Oral delivery of nanomedicine for genetic kidney disease.

Chronic and genetic kidney diseases such as autosomal dominant polycystic kidney disease (ADPKD) have few therapeutic options, and clinical trials testing small molecule drugs have been unfavorable due to low kidney bioavailability and adverse side effects. Although nanoparticles can be designed to deliver drugs directly to the diseased site, there are no kidney-targeted nanomedicines clinically available, and most FDA-approved nanoparticles are administered intravenously which is not ideal for chronic diseases. To meet these challenges of chronic diseases, we developed a biomaterials-based strategy using chitosan particles (CP) for oral delivery of therapeutic, kidney-targeting peptide amphiphile micelles (KMs). We hypothesized that encapsuling KMs into CP would enhance the bioavailability of KMs upon oral administration given the high stability of chitosan in acidic conditions and mucoadhesive properties enabling absorption within the intestines. To test this, we evaluated the mechanism of KM access to the kidneys via intravital imaging and investigated the KM biodistribution in a porcine model. Next, we loaded KMs carrying the ADPKD drug metformin into CP (KM-CP-met) and measured in vitro therapeutic effect. Upon oral administration in vivo, KM-CP-met showed significantly greater bioavailability and accumulation in the kidneys as compared to KM only or free drug. As such, KM-CP-met treatment in ADPKD mice (Pkd1fl/fl;Pax8-rtTA;Tet-O-Cre which develops the disease over 120 days and mimics the slow development of ADPKD) showed enhanced therapeutic efficacy without affecting safety despite repeated treatment. Herein, we demonstrate the potential of KM-CP as a nanomedicine strategy for oral delivery for the long-term treatment of chronic kidney diseases.

Long-term expandable mouse and human-induced nephron progenitor cells enable kidney organoid maturation and modeling of plasticity and disease.

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.

Female Protection Against Diabetic Kidney Disease Is Regulated by Kidney-Specific AMPK Activity.

Reduced kidney AMPK activity is associated with nutrient stress-induced chronic kidney disease (CKD) in male mice. In contrast, female mice resist nutrient stress-induced CKD. The role of kidney AMPK in sex-related organ protection against nutrient stress and metabolite changes was evaluated in diabetic kidney tubule-specific AMPKγ2KO (KTAMPKγ2ΚΟ) male and female mice. In wild-type (WT) males, diabetes increased albuminuria, urinary kidney injury molecule-1, hypertension, kidney p70S6K phosphorylation, and kidney matrix accumulation; these features were not exacerbated with KTAMPKγ2ΚΟ. Whereas WT females had protection against diabetes-induced kidney injury, KTAMPKγ2ΚΟ led to loss of female protection against kidney disease. The hormone 17ß-estradiol ameliorated high glucose-induced AMPK inactivation, p70S6K phosphorylation, and matrix protein accumulation in kidney tubule cells. The mechanism for female protection against diabetes-induced kidney injury is likely via an estrogen-AMPK pathway, as inhibition of AMPK led to loss of estrogen protection to glucose-induced mTORC1 activation and matrix production. RNA sequencing and metabolomic analysis identified a decrease in the degradation pathway of phenylalanine and tyrosine resulting in increased urinary phenylalanine and tyrosine levels in females. The metabolite levels correlated with loss of female protection. The findings provide new insights to explain evolutionary advantages to females during states of nutrient challenges.

Investigation of Basolateral Targeting Micelles for Drug Delivery Applications in Polycystic Kidney Disease.

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/-:TSLargeT mice. Next, we synthesized and characterized the RGD micelles encapsulated with drug combinations and measured their in vitro therapeutic 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.

Modeling kidney development, disease, and plasticity with clonal expandable nephron progenitor cells and nephron organoids.

Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here we report manipulation of p38 and YAP activity creates a synthetic niche that allows the long-term clonal expansion of primary mouse and human NPCs, and induced NPCs (iNPCs) from human pluripotent stem cells. Cultured iNPCs resemble closely primary human NPCs, generating nephron organoids with abundant distal convoluted tubule cells, which are not observed in published kidney organoids. The synthetic niche reprograms differentiated nephron cells into NPC state, recapitulating the plasticity of developing nephron in vivo. Scalability and ease of genome-editing in the cultured NPCs allow for genome-wide CRISPR screening, identifying novel genes associated with kidney development and disease. A rapid, efficient, and scalable organoid model for polycystic kidney disease was derived directly from genome-edited NPCs, and validated in drug screen. These technological platforms have broad applications to kidney development, disease, plasticity, and regeneration.

Association of Longitudinal Urinary Metabolic Biomarkers With ADPKD Severity and Response to Metformin in TAME-PKD Clinical Trial Participants.

