The outcome of renal ischemia-reperfusion injury is unchanged in AMPK-β1 deficient mice

AIM: Activation of the master energy-regulator AMP-activated protein kinase (AMPK) in the heart reduces the severity of ischemia-reperfusion injury (IRI) but the role of AMPK in renal IRI is not known. The aim of this study was to determine whether activation of AMPK by acute renal ischemia influences the severity of renal IRI. METHODS: AMPK expression and activation and the severity of renal IRI was studied in mice lacking the AMPK β1 subunit and compared to wild type (WT) mice. RESULTS: Basal expression of activated AMPK, phosphorylayed at αThr¹⁷², was markedly reduced by 96% in AMPK-β1⁻/⁻ mice. Acute renal ischaemia caused a 3.2-fold increase in α1-AMPK activity and a 2.5-fold increase in α2-AMPK activity (P<0.001) that was associated with an increase in AMPK phosphorylation of the AMPK-α subunit at Thr¹⁷² and Ser⁴⁸⁵, and increased inhibitory phosphorylation of the AMPK substrate acetyl-CoA carboxylase. After acute renal ischemia AMPK activity was reduced by 66% in AMPK-β1⁻/⁻ mice compared with WT. There was no difference, however, in the severity of renal IRI at 24-hours between AMPK-β1⁻/⁻ and WT mice, as measured by serum urea and creatinine and histological injury score. In the heart, macrophage migration inhibitory factor (MIF) released during IRI contributes to AMPK activation and protects from injury. In the kidney, however, no difference in AMPK activation by acute ischemia was observed between MIF⁻/⁻ and WT mice. Compared with the heart, expression of the MIF receptor CD74 was found to be reduced in the kidney. CONCLUSION: The failure of AMPK activation to influence the outcome of IRI in the kidney contrasts with what is reported in the heart. This difference might be due to a lack of effect of MIF on AMPK activation and lower CD74 expression in the kidney

Salt and energy balance in the kidney: the role of AMP-activated protein kinase (AMPK)

© 2013 Dr. Suet-Wan ChoyMetabolic diseases such as obesity and diabetes are an important and growing cause of mortality and morbidity. Inhibition of the renin-angiotensin system is important in avoiding complications of these diseases. In the kidney, transepithelial sodium reabsorption is a highly energy consuming process, as it relies on active transport of ions against electrochemical gradients. In the broader clinical context disordered renal sodium handling likely underlies the pathophysiology of hypertension. The association between hypertension and disorders of energy excess such as obesity and type II diabetes mellitus is well established clinically and epidemiologically. The ubiquitously expressed metabolic sensor, AMP-activated protein kinase (AMPK), has emerged as an important homeostatic regulator of energy balance and is involved in integrating a wide variety of cellular processes including the function of membrane-transport proteins. One of the best-known substrates for AMPK is acetyl-Coenzyme A carboxylase (ACC), where phosphorylation by AMPK increases fatty acid oxidation. The renal specific Na/K/2Cl- co-transporter, NKCC2, located in the macula densa within the thick ascending limb of the renal tubule, plays a crucial role in sensing luminal salt concentrations. This underlies the tubuloglomerular feedback system that regulates the glomerular filtration rate through control of afferent arteriole tone and renin release via a number of signalling molecules, including prostaglandins, adenosine, nitric oxide and ATP. Previous work has demonstrated that AMPK is activated by iso-osmolar low salt conditions in a macula densa cell line, phosphorylates NKCC2 and co-localises with NKCC2 in rat kidneys. The underlying cellular and molecular mechanisms involved in AMPK-dependent regulation of metabolism-transport coupling have not been well defined and this thesis examined the role of AMPK in renal tubular salt transport as well as renin synthesis and secretion. The chronic salt restriction model was used as a stimulus for renin secretion in both AMPK β1-/- (mice with a null mutation of the β1 subunit) and ACC1S79A Knock-In mice, which have a Serine →Alanine mutation at the inhibitory phosphosite for AMPK. AMPK activity is increased in response to salt restriction in vivo. It was found that AMPK β1-/- mice have a salt losing phenotype largely due to reduction of NKCC2 surface localisation. Both AMPK β1-/- and ACC1S79A KI mice had an augmented renin response when salt restricted, suggesting that AMPK has a role in negative regulation of renin and that AMPK may regulate renin through ACC. Co-immunoprecipitation studies demonstrated that AMPK, NKCC2 and ACC physically associate, suggesting formation of a multi-molecular signalling complex that senses salt and has a role in renin secretion and fatty acid metabolism. Given that abnormalities in energy metabolism are associated with diseases of energy excess such as obesity and diabetes, the effect of AMPK on glucose homeostasis and diabetic kidney disease was also explored. Overall the work in this thesis substantiates the evidence for AMPK linking energy regulation and sodium reabsorption in the kidney, through renin production. This may be an important contributor to hypertension and fluid retention in metabolic diseases such as diabetes and may prove clinically relevant in the future development of antihypertensive therapies

