3 ⫻ 5 min). Afterwards, the secondary antibody (goat anti-guinea pig, Alexa, 1:250, Molecular Probes) was applied for 30 min at room temperature followed by three Tris 䡠 CSA washings. DAPI was used to counterstain nuclei (1:1,000 in distilled water for 5 min) followed by rinsing in Tris 䡠 CSA buffer (3 ⫻ 5 min). Finally, sections were covered with mowiol and stored in darkness at 4°C until analysis. Data are provided as means ⫾ SE; n represents the number of independent experiments. All data were tested for significance using Fig. 7. Nephrin expression in gsk3KI and gsk3WT mice before and following DOCA/salt treatment. Nephrin immunofluorescence of kidney sections from 8-mo-old gsk3KI (right) and gsk3WT (left) mice before (top) and following (bottom) a 21-day DOCA/ salt treatment. Antibodies are directed against nephrin (green fluorescence). Nuclei are counterstained in blue with DAPI. AJP-Renal Physiol • VOL 296 • JANUARY 2009 • www.ajprenal.org Downloaded from journals.physiology.org/journal/ajprenal (054.083.151.133) on January 26, 2022. F157 GSK-SENSITIVE PROTEIN EXCRETION Fig. 8. Histological evaluation of kidneys from gsk3KI and gsk3WT mice following DOCA/salt treatment. Histomorphological data after 21 days of DOCA/salt treatment (paraffin sections). Arithmetic means ⫾ SE (n ⫽ 5– 6 each group) of glomerular sclerosis index (GSI), mesangiolysis score (MSI), and tubulointerstitial damage index (TSI) in gsk3KI (filled bars) and gsk3WT (open bars) mice following a 21-day DOCA/salt treatment. *Significant difference between gsk3KI and gsk3WT mice. ANOVA, paired or unpaired Student’s t-test, and only results with P ⬍ 0.05 were considered statistically significant. RESULTS As reported earlier (5), urinary flow rate and fluid intake were significantly larger in gsk3KI than in gsk3WT mice, an effect persisting under treatment with DOCA (Fig. 1). Creatinine clearance was again significantly higher in gsk3KI than in gsk3WT mice (Fig. 1). The 18-day treatment with DOCA/high salt was followed by a significant increase of both fluid intake and urinary flow rate in both phenotypes. Both values remained significantly larger in gsk3KI than in gsk3WT mice. The treatment tended to decrease creatinine clearance, an effect, however, not reaching statistical significance (Fig. 1). Urinary protein excretion was significantly enhanced in gsk3KI compared with gsk3WT mice (Fig. 2). Similarly, urinary excretion of albumin was larger in gsk3KI than in gsk3WT mice (Fig. 2). Urine electrophoresis again confirmed that urinary protein was largely due to albumin, an observation pointing to glomerular origin of the excreted proteins (Fig. 2). Under control conditions, plasma albumin concentration was not significantly different between the two genotypes. The 18-day treatment with DOCA/high salt resulted in a significant increase of plasma albumin concentration in both genotypes. Following DOCA/high-salt treatment, the plasma albumin concentration was significantly lower in gsk3KI than in gsk3WT mice (Fig. 3). As demonstrated earlier (5), blood pressure was significantly higher in gsk3KI than in gsk3WT mice before DOCA treatment. DOCA/high-salt treatment significantly increased the blood pressure in both genotypes. Following DOCA/high-salt treatment, the blood pressure was again significantly higher in gsk3KI than in gsk3WT mice (Fig. 4). As proteinuria could at least partially have been due to increased blood pressure, a further series of experiments aimed to define the influence of systolic blood pressure on proteinuria. To this end, blood pressure and proteinuria were determined following administration of the ␣1-blocker prazosin. Before the prazosin treatment, the systolic blood pressure was in this series again significantly higher in gsk3KI mice (108.2 ⫾ 0.8 mmHg, n ⫽ 6) than in gsk3WT mice (90.0 ⫾ 1.7 mmHg, AJP-Renal Physiol • VOL n ⫽ 6). Prazosin treatment was followed by a decrease of blood pressure, which was significantly more pronounced in gsk3KI mice (90.7 ⫾ 0.7 mmHg, n ⫽ 5) than in gsk3WT mice (86.4 ⫾ 1.5 mmHg, n ⫽ 4). Before the prazosin treatment, urinary flow rate and urinary protein excretion were in this series again significantly higher in gsk3KI than in gsk3WT