Kurt Gleich

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.

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.

Limited capacity of proximal tubular proteolysis in mice with proteinuria.

Albuminuria is associated with the additional loss in the urine of small molecular weight proteins normally degraded by the proximal convoluted tubule (PCT), and competition for binding to the megalin/cubilin reuptake system has been considered the likely cause. We have previously reported that deficiency of the intrinsic lysosomal protein Limp-2 causes tubular proteinuria due to reduced fusion of endosomes with lysosomes in the PCT leading to inadequate proteolysis. To determine whether this mechanism also contributes to the tubular proteinuria induced by albumin overload in normal mice, wild-type (WT) mice received daily BSA injections intraperitoneally for 10 days, using untreated Limp-2(-/-) mice as positive controls for inadequate proteolysis. BSA overload induced significant urinary loss of megalin and cubilin ligands in WT mice. Tubular uptake of Alexa-conjugated BSA, administered by intravenous injection, was not reduced in the PCT of mice receiving intraperitoneal BSA. Expression of the tubular protein receptor megalin was also unchanged. There was a delay in proteolysis of reabsorbed proteins in WT mice receiving BSA, evidenced by an increased quantity of retinol-binding protein (RBP) in the kidney cortex, increased basal distribution of endocytosed RBP in cells of the PCT, and persistence of exogenous Alexa-conjugated BSA and RBP after injection. Upregulation of cathepsin L and normal fusion of lysosomes with endosomes were apparently not sufficient to maintain normal clearance of endocytosed proteins. The data suggest that in the presence of competition from albumin overload, reabsorption of filtered proteins is limited by the capacity of lysosomal degradation rather than receptor-mediated endocytosis.

Tubular proteinuria in mice and humans lacking the intrinsic lysosomal protein SCARB2/Limp-2

Deficiency of the intrinsic lysosomal protein human scavenger receptor class B, member 2 (SCARB2; Limp-2 in mice) causes collapsing focal and segmental glomerular sclerosis (FSGS) and myoclonic epilepsy in humans, but patients with no apparent kidney damage have recently been described. We now demonstrate that these patients can develop tubular proteinuria. To determine the mechanism, mice deficient in Limp-2, the murine homolog of SCARB2, were studied. Most low-molecular-weight proteins filtered by the glomerulus are removed in the proximal convoluted tubule (PCT) by megalin/cubilin-dependent receptor-mediated endocytosis. Expression of megalin and cubilin was unchanged in Limp-2(-/-) mice, however, and the initial uptake of injected Alexa Fluor 555-conjugated bovine serum albumin (Alexa-BSA) was similar to wild-type mice, indicating that megalin/cubilin-dependent, receptor-mediated endocytosis was unaffected. There was a defect in proteolysis of reabsorbed proteins in the Limp-2(-/-) mice, demonstrated by the persistence of Alexa-BSA in the PCT compared with controls. This was associated with the failure of the lysosomal protease cathepsin B to colocalize with Alexa-BSA and endogenous retinol-binding protein in kidneys from Limp-2(-/-) mice. The data suggest that tubular proteinuria in Limp-2(-/-) mice is due to failure of endosomes containing reabsorbed proteins to fuse with lysosomes in the proximal tubule of the kidney. Failure of proteolysis is a novel mechanism for tubular proteinuria.

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-KI(S79A) 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-KI(S79A) 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.

Low salt concentrations activate AMP-activated protein kinase in mouse macula densa cells

The energy-sensing kinase AMP-activated protein kinase (AMPK) is associated with the sodium-potassium-chloride cotransporter NKCC2 in the kidney and phosphorylates it on a regulatory site in vitro. To identify a potential role for AMPK in salt sensing at the macula densa, we have used the murine macula densa cell line MMDD1. In this cell line, AMPK was rapidly activated by isosmolar low-salt conditions. In contrast to the known salt-sensing pathway in the macula densa, AMPK activation occurred in the presence of either low sodium or low chloride and was unaffected by inhibition of NKCC2 with bumetanide. Assays using recombinant AMPK demonstrated activation of an upstream kinase by isosmolar low salt. The specific calcium/calmodulin-dependent kinase kinase inhibitor STO-609 failed to suppress AMPK activation, suggesting that it was not part of the signal pathway. AMPK activation was associated with increased phosphorylation of the specific substrate acetyl-CoA carboxylase (ACC) at Ser(79), as well as increased NKCC2 phosphorylation at Ser(126). AMPK activation due to low salt concentrations was inhibited by an adenovirus construct encoding a kinase dead mutant of AMPK, leading to reduced ACC Ser(79) and NKCC2 Ser(126) phosphorylation. This work demonstrates that AMPK activation in macula densa-like cells occurs via isosmolar changes in sodium or chloride concentration, leading to phosphorylation of ACC and NKCC2. Phosphorylation of these substrates in vivo is predicted to increase intracellular chloride and so reduce the effect of salt restriction on tubuloglomerular feedback and renin secretion.

