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Autosomal dominant polycystic kidney disease (ADPKD) is a complex disorder characterized by uncontrolled renal cyst growth, leading to kidney function decline. The multifaceted nature of ADPKD suggests that single-pathway interventions using individual small molecule drugs may not be optimally effective. As such, a strategy encompassing combination therapy that addresses multiple ADPKD-associated signaling pathways could offer synergistic therapeutic results. However, severe off-targeting side effects of small molecule drugs pose a major hurdle to their clinical transition. To address this, we identified four drug candidates from ADPKD clinical trials, bardoxolone methyl (Bar), octreotide (Oct), salsalate (Sal), and pravastatin (Pra), and incorporated them into peptide amphiphile micelles containing the RGD peptide (GRGDSP), which binds to the basolateral surface of renal tubules via integrin receptors on the extracellular matrix. We hypothesized that encapsulating drug combinations into RGD micelles would enable targeting to the basolateral side of renal tubules, which is the site of disease, via renal secretion, leading to superior therapeutic benefits compared to free drugs. To test this, we first evaluated the synergistic effect of drug combinations using the 20% inhibitory concentration for each drug (IC20) on renal proximal tubule cells derived from Pkd1flox/-:TSLargeTmice. Next, we synthesized and characterized the RGD micelles encapsulated with drug combinations and measured their in vitrotherapeutic effects via a 3D PKD growth model. Upon both IV and IP injections in vivo, RGD micelles showed a significantly higher accumulation in the kidneys compared to NT micelles, and the renal access of RGD micelles was significantly reduced after the inhibition of renal secretion. Specifically, both Bar+Oct and Bar+Sal in the RGD micelle treatment showed enhanced therapeutic efficacy in ADPKD mice (Pkd1fl/fl;Pax8-rtTA;Tet-O-Cre) with a significantly lower KW/BW ratio and cyst index as compared to PBS and free drug-treated controls, while other combinations did not show a significant difference. Hence, we demonstrate that renal targeting through basolateral targeting micelles enhances the therapeutic potential of combination therapy in genetic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic kidney disease and is characterized by the formation of renal cysts and the eventual development of end-stage kidney disease. One approach to treating ADPKD is through inhibition of the mammalian target of rapamycin (mTOR) pathway, which has been implicated in cell overproliferation, contributing to renal cyst expansion. However, mTOR inhibitors, including rapamycin, everolimus, and RapaLink-1, have off-target side effects including immunosuppression. Thus, we hypothesized that the encapsulation of mTOR inhibitors in drug delivery carriers that target the kidneys would provide a strategy that would enable therapeutic efficacy while minimizing off-target accumulation and associated toxicity. Toward eventual in vivo application, we synthesized cortical collecting duct (CCD) targeted peptide amphiphile micelle (PAM) nanoparticles and show high drug encapsulation efficiency (>92.6%). In vitroanalysis indicated that drug encapsulation into PAMs enhanced the anti-proliferative effect of all three drugs in human CCD cells. Analysis of in vitrobiomarkers of the mTOR pathway via western blotting confirmed that PAM encapsulation of mTOR inhibitors did not reduce their efficacy. These results indicate that PAM encapsulation is a promising way to deliver mTOR inhibitors to CCD cells and potentially treat ADPKD. Future studies will evaluate the therapeutic effect of PAM-drug formulations and ability to prevent off-target side effects associated with mTOR inhibitors in mouse models of ADPKD.

Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here, manipulation of p38 and YAP activity allowed for long-term clonal expansion of primary mouse and human NPCs and induced NPCs (iNPCs) from human pluripotent stem cells (hPSCs). Molecular analyses demonstrated that cultured iNPCs closely resemble primary human NPCs. iNPCs generated nephron organoids with minimal off-target cell types and enhanced maturation of podocytes relative to published human kidney organoid protocols. Surprisingly, the NPC culture medium uncovered plasticity in human podocyte programs, enabling podocyte reprogramming to an NPC-like state. Scalability and ease of genome editing facilitated genome-wide CRISPR screening in NPC culture, uncovering genes associated with kidney development and disease. Further, NPC-directed modeling of autosomal-dominant polycystic kidney disease (ADPKD) identified a small-molecule inhibitor of cystogenesis. These findings highlight a broad application for the reported iNPC platform in the study of kidney development, disease, plasticity, and regeneration.