Absence of the β1 subunit of AMP

Activation of the heterotrimeric energy‐sensing kinase AMP‐activated protein kinase (AMPK) has been reported to improve experimental diabetic kidney disease. We examined the effect of type 1 diabetes in wild‐type (WT) mice and mice lacking the β1 subunit of AMPK (AMPK β1−/− mice), which have reduced AMPK activity in kidneys and other organs. Diabetes was induced using streptozotocin (STZ) and the animals followed up for 4 weeks. Hyperglycaemia was more severe in diabetic AMPK β1−/− mice, despite the absence of any difference in serum levels of insulin, adiponectin and leptin. There was no change in AMPK activity in the kidneys of diabetic WT mice by AMPK activity assay, or phosphorylation of either the αT172 activation site on the α catalytic subunit of AMPK or the AMPK‐specific phosphosite S79 on acetyl CoA carboxylase 1 (ACC1). Phosphorylation of the inhibitory αS485 site on the α subunit of AMPK was significantly increased in the WT diabetic mice compared to non‐diabetic controls. Despite increased plasma glucose levels in the diabetic AMPK β1−/− mice, there were fewer myofibroblasts in the kidneys compared to diabetic WT mice, as evidenced by reduced α‐smooth muscle actin (α‐SMA) protein by Western blot, mRNA by qRT‐PCR and fewer α‐SMA‐positive cells by immunohistochemical staining. Albuminuria was also reduced in the AMPK β1−/− mice. In contrast to previous studies, therefore, myofibroblasts were reduced in the kidneys of AMPK β1−/− diabetic mice compared to diabetic WT mice, despite increased circulating glucose, suggesting that AMPK can worsen renal fibrosis in type 1 diabetes

Absence of the β1 subunit of AMP-activated protein kinase reduces myofibroblast infiltration of the kidneys in early diabetes

Activation of the heterotrimeric energy‐sensing kinase AMP‐activated protein kinase (AMPK) has been reported to improve experimental diabetic kidney disease. We examined the effect of type 1 diabetes in wild‐type (WT) mice and mice lacking the β1 subunit of AMPK (AMPK β1−/− mice), which have reduced AMPK activity in kidneys and other organs. Diabetes was induced using streptozotocin (STZ) and the animals followed up for 4 weeks. Hyperglycaemia was more severe in diabetic AMPK β1−/− mice, despite the absence of any difference in serum levels of insulin, adiponectin and leptin. There was no change in AMPK activity in the kidneys of diabetic WT mice by AMPK activity assay, or phosphorylation of either the αT172 activation site on the α catalytic subunit of AMPK or the AMPK‐specific phosphosite S79 on acetyl CoA carboxylase 1 (ACC1). Phosphorylation of the inhibitory αS485 site on the α subunit of AMPK was significantly increased in the WT diabetic mice compared to non‐diabetic controls. Despite increased plasma glucose levels in the diabetic AMPK β1−/− mice, there were fewer myofibroblasts in the kidneys compared to diabetic WT mice, as evidenced by reduced α‐smooth muscle actin (α‐SMA) protein by Western blot, mRNA by qRT‐PCR and fewer α‐SMA‐positive cells by immunohistochemical staining. Albuminuria was also reduced in the AMPK β1−/− mice. In contrast to previous studies, therefore, myofibroblasts were reduced in the kidneys of AMPK β1−/− diabetic mice compared to diabetic WT mice, despite increased circulating glucose, suggesting that AMPK can worsen renal fibrosis in type 1 diabetes