mice (Fig. 5). Prazosin treatment significantly decreased the urinary flow rate and urinary protein excretion in gsk3KI mice and abrogated the significant difference between gsk3KI and gsk3WT mice (Fig. 5). Immunofluorescence revealed that both glomerular nephrin and podocin expression were less pronounced in 3-mo-old gsk3KI mice than in 3-mo-old gsk3WT mice (Fig. 6). Nephrin expression has further been determined in 8-mo-old animals without or with DOCA/high-salt treatment. As illustrated in Fig. 7, the DOCA/high-salt treatment decreased the nephrin abundance in both genotypes. Following DOCA/high-salt treatment, nephrin abundance was again lower in gsk3KI than in gsk3WT mice (Fig. 7). Histomorphological analysis was performed following DOCA/high-salt treatment to define the renal injury due to mineralocorticoid/salt excess. The results are illustrated in Fig. 8 and listed in Table 1. Volume densities of the medulla and cortex and mean and total glomerular volume were similar in gsk3KI and gsk3WT mice. Mesangiolysis score (MSI) tended to be higher, and kidney weight, kidney volume, medulla and cortex volume tended to be lower in gsk3KI than in gsk3WT mice. None of these parameters were significantly different between the two genotypes. Glomerular number was moderately but significantly smaller and volume density of the glomerula was significantly higher in gsk3KI than in gsk3WT mice. Most importantly, the glomerular sclerosis index and tubulointerstitial damage index were significantly higher in gsk3KI mice than in gsk3WT mice (Fig. 8). DISCUSSION The present study reveals that mice lacking PKB/SGKdependent regulation of GSK3 activity are not protected against glomerular injury following mineralocorticoid and salt excess. Instead, glomerular integrity is compromised in mice Table 1. Histomorphological data after 18 days of DOCA/ high-salt treatment Parameter Gsk3WT Gsk3KI P GSI 0.86⫾0.04 1.31⫾0.08 0.001* MSI 1.46⫾0.01 1.56⫾0.08 0.234 TSI 0.30⫾0.05 0.58⫾0.03 0.001* Volume density of the medulla, % 35.2⫾0.9 35.8⫾1.3 0.712 Volume density of the cortex, % 64.8⫾0.9 64.2⫾1.3 0.712 Volume density of the glomerula, % 12.7⫾0.5 14.8⫾0.6 0.033* Kidney wt, g 0.26⫾0.02 0.20⫾0.02 0.091 Kidney volume, mm3 250⫾15 197⫾22 0.091 Volume cortex, mm3 163⫾12 127⫾15 0.109 3 Volume medulla, mm 87.5⫾3.2 70.1⫾7.4 0.075 Volume glomerula, mm3 20.5⫾1.7 18.7⫾2.1 0.519 Glomerular number per kidney 20,024⫾470 16,331⫾1,036 0.014* Total glomerular volume, mm3 20.5⫾1.7 18.7⫾2.1 0.519 Mean glomerular volume, 103 ␮m3 1,028⫾84 1,137⫾101 0.438 Arithmetic means ⫾ SE (paraffin sections), n ⫽ 5– 6 each group. *The P value indicates significant difference between GSK3 knockin mice (gskKI ) and their wild-type littermates (gsk3WT). GSI, glomerular sclerosis index; MSI, mesangiolysis score; TSI, tubulointerstitial damage index. 296 • JANUARY 2009 • www.ajprenal.org Downloaded from journals.physiology.org/journal/ajprenal (054.083.151.133) on January 26, 2022. F158 GSK-SENSITIVE PROTEIN EXCRETION carrying a mutation of GSK3␤, in which the serine of the PKB phosphorylation site was replaced by an alanine (GSK3␤9A/9A) and at the same time carrying a mutation of GSK3␣, in which the serine of the PKB phosphorylation site was replaced by an alanine (GSK3␤21A/21A). The mice carrying the PKB/SGKresistant GSK mutants (gsk3KI) have significantly enhanced glomerular injury compared with their wild-type littermates (gsk3WT). Glycogen synthase 3 (GSK3) is phosphorylated and thus inhibited by SGK1 (32) and protein kinase B (8, 33). Inhibition of GSK3 mediates the effect of insulin on glycogen synthase (6, 19). Accordingly, the effect of insulin on muscle glycogen synthase is abrogated in gsk3KI mice (19). Glycogen synthase kinase GSK3␤ further phosphorylates and thus inactivates Snail, a repressor of nephrin transcription (18). Loss of inhibition by PKB/SGK would be expected to rather enhance GSK activity and thus to repress Snail leading to increased expression of nephrin. The present data show the opposite, i.e., a decreased expression of nephrin in gsk3KI mice. Accordingly, the animals are proteinuric even in the absence of DOCA/highsalt treatment. The DOCA/high-salt treatment enhances proteinuria in both