The metabolic sensor AMP-activated protein kinase (AMPK) inhibits the epithelial Na+channel (ENaC), a key regulator of salt reabsorption by the kidney and thus total body volume and blood pressure. Recent studies have suggested that AMPK promotes the association of p21-activated kinase-interacting exchange factor-β1β1Pix, 14-3-3 proteins, and the ubiquitin ligase neural precursor cell expressed developmentally downregulated protein (Nedd)4-2 into a complex that inhibits ENaC by enhancing Nedd4-2 binding to ENaC and ENaC degradation. Functional β1Pix is required for ENaC inhibition by AMPK and promotes Nedd4-2 phosphorylation and stability in mouse kidney cortical collecting duct cells. Here, we report that AMPK directly phosphorylates β1Pix in vitro. Among several AMPK phosphorylation sites on β1Pix detected by mass spectrometry, Ser71was validated as functionally significant. Compared with wild-type β1Pix, overexpression of a phosphorylation-deficient β1Pix-S71A mutant attenuated ENaC inhibition and the AMPK-activated interaction of both β1Pix and Nedd4-2 to 14-3-3 proteins in cortical collecting duct cells. Similarly, overexpression of a β1Pix-Δ602–611 deletion tract mutant unable to bind 14-3-3 proteins decreased the interaction between Nedd4-2 and 14-3-3 proteins, suggesting that 14-3-3 binding to β1Pix is critical for the formation of a β1Pix/Nedd4-2/14-3-3 complex. With expression of a general peptide inhibitor of 14-3-3-target protein interactions (R18), binding of both β1Pix and Nedd4-2 to 14-3-3 proteins was reduced, and AMPK-dependent ENaC inhibition was also attenuated. Altogether, our results demonstrate the importance of AMPK-mediated phosphorylation of β1Pix at Ser71, which promotes 14-3-3 interactions with β1Pix and Nedd4-2 to form a tripartite ENaC inhibitory complex, in the mechanism of ENaC regulation by AMPK.

Aquaporin-2 (AQP2) is essential to maintain body water homeostasis. AQP2 traffics from intracellular vesicles to the apical membrane of kidney collecting duct principal cells in response to vasopressin [arginine vasopressin (AVP)], a hormone released with low intravascular volume, which causes decreased kidney perfusion. Decreased kidney perfusion activates AMP-activated kinase (AMPK), a metabolic sensor that inhibits the activity of several transport proteins. We hypothesized that AMPK activation also inhibits AQP2 function. These putative AMPK effects could protect interstitial ionic gradients required for urinary concentration during metabolic stress when low intravascular volume induces AVP release. Here we found that short-term AMPK activation by treatment with 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR; 75 min) in kidney tissue prevented baseline AQP2 apical accumulation in principal cells, but did not prevent AQP2 apical accumulation in response to the AVP analog desmopressin (dDAVP). Prolonged AMPK activation prevented AQP2 cell membrane accumulation in response to forskolin in mouse collecting duct mpkCCDc14cells. Moreover, AMPK inhibition accelerated hypotonic lysis of Xenopusoocytes expressing AQP2. We performed phosphorylation assays to elucidate the mechanism by which AMPK regulates AQP2. Although AMPK weakly phosphorylated immunoprecipitated AQP2 in vitro, no direct AMPK phosphorylation of the AQP2 COOH-terminus was detected by mass spectrometry. AMPK promoted Ser-261 phosphorylation and antagonized dDAVP-dependent phosphorylation of other AQP2 COOH-terminal sites in cells. Our findings suggest an increasing, time-dependent antagonism of AMPK on AQP2 regulation with AICAR-dependent inhibition of cAMP-dependent apical accumulation and AVP-dependent phosphorylation of AQP2. This inhibition likely occurs via a mechanism that does not involve direct AQP2 phosphorylation by AMPK.