Novel mechanisms of Na+ retention in obesity: Phosphorylation of NKCC2 and regulation of SPAK/OSR1 by AMPK

Enhanced tubular reabsorption of salt is important in the pathogenesis of obesity-related hypertension, but the mechanisms remain poorly defined. To identify changes in the regulation of salt transporters in the kidney, C57BL/6 mice were fed a 40% fat diet [high-fat diet ( HFD )] or a 12% fat diet ( control diet ) for 14 wk. Compared with control diet-fed mice, HFD-fed mice had significantly greater elevations in weight, blood pressure, and serum insulin and leptin levels. When we examined Na+ transporter expression, Na+-K+-2Cl− cotransporter ( NKCC2 ) was unchanged in whole kidney and reduced in the cortex, Na+-Cl− cotransporter ( NCC ) and α-epithelial Na+ channel ( ENaC ) and γ-ENaC were unchanged, and β-ENaC was reduced. Phosphorylation of NCC was unaltered. Activating phosphorylation of NKCC2 at S126 was increased 2.5-fold. Activation of STE-20/SPS1-related proline-alanine-rich protein kinase ( SPAK )/oxidative stress responsive 1 kinase ( OSR1 ) was increased in kidneys from HFD-fed mice, and enhanced phosphorylation of NKCC2 at T96/T101 was evident in the cortex. Increased activity of NKCC2 in vivo was confirmed with diuretic experiments. HFD-fed mice had reduced activating phosphorylation of AMP-activated protein kinase ( AMPK ) in the renal cortex. In vitro, activation of AMPK led to a reduction in phospho-SPAK/phospho-OSR1 in AMPK+/+ murine embryonic fibroblasts ( MEFs ), but no effect was seen in AMPK−/− MEFs, indicating an AMPK-mediated effect. Activation of the with no lysine kinase/SPAK/OSR1 pathway with low-NaCl solution invoked a greater elevation in phospho-SPAK/phospho-OSR1 in AMPK−/− MEFs than in AMPK+/+ MEFs, consistent with a negative regulatory effect of AMPK on SPAK/OSR1 phosphorylation. In conclusion, this study identifies increased phosphorylation of NKCC2 on S126 as a hitherto-unrecognized mediator of enhanced Na+ reabsorption in obesity and identifies a new role for AMPK in regulating the activity of SPAK/OSR1

Activation of AMPK reduces the co-transporter activity of NKCC1

The co-transporter activity of Na+ -K+ -2Cl 1 (NKCC1) is dependent on phosphorylation. In this study we show the energy-sensing kinase AMPK inhibits NKCC1 activity. Three separate AMPK activators (AICAR, Phenformin and A-769662) inhibited NKCC1 flux in a variety of nucleated cells. Treatment with A-769662 resulted in a reduction of NKCC1T212/T217 phosphorylation, and this was reversed by treatment with the non-selective AMPK inhibitor Compound C. AMPK dependence was confirmed by treatment of AMPK null mouse embryonic fibroblasts, where A-769662 had no effect on NKCC1 mediated transport. AMPK was found to directly phosphorylate a recombinant human-NKCC1 N-terminal fragment (1–293) with the phosphorylated site identified as S77. Mutation of Serine 77 to Alanine partially prevented the inhibitory effect of A-769662 on NKCC1 activity. In conclusion, AMPK can act to reduce NKCC1-mediated transport. While the exact mechanism is still unclear there is evidence for both a direct effect on phosphorylation of S77 and reduced phosphorylation of T212/217