genotypes, but again, proteinuria is more pronounced in gsk3KI than in gsk3WT mice. The enhanced proteinuria under DOCA/high-salt treatment again correlates with decreased glomerular nephrin expression in gsk3KI mice. The proteinuria, in turn, correlates with renal glomerular and tubular injury apparent from histomorphological analysis. Even though the decreased nephrin expression in glomerular tissue is suggestive for an intrarenal cause of proteinuria (42), enhanced salt intake and increased blood pressure may, at least under DOCA and high-salt treatment, have contributed to the development of proteinuria. As reported earlier (5), gsk3KI mice have significantly enhanced blood pressure as early as at an age of 3 mo. Lowering of blood pressure by the ␣1antagonist prazosin was followed by a significant decrease of proteinuria in gsk3KI mice, tended to decrease blood pressure in gsk3WT mice, and dissipated the differences in blood pressure as well as proteinuria between gsk3KI and gsk3WT mice. Thus, the increased blood pressure of gsk3KI mice at least contributes to the proteinuria in those mice. Along those lines, proteinuria, paralleling a decrease of glomerular nephrin expression, has been observed in spontaneous hypertensive rats (11). Accordingly, the decreased nephrin expression and excessive proteinuria of gsk3KI mice during DOCA/high-salt treatment may at least partially be due to hypertension. Under control diet, the creatinine clearance was significantly higher in gsk3KI than in gsk3WT mice, a finding which may again be related to the enhanced blood pressure in those mice. However, in those mice, DOCA/high-salt intake tended to decrease creatinine clearance despite a further increase of blood pressure. The effect did, however, not reach statistical significance. A fall in creatinine clearance in response to a DOCA/high salt would be counterintuitive with regard to salt balance. Such a “salt paradox” has been observed previously in rats (39) and patients (22) with type I diabetes. According to micropuncture experiments, the salt paradox in those animals is due to altered tubuloglomerular feedback (37). The mechanisms are independent of renal nerves (4) and ANG II receptor activation (39). The present study did not aim to elucidate the cause for the enhanced blood pressure in gsk3KI mice. Inhibition of GSK3 by AJP-Renal Physiol • VOL lithium has been reported to upregulate endothelial nitric oxide synthase (eNOS) (24). Accordingly, resistance of GSK to the inhibitory effect of PKB/SGK could, at least in theory, decrease eNOS activity and thus favor an increase of blood pressure. Moreover, NOS participates in the regulation of thirst (30) and reduced NOS activity could, in theory, contribute to the increased fluid intake of the gsk3KI mice. On the other hand, the exquisite sensitivity of blood pressure in gsk3KI mice to ␣1-blockade may point to enhanced peripheral sympathetic nerve tone in those mice. In any case, PKB/SGK resistance of GSK does not confer protection of glomerula against injury caused by mineralocorticoid/salt excess. In contrast, lack of PKB/SGK-dependent regulation of GSK3 leads to glomerular and tubular injury. Gene-targeted mice lacking SGK1, on the other hand, are protected against DOCA/high salt-induced renal (1) and cardiac (40) injury. According to the present observations, GSK resistance does not protect against SGK1-mediated renal injury. Along those lines, GSK3␤ phosphorylation did not correlate with cardiac injury following DOCA/high-salt treatment (41). ACKNOWLEDGMENTS The authors acknowledge the technical assistance of E. Faber, M. Klewer, M. Reutelshöfer, S. Söllner, and the meticulous preparation of the manuscript by T. Loch and L. Subasic. GRANTS This study was supported by the Deutsche Forschungsgemeinschaft (GRK 1302/1 and SFB 423, project Z2). REFERENCES 1. Artunc F, Amann K, Nasir O, Friedrich B, Sandulache D, Jahovic N, Risler T, Vallon V, Wulff P, Kuhl D, Lang F. Blunted DOCA/high salt induced albuminuria and renal tubulointerstitial damage in gene-targeted mice lacking SGK1. J Mol Med 84: 737–746, 2006. 2. Asanuma K, Campbell KN, Kim K, Faul C, Mundel P. 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