Extracellular proton-secreting transport systems that contribute to extracellular pH include the vacuolar H+-ATPase (V-ATPase). This pump, which mediates ATP-driven transport of H+across membranes, is involved in metastasis. We previously showed (Alzamora R, Thali RF, Gong F, Smolak C, Li H, Baty CJ, Bertrand CA, Auchli Y, Brunisholz RA, Neumann D, Hallows KR, Pastor-Soler NM. J Biol Chem285: 24676–24685, 2010) that V-ATPase A subunit phosphorylation at Ser-175 is important for PKA-induced V-ATPase activity at the membrane of kidney intercalated cells. However, Ser-175 is also located within a larger phosphorylation consensus sequence for Aurora kinases, which are known to phosphorylate proteins that contribute to the pathogenesis of metastatic carcinomas. We thus hypothesized that Aurora kinase A (AURKA), overexpressed in aggressive carcinomas, regulates the V-ATPase in human kidney carcinoma cells (Caki-2) via Ser-175 phosphorylation. We found that AURKA is abnormally expressed in Caki-2 cells, where it binds the V-ATPase A subunit in an AURKA phosphorylation-dependent manner. Treatment with the AURKA activator anacardic acid increased V-ATPase expression and activity at the plasma membrane of Caki-2 cells. In addition, AURKA phosphorylates the V-ATPase A subunit at Ser-175 in vitro and in Caki-2 cells. Immunolabeling revealed that anacardic acid induced marked membrane accumulation of the V-ATPase A subunit in transfected Caki-2 cells. However, anacardic acid failed to induce membrane accumulation of a phosphorylation-deficient Ser-175-to-Ala (S175A) A subunit mutant. Finally, S175A-expressing cells had decreased migration in a wound-healing assay compared with cells expressing wild-type or a phospho-mimetic Ser-175-to-Asp (S175D) mutant A subunit. We conclude that AURKA activates the V-ATPase in kidney carcinoma cells via phosphorylation of Ser-175 in the V-ATPase A subunit. This regulation contributes to kidney carcinoma V-ATPase-mediated extracellular acidification and cell migration.

Profilin-1 (Pfn1) is an important regulator of actin polymerization that is downregulated in human breast cancer. Previous studies have shown Pfn1 has a tumor-suppressive effect on mesenchymal-like triple-negative breast cancer cells, and Pfn1-induced growth suppression is partly mediated by upregulation of cell-cycle inhibitor p27kip1(p27). In this study, we demonstrate that Pfn1 overexpression leads to accumulation of p27 through promoting AMPK activation and AMPK-dependent phosphorylation of p27 on T198 residue, a post-translational modification that leads to increased protein stabilization of p27. This pathway is mediated by Pfn1-induced epithelial morphological reversion of mesenchymal breast cancer through cadherin-mediated restoration of adherens junctions. These findings not only elucidate a potential mechanism of how Pfn1 may inhibit proliferation of mesenchymal breast cancer cells, but also highlight a novel pathway of cadherin-mediated p27 induction and therefore cell-cycle control in cells.

Renal hypoxia contributes to chronic kidney disease (CKD) progression, as validated in experimental and human CKD. In the early stages, increased oxygen consumption causes oxygen demand/supply mismatch, leading to hypoxia. Hence, early targeting of the determinants and regulators of oxygen consumption in CKD may alter the disease course before permanent damage ensues. Here, we focus on hypoxia inducible factor-1α (HIF-1α) and AMP-activated protein kinase (AMPK) and on the mechanisms by which they may facilitate cellular hypoxia adaptation. We found that HIF-1α activation in the subtotal nephrectomy (STN) model of CKD limits protein synthesis, inhibits apoptosis, and activates autophagy, presumably for improved cell survival. AMPK activation was diminished in the STN kidney and was remarkably restored by HIF-1α activation, demonstrating a novel role for HIF-1α in the regulation of AMPK activity. We also investigated the independent and combined effects of HIF-1α and AMPK on cell survival and death pathways by utilizing pharmacological and knockdown approaches in cell culture models. We found that the effect of HIF-1α activation on autophagy is independent of AMPK, but on apoptosis it is partially AMPK dependent. The effects of HIF-1α and AMPK activation on inhibiting protein synthesis via the mTOR pathway appear to be additive. These various effects were also observed under hypoxic conditions. In conclusion, HIF-1α and AMPK appear to be linked at a molecular level and may act as components of a concerted cellular response to hypoxic stress in the pathophysiology of CKD.