The Thiazide-Sensitive Co-Transporter Promotes the Development of Sodium Retention in Mice with Diet-Induced Obesity.

Background/Aims: Intravascular volume expansion due to sodium retention is involved in the pathogenesis of obesity-related hypertension. Institution of high fat diet (HFD) feeding leads to an initial state of positive sodium balance due to enhanced tubular reabsorption of sodium, but which tubular sodium transporters are responsible for this remains undefined. Methods: C57/Bl6 mice were fed control or HFD for 3 weeks. Blood pressures were recorded by tail cuff method. Sodium transporter expression and phosphorylation were determined by Western blotting. In vivo activity of NCC was determined using natriuretic responses to hydrochlorothiazide. Expression of NCC mRNA was determined using qPCR. Results: At 3 weeks HFD mice had significant weight gains compared to control mice, but blood pressures were not yet elevated. There were no changes in expression or phosphorylation of the bumetanide-sensitive cotransporter, NKCC2, or in expression of subunits of the amiloride-sensitive ion channel, ENaC. However, there were significant increases in mRNA and protein expression of the thiazide-sensitive co-transporter, NCC, in kidneys from HFD mice. Consistent with this, HFD mice had increased in vivo activity of NCC. Conclusions: Increased expression of NCC promotes the sodium loading response to institution of HFD feeding before onset of hypertension.

Activation of AMPK reduces the co-transporter activity of NKCC1.

Abstract 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.

Expression of the transmembrane lysosomal protein SCARB2/Limp-2 in renin secretory granules controls renin release.

Background/Aims: Renin processing and storage is believed to occur in lysosome-like structures in the afferent arteriole. SCARB2/Limp-2 is a transmembrane lysosomal protein responsible for the intracellular trafficking of β-glucocerebrosidase. This study aimed to confirm the expression of SCARB2/Limp-2 in renin secretory granules, and explore its role in renin processing and secretion. Methods: Co-localisation studies of (pro)renin with lysosomal membrane proteins, SCARB2/Limp-2, LAMP-1 and LAMP-2, were performed in mouse and human kidney sections. Intrarenal expression and secretion of (pro)renin in wild-type (WT) and Limp-2-/- mice were compared with and without stimulation. Results: SCARB2/Limp-2, LAMP-1 and LAMP-2 co-localised with (pro)- renin in mouse and human kidney. Plasma renin concentration was increased in Limp-2-/- mice when compared to WT littermates. No change in (pro)renin expression, however, was observed in Limp-2-/- mouse kidney cortex by immunofluorescence microscopy, Western blotting, quantitative RT-PCR or the ultrastructural appearance of renin secretory granules. Acute stimulation of renin release by isoprenaline or hydralazine was similar in WT and Limp-2-/- mice. Following chronic salt restriction, however, immunofluorescence microscopy showed less (pro)renin expressed in Limp-2-/- compared with WT mouse kidneys, and there was significantly less prorenin but not renin by Western blotting in Limp-2-/- mouse kidney cortex, despite no difference in circulating renin levels. Conclusion: Renin secretory granules possess integral lysosomal proteins, confirming that they are indeed modified lysosomes. Limp-2 deficiency leads to a minor increase in circulating renin. Limp-2, however, is not required for acute or chronic stimulation of renin release.

Abnormal Processing of Autophagosomes in Transformed B Lymphocytes from SCARB2-Deficient Subjects.

Abstract Mutations of the intrinsic lysosomal membrane protein SCARB2 cause action myoclonus-renal failure syndrome (AMRF syndrome), a rare disease characterized by renal and neurological manifestations. In this study, examination of Cos7 cells transfected with SCARB2 cDNA derived from two patients with AMRF syndrome showed that the resultant protein was truncated and was not incorporated into vesicular structures, as occurred with full-length SCARB2 cDNA. Mutant SCARB2 protein failed to colocalize with lysosomes and was found in the endoplasmic reticulum or the cytosol indicating a loss of function. Cultured skin fibroblast and Epstein–Barr virus–transformed lymphoblastoid B cell lines (LCLs) were created from these two patients. Despite the loss of SCARB2 function, studies with lysosomal-associated membrane protein (LAMP) 1 and LAMP2 demonstrated normal lysosomal numbers in fibroblasts and LCLs. Immunofluorescence microscopy using anti-LAMP1 and anti-LAMP2 antibodies also showed normal lysosomal structures in fibroblasts. There was no change in the morphology of fibroblasts examined by electron microscopy compared with cells from unaffected individuals. By contrast, LCLs from individuals bearing SCARB2 mutations had large intracellular vesicles that resembled autophagosomes and contained heterogeneous cellular debris. Some of the autophagosomes were seen to be extruding cellular contents into the media. Furthermore, LCLs had elevated levels of microtubule-associated protein light chain 3-II, consistent with increased autophagy. These data demonstrate that SCARB2 mutations are associated with an inability to process autophagosomes in B lymphocytes, suggesting a novel function for SCARB2 in immune function.