AMP-Activated Protein Kinase (AMPK)-Dependent Regulation of Renal Transport

AMP-activated kinase (AMPK) is a serine/threonine kinase that is expressed in most cells and activated by a high cellular AMP/ATP ratio (indicating energy deficiency) or by Ca2+. In general, AMPK turns on energy-generating pathways (e.g., glucose uptake, glycolysis, fatty acid oxidation) and stops energy-consuming processes (e.g., lipogenesis, glycogenesis), thereby helping cells survive low energy states. The functional element of the kidney, the nephron, consists of the glomerulus, where the primary urine is filtered, and the proximal tubule, Henle’s loop, the distal tubule, and the collecting duct. In the tubular system of the kidney, the composition of primary urine is modified by the reabsorption and secretion of ions and molecules to yield final excreted urine. The underlying membrane transport processes are mainly energy-consuming (active transport) and in some cases passive. Since active transport accounts for a large part of the cell’s ATP demands, it is an important target for AMPK. Here, we review the AMPK-dependent regulation of membrane transport along nephron segments and discuss physiological and pathophysiological implications.


Proximal Tubule
A wide variety of luminal Na + -dependent cotransporters, which are secondary active, are involved in epithelial transport in the proximal tubule. Secondary active transporters utilize the energy of the transmembrane Na + gradient generated by the primary active ATP-consuming Na + /K + -ATPase to facilitate transport of a substrate against its concentration gradient [105,106]. These transporters and the basolateral Na + /K + -ATPase consume substantial amounts of total cellular energy [74,75,107].
Hence, AMPK has been demonstrated to be an important regulator of proximal tubule transport [74,75].

Glucose Transport
Since glucose is freely filtered by the glomerulus, glucose concentration in primary urine is similar to the plasma glucose concentration, whereas excreted urine is usually free of glucose [108][109][110]. The sugar is reabsorbed in the proximal tubule by the Na + -dependent glucose cotransporter 1 and 2 (SGLT1 and 2), the different expression patterns and properties of which ensure total glucose reabsorption as long as the plasma glucose concentration is not abnormally high [89,108]. SGLT2 has a high transport capacity but low affinity for glucose and is predominantly expressed in the kidney, while SGLT1 is also expressed in other tissues including the small intestine. SGLT2 contributes to the reabsorption of up to 90% of filtered glucose [108,109,111,112]. On the other hand, AMPK-regulated SGLT1 [7,92,113] has a low transport capacity but high affinity for glucose and reabsorbs the remaining glucose [108][109][110]114,115]. Glucose leaves the basolateral membrane through passive glucose carriers GLUT1 and GLUT2 [108,[116][117][118]. AMPK activates SGLT1-dependent glucose transport, presumably by stimulating membrane insertion of the cotransporter as observed in colorectal Caco-2 cells [92,119]. In line with this, AMPK activation is associated with increased SGLT1 expression and glucose uptake in cardiomyocytes [113,120]. Although the AMPK-dependent regulation of SGLT1 in the proximal tubule has not explicitly been addressed, it is tempting to speculate that it is similar to other cell types [92,113,119,120]. The regulation of SGLT by AMPK is a doubled-edged sword: on the one hand, SGLT1-dependent reabsorption of glucose in proximal tubular cells requires energy which is generated by β-oxidation of fatty acids to a large extent [121,122]. On the other hand, it prevents the loss of energy-rich glucose [122,123], thereby maintaining the Na + /K + -ATPase-facilitated Na + gradient for Na + -dependent transport and many other cellular processes [75,76]. SGLT1-mediated glucose uptake is linked to the GLUT1-dependent efflux at the basolateral side [108,116]. GLUT1 activity is stimulated by AMPK in various cell types [124][125][126][127][128][129][130][131]. Therefore, it is conceivable that renal GLUT1 might also be regulated by AMPK in order to save energy-providing glucose. In line with this, Baldwin et al. (1997) showed enhanced glucose uptake via GLUT1 in baby hamster kidney cells treated with AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) [132]. Moreover, Sokolovska et al. (2010) reported that metformin, another pharmacological AMPK activator, increased GLUT1 gene expression in rat kidneys [133]. Also, AMPK activation was associated with enhanced activity of GLUT2. These studies, however, found reduced SGLT1 membrane abundance upon AMPK activation, at least in the case of murine intestinal tissue [134,135].

Na + /H + Exchanger Isoform 1
The ubiquitous Na + /H + exchanger isoform 1 (NHE1) participates in cell volume and pH regulation by extruding one cytosolic H + in exchange for one extracellular Na + [136,137]. NHE1 is expressed in all parts of the nephron, including the proximal tubule. However, it cannot be detected in the macula densa and intercalated cells of the distal nephron [136,138,139]. In the proximal tubule, NHE1 is particularly important for HCO 3 − reabsorption [140]. In hypoxia, anaerobic glycolysis is predominant, which results in intracellular accumulation of lactate and H + [90]. Acidosis, however, inhibits glycolysis [90,141,142] and would jeopardize cellular energy generation. AMPK-dependent stimulation of NHE1 activity in human embryonic kidney (HEK) cells therefore helps cells keep up anaerobic glycolysis in oxygen deficiency, as demonstrated by Rotte et al. (2010) [90]. Given that NHE1 is needed for proximal tubular HCO 3 − reabsorption [140], AMPK may help retain HCO 3 − , thereby alleviating acidosis in energy deficiency and hypoxia.