Ischemia-reperfusion injury (IRI) due to hypotension is a common cause of human acute kidney injury (AKI). Hypoxia-inducible transcription factors (HIFs) orchestrate a protective response in renal endothelial and epithelial cells in AKI models. As human mucin 1 (MUC1) is induced by hypoxia and enhances HIF-1 activity in cultured epithelial cells, we asked whether mouse mucin 1 (Muc1) regulates HIF-1 activity in kidney tissue during IRI. Whereas Muc1 was localized on the apical surface of the thick ascending limb, distal convoluted tubule, and collecting duct in the kidneys of sham-treated mice, Muc1 appeared in the cytoplasm and nucleus of all tubular epithelia during IRI. Muc1 was induced during IRI, and Muc1 transcripts and protein were also present in recovering proximal tubule cells. Kidney damage was worse and recovery was blocked during IRI in Muc1 knockout mice compared with congenic control mice. Muc1 knockout mice had reduced levels of HIF-1α, reduced or aberrant induction of HIF-1 target genes involved in the shift of glucose metabolism to glycolysis, and prolonged activation of AMP-activated protein kinase, indicating metabolic stress. Muc1 clearly plays a significant role in enhancing the HIF protective pathway during ischemic insult and recovery in kidney epithelia, providing a new target for developing therapies to treat AKI. Moreover, our data support a role specifically for HIF-1 in epithelial protection of the kidney during IRI as Muc1 is present only in tubule epithelial cells.

Proximal tubule epithelial cells have a highly sophisticated endocytic machinery to retrieve the albumin in the glomerular filtrate. The megalin-cubilin complex and the endocytic adaptor disabled-2 (Dab2) play a pivotal role in albumin endocytosis. We previously demonstrated that protein kinase B (Akt) regulates albumin endocytosis in the proximal tubule through an interaction with Dab2. Here, we examined the nature of Akt-Dab2 interaction. The pleckstrin homology (PH) and catalytic domains (CD) of Akt interacted with the proline-rich domain (PRD) of Dab2 based on yeast-two hybrid (Y2H) experiments. Pull-down experiments utilizing the truncated constructs of Dab2 demonstrated that the initial 11 amino acids of Dab2-PRD were sufficient to mediate the interaction between Akt and Dab2. Endocytosis experiments utilizing Akt1- and Akt2-silencing RNA revealed that both Akt1 and Akt2 mediate albumin endocytosis in proximal tubule epithelial cells; therefore, Akt1 and Akt2 may play a compensatory role in albumin endocytosis. Furthermore, both Akt isoforms phosphorylated Dab2 at Ser residues 448 and 449. Ser-to-Ala mutations of these Dab2 residues inhibited albumin endocytosis and resulted in a shift in location of Dab2 from the peripheral to the perinuclear area, suggesting the physiological relevance of these phosphorylation sites in albumin endocytosis. We conclude that both Akt1 and Akt2 are involved in albumin endocytosis, and phosphorylation of Dab2 by Akt induces albumin endocytosis in proximal tubule epithelial cells. Further delineation of how Akt affects expression/phosphorylation of endocytic adaptors and receptors will enhance our understanding of the molecular network triggered by albumin overload in the proximal tubule.

The role of phosphorylation in ADAM17-dependent shedding is controversial. We show that the A1adenosine receptor stimulates exocytosis in umbrella cells by a pathway that requires phosphorylation of ADAM17–Ser-811, followed by HB-EGF shedding and EGF receptor transactivation. Preventing ADAM17 phosphorylation blocks these downstream events.