Activation of AMPK reduces the co-transporter activity of NKCC1

The co-transporter activity of Na+-K+-2Cl− 1 ( NKCC1 ) is dependent on phosphorylation. In this study we show the energy-sensing kinase AMPK inhibits NKCC1 activity. Three separate AMPK activators ( AICAR, Phenformin and A-769662 ) inhibited NKCC1 flux in a variety of nucleated cells. Treatment with A-769662 resulted in a reduction of NKCC1T212/T217 phosphorylation, and this was reversed by treatment with the non-selective AMPK inhibitor Compound C. AMPK dependence was confirmed by treatment of AMPK null mouse embryonic fibroblasts, where A-769662 had no effect on NKCC1 mediated transport. AMPK was found to directly phosphorylate a recombinant human-NKCC1 N-terminal fragment ( 1–293 ) with the phosphorylated site identified as S77. Mutation of Serine 77 to Alanine partially prevented the inhibitory effect of A-769662 on NKCC1 activity. In conclusion, AMPK can act to reduce NKCC1-mediated transport. While the exact mechanism is still unclear there is evidence for both a direct effect on phosphorylation of S77 and reduced phosphorylation of T212/217

AMPK couples plasma renin to cellular metabolism by phosphorylation of ACC1

Salt reabsorption is the major energy-requiring process in the kidney, and AMP-activated protein kinase (AMPK) is an important regulator of cellular metabolism. Mice with targeted deletion of the β1-subunit of AMPK (AMPK-β1−/− mice) had significantly increased urinary Na+ excretion on a normal salt diet. This was associated with reduced expression of the β-subunit of the epithelial Na+ channel (ENaC) and increased subapical tubular expression of kidney-specific Na+-K+-2Cl− cotransporter 2 (NKCC2) in the medullary thick ascending limb of Henle. AMPK-β1−/− mice fed a salt-deficient diet were able to conserve Na+, but renin secretion increased 180% compared with control mice. Cyclooxygenase-2 mRNA also increased in the kidney cortex, indicating greater signaling through the macula densa tubular salt-sensing pathway. To determine whether the increase in renin secretion was due to a change in regulation of fatty acid metabolism by AMPK, mice with a mutation of the inhibitory AMPK phosphosite in acetyl-CoA carboxylase 1 [ACC1-knockin (KI)S79A mice] were examined. ACC1-KIS79A mice on a normal salt diet had no increase in salt loss or renin secretion, and expression of NKCC2, Na+-Cl− cotransporter, and ENaC-β were similar to those in control mice. When mice were placed on a salt-deficient diet, however, renin secretion and cortical expression of cyclooxygenase-2 mRNA increased significantly in ACC1-KIS79A mice compared with control mice. In summary, our data suggest that renin synthesis and secretion are regulated by AMPK and coupled to metabolism by phosphorylation of ACC1

AMPK activation by acute renal ischemia in wild type, AMPK-α1^−/− and AMPK-β1^−/− mice.

<p>Lysates (1 mg protein) from control (□ no ischemia) and ischemic (▪ 10 min ischemia) kidneys of WT (C57Bl/6), AMPK-α1^−/− and AMPK-β1^−/− mice were immunoprecipitated with antibodies specific for the α1 and α2 AMPK catalytic subunits (n = 6–8 per group). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029887#s3" target="_blank">Results</a> are shown as mean ± SD. In WT mice AMPK activity was increased by acute renal ischaemia (P<0.001). In AMPK-β1^−/− mice, AMPK activity after acute renal ischaemia was reduced compared to WT for both AMPK-α1 (P<0.001) and AMPK-α2 (P<0.001) isoforms. In AMPK-α1^−/− mice there was no activation of AMPK-α1 by acute renal ischemia, whereas AMPK-α2 was activated by acute renal ischemia (P<0.01) by an amount not different to WT. * P<0.001, ** P<0.01.</p