Creatine Transporter
In some organs with high metabolic activity, including skeletal muscle, heart, and brain, creatine is used to refuel cellular ATP levels [143][144][145]. In the proximal tubule, creatine, a small molecule that is freely filtered, is also reabsorbed through secondary active Na + -dependent creatine transporter (CRT) (SLC6A8) [7,75,143,146]. AMPK has been demonstrated to downregulate CRT activity and apical membrane expression in a polarized mouse S3 proximal tubule cell line, presumably through mammalian target of rapamycin signaling [147]. The AMPK-dependent inhibition of CRT may help reduce unnecessary energy expenditure [75]. Conversely, AMPK stimulates CRT-mediated creatine transport in cardiomyocytes [148,149]. This again demonstrates that AMPK effects are tissue-specific [148].
The regulation of phosphate metabolism by AMPK is not restricted to NaPi-IIa: Recently, AMPK was demonstrated to control the formation of bone-derived hormone fibroblast growth factor 23 (FGF23) [56], which induces renal phosphate excretion by extracellular-signal regulated kinases 1/2 (ERK1/2)-mediated degradation of membrane NaPi-IIa [150]. AMPK inhibits FGF23 production in cell culture and in mice [56]. Despite markedly elevated FGF23 serum levels in AMPKα1-deficient mice, renal phosphate excretion was not different from wild-type animals [56]. The same holds true for cellular localization of NaPi-IIa and renal ERK1/2 [56]. Thus, it is possible that AMPK deficiency is paralleled with some FGF23 resistance.

Loop of Henle
2.3.1. Na + -K + -2Cl − Cotransporter The Na + -K + -2Cl − cotransporter (NKCC2), expressed in the thick ascending limb (TAL) of the loop of Henle and macula densa, is required for the generation of a hypertonic medullary interstitium, a mechanism needed for concentrating urine [75,76,88,91]. NKCC2 is a direct substrate of AMPK which phosphorylates it at its stimulatory serine residue Ser-126 [153]. Moreover, exposure of murine macula densa-like cells to low salt leads to AMPK activation and increased NKCC2 phosphorylation [154]. In addition, increased subapical expression (and apparent reduced apical expression) of NKCC2 in the medullary TAL of the loop of Henle along with elevated urinary Na + excretion in AMPKβ1-deficient mice on a normal salt diet were observed [155]. This is in line with AMPK being an important regulator of NKCC2-mediated salt retention in the medullary TAL of Henle [155]. Efe et al. (2016) recently observed markedly increased outer medullary expression of NKCC2 in rats treated with the AMPK activator metformin [156]. However, according to a recent in vivo study by Udwan et al. (2017), a low salt diet induced upregulation of NKCC2 surface expression in mouse kidneys but left AMPK activity unchanged [157]. Therefore, the exact role of AMPK in stimulating NKCC2 remains to be established.

Renal Outer Medullary K + Channel
The apical renal outer medullary K + channel (ROMK) is required for NKCC2 to work properly, as it allows the recirculation of K + ions taken up by NKCC2 into the lumen [75,88]. AMPK is an inhibitor of ROMK by downregulating both channel activity and membrane abundance of the channel protein in a heterologous expression system using Xenopus oocytes [158]. In vivo studies revealed that the AMPK effect on ROMK is relevant for the renal excretion of K + after an acute K + challenge, as upregulation of renal ROMK1 protein expression and the ability of K + elimination were more pronounced in AMPKα1-deficient than in wild-type mice [158].

Cystic Fibrosis Transmembrane Conductance Regulator
The ATP-gated and cyclic AMP (cAMP)-dependent Cl − channel cystic fibrosis transmembrane conductance regulator (CFTR) participates in Cl − secretion and is broadly known for its role in cystic fibrosis, the pathophysiology of which is due to channel malfunction [74][75][76]159]. In the kidney, CFTR contributes to Cl − secretion in the distal tubule and the principal cells of the cortical and medullary collecting ducts [74,75,160]. AMPK has been demonstrated to inhibit CFTR-dependent Cl − conductance in Xenopus oocytes [159] and to decrease CFTR channel activity in the lung [161,162] and colon [163]. cAMP-stimulated cell proliferation and CFTR-dependent Cl − secretion play a decisive role for epithelial cyst enlargement in autosomal dominant polycystic kidney disease (ADPKD) [164]. In line with this, AMPK activation inhibits CFTR in Madin-Darby canine kidney (MDCK) cells [165] as well as decreases cystogenesis in murine models of ADPKD [165,166], suggesting a potential role for pharmacological AMPK activation in the treatment of ADPKD [165,166].