The vacuolar H+-ATPase (V-ATPase) in intercalated cells contributes to luminal acidification in the kidney collecting duct and nonvolatile acid excretion. We previously showed that the A subunit in the cytoplasmic V1sector of the V-ATPase (ATP6V1A) is phosphorylated by the metabolic sensor AMP-activated protein kinase (AMPK) in vitro and in kidney cells. Here, we demonstrate that treatment of rabbit isolated, perfused collecting ducts with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) inhibited V-ATPase-dependent H+secretion from intercalated cells after an acid load. We have identified by mass spectrometry that Ser-384 is a major AMPK phosphorylation site in the V-ATPase A subunit, a result confirmed by comparing AMPK-dependent phosphate labeling of wild-type A-subunit (WT-A) with that of a Ser-384-to-Ala A subunit mutant (S384A-A) in vitro and in intact HEK-293 cells. Compared with WT-A-expressing HEK-293 cells, S384A-A-expressing cells exhibited greater steady-state acidification of HCO3−-containing media. Moreover, AICAR treatment of clone C rabbit intercalated cells expressing the WT-A subunit reduced V-ATPase-dependent extracellular acidification, an effect that was blocked in cells expressing the phosphorylation-deficient S384A-A mutant. Finally, expression of the S384A-A mutant prevented cytoplasmic redistribution of the V-ATPase by AICAR in clone C cells. In summary, direct phosphorylation of the A subunit at Ser-384 by AMPK represents a novel regulatory mechanism of the V-ATPase in kidney intercalated cells. Regulation of the V-ATPase by AMPK may couple V-ATPase activity to cellular metabolic status with potential relevance to ischemic injury in the kidney and other tissues.

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)S79Amice] were examined. ACC1-KIS79Amice 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-KIS79Amice 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.

The coupling of epithelial transport to underlying metabolic status is critical because solute transport processes normally consume a large proportion of total cellular energy. Recently, AMP-activated protein kinase (AMPK) has emerged as a critical transport regulator in tissues throughout the body. This review summarizes the role of AMPK in the regulation of renal epithelial transport, updates the growing list of AMPK transport protein targets and regulatory mechanisms, and discusses the potential clinical significance of this regulation in normal and disease states.

Galectins (Gal) are β-galactoside-binding proteins that function in epithelial development and homeostasis. An overlapping role for Gal-3 and Gal-7 in wound repair was reported in stratified epithelia. Although Gal-7 was thought absent in simple epithelia, it was reported in a proteomic analysis of cilia isolated from cultured human airway, and we recently identified Gal-7 transcripts in Madin-Darby canine kidney (MDCK) cells (Poland PA, Rondanino C, Kinlough CL, Heimburg-Molinaro J, Arthur CM, Stowell SR, Smith DF, Hughey RP. J Biol Chem286: 6780–6790, 2011). We now report that Gal-7 is localized exclusively on the primary cilium of MDCK, LLC-PK1(pig kidney), and mpkCCDc14(mouse kidney) cells as well as on cilia in the rat renal proximal tubule. Gal-7 is also present on most cilia of multiciliated cells in human airway epithelia primary cultures. Interestingly, exogenous glutathione S-transferase (GST)-Gal-7 bound the MDCK apical plasma membrane as well as the cilium, while the lectin Ulex europeausagglutinin, with glycan preferences similar to Gal-7, bound the basolateral plasma membrane as well as the cilium. In pull-down assays, β1-integrin isolated from either the basolateral or apical/cilia membranes of MDCK cells was similarly bound by GST-Gal-7. Selective localization of Gal-7 to cilia despite the presence of binding sites on all cell surfaces suggests that intracellular Gal-7 is specifically delivered to cilia rather than simply binding to surface glycoconjugates after generalized secretion. Moreover, depletion of Gal-7 using tetracycline-induced short-hairpin RNA in mpkCCDc14cells significantly reduced cilia length and slowed wound healing in a scratch assay. We conclude that Gal-7 is selectively targeted to cilia and plays a key role in surface stabilization of glycoconjugates responsible for integrating cilia function with epithelial repair.

The KCNQ1 K+channel plays a key role in the regulation of several physiological functions, including cardiac excitability, cardiovascular tone, and body electrolyte homeostasis. The metabolic sensor AMP-activated protein kinase (AMPK) has been shown to regulate a growing number of ion transport proteins. To determine whether AMPK regulates KCNQ1, we studied the effects of AMPK activation on KCNQ1 currents in Xenopus laevisoocytes and collecting duct epithelial cells. AMPK activation decreased KCNQ1 currents and channel surface expression in X. laevisoocytes, but AMPK did not phosphorylate KCNQ1 in vitro, suggesting an indirect regulatory mechanism. As it has been recently shown that the ubiquitin-protein ligase Nedd4-2 inhibits KCNQ1 plasma membrane expression and that AMPK regulates epithelial Na+channels via Nedd4-2, we examined the role of Nedd4-2 in the AMPK-dependent regulation of KCNQ1. Channel inhibition by AMPK was blocked in oocytes coexpressing either a dominant-negative or constitutively active Nedd4-2 mutant, or a Nedd4-2 interaction-deficient KCNQ1 mutant, suggesting that Nedd4-2 participates in the regulation of KCNQ1 by AMPK. KCNQ1 is expressed at the basolateral membrane in mouse polarized kidney cortical collecting duct (mpkCCDc14) cells and in rat kidney. Treatment with the AMPK activators AICAR (2 mM) or metformin (1 mM) reduced basolateral KCNQ1 currents in apically permeabilized polarized mpkCCDc14cells. Moreover, AICAR treatment of rat kidney slices ex vivo induced AMPK activation and intracellular redistribution of KCNQ1 from the basolateral membrane in collecting duct principal cells. AICAR treatment also induced increased ubiquitination of KCNQ1 immunoprecipitated from kidney slice homogenates. These results indicate that AMPK inhibits KCNQ1 activity by promoting Nedd4-2-dependent channel ubiquitination and retrieval from the plasma membrane.

The metabolic sensor AMP-activated protein kinase (AMPK) regulates several transport proteins, potentially coupling transport activity to cellular stress and energy levels. The creatine transporter (CRT; SLC6A8) mediates creatine uptake into several cell types, including kidney epithelial cells, where it has been proposed that CRT is important for reclamation of filtered creatine, a process critical for total body creatine homeostasis. Creatine and phosphocreatine provide an intracellular, high-energy phosphate-buffering system essential for maintaining ATP supply in tissues with high energy demands. To test our hypothesis that CRT is regulated by AMPK in the kidney, we examined CRT and AMPK distribution in the kidney and the regulation of CRT by AMPK in cells. By immunofluorescence staining, we detected CRT at the apical pole in a polarized mouse S3 proximal tubule cell line and in native rat kidney proximal tubules, a distribution overlapping with AMPK. Two-electrode voltage-clamp (TEV) measurements of Na+-dependent creatine uptake into CRT-expressing Xenopus laevisoocytes demonstrated that AMPK inhibited CRT via a reduction in its Michaelis-Menten Vmaxparameter. [14C]creatine uptake and apical surface biotinylation measurements in polarized S3 cells demonstrated parallel reductions in creatine influx and CRT apical membrane expression after AMPK activation with the AMP-mimetic compound 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside. In oocyte TEV experiments, rapamycin and the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranosyl 5′-monophosphate (ZMP) inhibited CRT currents, but there was no additive inhibition of CRT by ZMP, suggesting that AMPK may inhibit CRT indirectly via the mammalian target of rapamycin pathway. We conclude that AMPK inhibits apical membrane CRT expression in kidney proximal tubule cells, which could be important in reducing cellular energy expenditure and unnecessary creatine reabsorption under conditions of local and whole body metabolic stress.