Ca 2+ Transport
Most Ca 2+ is reabsorbed by passive paracellular diffusion along with other ions and water through tight junctions in the proximal tubule and the more distal parts of the nephron [88,167]. Conversely, only 5-10% of filtered Ca 2+ is reabsorbed by transcellular transport involving the apical transient receptor potential vanilloid 5 channel TRPV5 in the distal convoluted tubule [88]: Ca 2+ enters the cell through TRPV5, whereas basolateral Ca 2+ efflux is accomplished by the Na + /Ca 2+ exchanger (NCX) and the Ca 2+ -ATPase [88,167,168]. AMPK has been shown to inhibit NCX and decrease Orai1-mediated SOCE in murine dendritic cells [169]. Therefore, it is tempting to speculate that Ca 2+ reabsorption may be downregulated in the distal tubule in ATP deficiency [169,170]. Indeed, AMPK downregulates Orai1-dependent SOCE in T-lymphocytes [171], endothelial cells [45], and in osteoblast-like cells [56]. Since renal Orai1 activity contributes to kidney fibrosis [172], AMPK-mediated Orai1 downregulation may also be therapeutically desirable.

Voltage-Gated K + Channel
The voltage-gated K + channel (KCNQ1) is important for the cardiovascular system as well as for electrolyte and fluid homeostasis and is expressed in the distal nephron including the collecting duct [170,[187][188][189]. Its exact role is ill-defined, although a contribution to cell volume regulation is postulated [75,187]. Similar to ENaC, AMPK inhibits KCNQ1 via Nedd4-2, as demonstrated in collecting duct principal cells of rat ex vivo kidney slices [187], MDCK cells [190], and Xenopus oocytes [191].

Vacuolar H + -ATPase
The primary active vacuolar H + -ATPase (V-ATPase) is located at the apical membrane of proximal tubule cells and collecting duct type A intercalated cells. It contributes to the regulation of acid-base homeostasis by secreting H + ions into the tubular lumen [76,192,193]. AMPK inhibits the protein kinase A (PKA)-dependent membrane expression of V-ATPase in collecting duct intercalated cells of rat ex vivo kidney slices [193]. Moreover, epididymal proton-secreting clear cells, developmentally related to intercalated cells, exhibit reduced apical membrane abundance of V-ATPase after in vivo perfusion with the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) into rats [194]. It appears to be likely that energy deficiency limits highly energy-consuming primary active H + excretion in the proximal tubule, whereas secondary active NHE1-dependent H + secretion is maintained, thereby keeping up at least anaerobic glycolysis [192]. The opposing effects of AMPK and PKA on V-ATPase expression and activity in kidney intercalated cells can be explained by different phosphorylation sites, as AMPK and PKA phosphorylate the A subunit at Ser-384 and Ser-175, respectively [195,196]. McGuire and Forgac (2018) further demonstrated that AMPK increases lysosomal V-ATPase assembly and activity in HEK293T cells under conditions of energy depletion [197]. In cells depleted of energy, acidification of autophagic intracellular compartments by V-ATPases enables the lysosomal degradation of proteins and lipids to generate energy substrates for ATP production [197,198]. Thus, it appears to be likely that AMPK-regulated V-ATPase activity depends on its concrete cellular localization and function [197].

Water and Urea Handling
AMPK also regulates renal urea and water handling [76,199]. In the inner medullary collecting duct, osmotic gradients are generated by NKCC2 and urea transporter UT-A1 and water is reabsorbed through aquaporin 2 (AQP2) [76,156,199,200]. The concentration of urine requires the antidiuretic hormone vasopressin, which binds to vasopressin type 2 receptors of collecting duct principal cells, resulting in cAMP-mediated activation of PKA and subsequent phosphorylation and apical membrane insertion of AQP2 and UT-A1 [76,156,199]. Congenital nephrogenic diabetes insipidus (NDI) is a disease primarily caused by mutations of vasopressin type 2 receptors that is characterized by renal resistance to vasopressin and limited urine concentrating capacity [156,201]. According to two in vivo studies using rodent models of congenital NDI, the metformin-stimulated AMPK activation ameliorates the ability of the kidney to concentrate urine by increasing the phosphorylation and apical membrane expression of inner medullary AQP2 and UT-A1 [156,202]. In contrast, an ex vivo treatment of rat kidney slices with AICAR led to reduced apical membrane insertion of AQP2 [203]. Moreover, AMPK antagonizes the desmopressin-induced AQP2 phosphorylation in vitro, thus also suggesting an inhibitory function of AMPK on AQP2 regulation [203]. It appears likely that AMPK-independent effects of the pharmacological AMPK agonists contribute to this discrepancy [156,202,203]. Thus, further studies are clearly required.

Reduction of channel activity
Xenopus oocytes [213] Human kidney inner medulla [215] and mouse kidney medulla (basolateral membranes of collecting ducts and TAL of Henle) [216] EAAT3

Conflicts of Interest:
The authors declare no conflict of interest.