The ultrasensitive energy sensor AMP-activated protein kinase (AMPK) orchestrates the regulation of energy-generating and energy-consuming pathways. AMPK is highly expressed in the kidney where it is reported to be involved in a variety of physiological and pathological processes including ion transport, podocyte function, and diabetic renal hypertrophy. Sodium transport is the major energy-consuming process in the kidney, and AMPK has been proposed to contribute to the coupling of ion transport with cellular energy metabolism. Specifically, AMPK has been identified as a regulator of several ion transporters of significance in renal physiology, including the cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial sodium channel (ENaC), the Na+-K+-2Cl−cotransporter (NKCC), and the vacuolar H+-ATPase (V-ATPase). Identified regulators of AMPK in the kidney include dietary salt, diabetes, adiponectin, and ischemia. Activation of AMPK in response to adiponectin is described in podocytes, where it reduces albuminuria, and in tubular cells, where it reduces glycogen accumulation. Reduced AMPK activity in the diabetic kidney is associated with renal accumulation of triglyceride and glycogen and the pathogenesis of diabetic renal hypertrophy. Acute renal ischemia causes a rapid and powerful activation of AMPK, but the functional significance of this observation remains unclear. Despite the recent advances, there remain significant gaps in the present understanding of both the upstream regulating pathways and the downstream substrates for AMPK in the kidney. A more complete understanding of the AMPK pathway in the kidney offers potential for improved therapies for several renal diseases including diabetic nephropathy, polycystic kidney disease, and ischemia-reperfusion injury.

The vacuolar H+-ATPase (V-ATPase) in type A kidney intercalated cells is a major contributor to acid excretion in the collecting duct. The mechanisms of V-ATPase-trafficking regulation in kidney intercalated cells have not been well-characterized. In developmentally related epididymal clear cells, we showed previously that PKA, acting downstream of soluble adenylyl cyclase (sAC), induces V-ATPase apical membrane accumulation. These PKA-mediated effects were inhibited by activators of the metabolic sensor AMP-activated kinase (AMPK) in clear cells. Here, we examined the regulation of V-ATPase subcellular localization in intercalated cells by PKA and AMPK in rat kidney tissue slices ex vivo. Immunofluorescence labeling of kidney slices revealed that the PKA activator N6-monobutyryl cAMP (6-MB-cAMP) induced V-ATPase apical membrane accumulation in collecting duct intercalated cells, whereas the V-ATPase had a more cytosolic distribution when incubated in Ringer buffer alone for 30 min. V-ATPase accumulated at the apical membrane in intercalated cells in kidney slices incubated in Ringer buffer for 75 min, an effect that was prevented by treatment with PKA inhibitor (mPKI). The V-ATPase distribution was cytosolic in intercalated cells treated with the carbonic anhydrase inhibitor acetazolamide or the sAC inhibitor KH7, effects that were overridden by 6-MB-cAMP. Preincubation of kidney slices with an AMPK activator blocked V-ATPase apical membrane accumulation induced by 6-MB-cAMP, suggesting that AMPK antagonizes cAMP/PKA effects on V-ATPase distribution. Taken together, our results suggest that in intercalated cells V-ATPase subcellular localization and therefore its activity may be coupled to acid-base status via PKA, and metabolic status via AMPK.

It has long been recognized that the coupling of membrane transport to underlying cellular metabolic status is critical because transport processes consume a large portion of total cellular energy. Recently, the finely tuned metabolic sensor AMP-activated protein kinase (AMPK) has emerged as a membrane transport regulator, which may permit sensitive transport-metabolism crosstalk. This review will discuss how AMPK may play an important role in the regulation of ion and solute transport across the plasma membrane under both physiological and pathological conditions in epithelia and other tissues.

We measured changes in the deformability of human pro myelocytic leukemic (HL-60) cells induced to differentiate for 5–6 days along the granulocyte pathway by 1.25 % dimethylsulfoxide (DMSO). Differentiation resulted in an approximately 90 % reduction in the transit times of the cells through capillary-sized pores over a range of aspiration pressures. Cell volume, as measured by two methods, decreased by an average of 35 %. To account for the contribution of the volume decrease to the decrease in transit time, the liquid drop model, developed to describe neutrophil deformability, was used to calculate an apparent viscosity of the cells during this deformation. The apparent viscosity of both uninduced and induced HL-60 cells was a function of aspiration pressure, and an approximately 80 % reduction in viscosity occurred with induction, as determined by regression analysis. The deformation rate-dependent viscosities of the induced cells were between 65 and 240 Pa-sec, values similar to those measured for circulating neutrophils. To assess the role of polymerized actin in these viscosity changes, intracellular F-actin content was measured, and the effect of dihydrocytochalasin B (DHB), an agent that disrupts actin polymerization, was determined. Despite the significant decrease in cellular viscosity, F-actin content per cell volume did not change significantly after induced differentiation. Treatment with 3 and 30 μM DHB lowered cellular F-actin content in a dose-dependent manner in both uninduced and induced cells. Cellular viscosity of both uninduced and induced cells decreased sharply with 3 μM DHB treatment (85 % and 76 % respectively). 30 μM DHB treatment caused a further significant reduction in the viscosity of uninduced cells, but for induced cells the additional decrease in viscosity was not significant. These data indicate that reductions in both cell volume and intrinsic viscosity contribute to the increased deformability of HL-60 cells with DMSO-induced differentiation. However, changes in the concentration of F-actin cannot account for the decrease in cellular viscosity that occurs.

Autosomal dominant polycystic kidney disease (ADPKD), caused by mutations in the PKD1 or PKD2 genes encoding polycystins, presents with progressive development of kidney cysts and eventual end-stage kidney disease (ESKD) with limited treatment options. Previous work showed that metformin reduces cyst growth in rapid ADPKD mouse models via inhibition of CFTR-mediated fluid secretion, mTOR, and cAMP pathways. The present study importantly tested the effectiveness of metformin as a therapy for ADPKD in a more clinically relevant Pkd1RC/RC mouse model, homozygous for the R3277C knock-in point mutation in the Pkd1 gene. This mutation causes ADPKD in humans. Pkd1RC/RC male and female mice, which have slow progression to ESKD, received metformin (300 mg/kg/day in drinking water vs. water alone) from 3 to 9 or 12 months of age. As previously reported, Pkd1RC/RC females had a more severe disease phenotype than males. Metformin treatment reduced the ratio of total kidney weight to body weight relative to age- and sex-matched untreated controls at both 9 and 12 months and reduced cystic index in females at 9 months. Metformin also increased glomerular filtration rate (GFR), lowered systolic blood pressure, improved anemia, and lowered blood urea nitrogen levels relative to controls in both sexes. Moreover, metformin reduced gene expression of key inflammatory markers and both gene and protein expression of kidney injury marker-1 and cyclin-dependent kinase-1 vs. untreated controls. Altogether, these findings suggest several beneficial effects of metformin in this highly relevant slowly progressive ADPKD mouse model, which may help inform new ADPKD therapies in patients.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by growth of kidney cysts and glomerular filtration rate (GFR) decline. Metformin was found to impact cystogenesis in preclinical models of polycystic disease, is generally considered safe and may be a promising candidate for clinical investigation in ADPKD. In this phase 2 two-year trial, we randomly assigned 97 patients, 18-60 years of age, with ADPKD and estimated GFR over 50 ml/min/1.73 m2, in a 1:1 ratio to receive metformin or placebo twice daily. Primary outcomes were medication safety and tolerability. Secondary outcomes included estimated GFR decline, and total kidney volume growth. Thirty-eight metformin and 39 placebo participants still received study product at 24-months. Twenty-one participants in the metformin arm reduced drug dose due to inability to tolerate, compared with 14 in the placebo arm (not significant). Proportions of participants experiencing serious adverse events was similar between the groups. The Gastrointestinal Symptoms Rating Scale score was low at baseline and did not significantly change over time. The annual change for estimated GFR was -1.71 with metformin and -3.07 ml/min/1.73m2per year with placebo (mean difference 1.37 {-0.70, 3.44} ml/min/1.73m2), while mean annual percent change in height-adjusted total kidney volume was 3.87% in metformin and 2.16% per year in placebo, (mean difference 1.68% {-2.11, 5.62}). Thus, metformin in adults with ADPKD was found to be safe and tolerable while slightly reducing estimated GFR decline but not to a significant degree. Hence, evaluation of efficacy requires a larger trial, with sufficient power to detect differences in endpoints.