Introduction: Dysregulated cellular metabolism contributes to autosomal dominant polycystic kidney disease (ADPKD) pathogenesis. The Trial of Administration of Metformin in Polycystic Kidney Disease (TAME-PKD) tested the effects of metformin treatment over 2 years in adult ADPKD patients with mild-moderate disease severity. Metformin was found to be safe and tolerable with an insignificant trend toward reduced estimated glomerular filtration rate (eGFR) decline compared to placebo. Here we tested whether targeted urinary metabolic biomarkers measured in TAME-PKD participants correlated with disease progression, severity, and metformin treatment in cross-sectional and longitudinal analyses. Methods: Concentrations of total protein, targeted metabolites (lactate, pyruvate, and succinate), and glycolytic enzymes (pyruvate kinase-M2, lactate dehydrogenase-A, and pyruvate dehydrogenase kinase-1) were measured and normalized by creatinine or osmolality in urine specimens and compared with height-adjusted total kidney volume (htTKV) and eGFR at the different study timepoints. Results: In cross-sectional analyses utilizing placebo group data, urinary succinate normalized by creatinine negatively correlated with ln (htTKV), whereas protein excretion strongly positively correlated with ln (htTKV), and negatively correlated with eGFR. Significant time-varying negative associations occurred with eGFR and the lactate/pyruvate ratio and with urine protein normalized by osmolality, indicating correlations of these biomarkers with disease progression. In secondary analyses, urinary pyruvate normalized by osmolality was preserved in metformin-treated participants but declined in placebo over the 2-year study period with a significant between-arm difference, suggesting time-dependent urinary pyruvate changes may serve as a discriminator for metformin treatment effects in this study population. Conclusion: Proteinuria with enhanced glycolytic and reduced oxidative metabolic markers generally correlated with disease severity and risk of progression in the TAME-PKD study population.

In vitro delivery of mTOR inhibitors by kidney-targeted micelles for autosomal dominant polycystic 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 vitro analysis indicated that drug encapsulation into PAMs enhanced the anti-proliferative effect of all three drugs in human CCD cells. Analysis of in vitro biomarkers 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.

Beneficial effects of bempedoic acid treatment in polycystic kidney disease cells and mice.

ADPKD has few therapeutic options. Tolvaptan slows disease but has side effects limiting its tolerability. Bempedoic acid (BA), an ATP citrate-lyase (ACLY) inhibitor FDA-approved for hypercholesterolemia, catalyzes a key step in fatty acid/sterol synthesis important for cell proliferation. BA is activated by very long-chain acyl-CoA synthetase (FATP2) expressed primarily in kidney and liver. BA also activates AMPK. We hypothesized that BA could be a novel ADPKD therapy by inhibiting cyst growth, proliferation, injury, and metabolic dysregulation via ACLY inhibition and AMPK activation. Pkd1-null kidney cell lines derived from mouse proximal tubule (PT) and collecting duct (IMCD) were grown in 2D or 3D Matrigel cultures and treated ± BA, ± SB-204990 (another ACLY inhibitor) or with Acly shRNA before cyst analysis, immunoblotting or mitochondrial assays using MitoSox and MitoTracker staining. Pkd1 fl/fl ; Pax8-rtTA; Tet-O-Cre C57BL/6J mice were induced with doxycycline injection on postnatal days 10 and 11 (P10-P11) and then treated ± BA (30 mg/kg/d) ± tolvaptan (30-100 mg/kg/d) by gavage from P12-21. Disease severity was determined by % total-kidney-weight-to-bodyweight (%TKW/BW) and BUN levels at euthanasia (P22). Kidney and liver homogenates were immunoblotted for expression of key biomarkers. ACLY expression and activity were upregulated in Pkd1-null PT and IMCD-derived cells vs. controls. Relative to controls, both BA and SB-204990 inhibited cystic growth in Pkd1-null kidney cells, as did Acly knockdown. BA inhibited mitochondrial superoxide production and promoted mitochondrial elongation, suggesting improved mitochondrial function. In ADPKD mice, BA reduced %TKW/BW and BUN to a similar extent as tolvaptan vs. untreated controls. Addition of BA to tolvaptan caused a further reduction in %TKW/BW and BUN vs. tolvaptan alone. BA generally reduced ACLY and stimulated AMPK activity in kidneys and livers vs. controls. BA also inhibited mTOR and ERK signaling and reduced kidney injury markers. In liver, BA treatment, both alone and together with tolvaptan, increased mitochondrial biogenesis while inhibiting apoptosis. We conclude that BA and ACLY inhibition inhibited cyst growth in vitro, and BA decreased ADPKD severity in vivo. Combining BA with tolvaptan further improved various ADPKD disease parameters. Repurposing BA may be a promising new ADPKD therapy, having beneficial effects alone and along with tolvaptan.

Effects of Hydrochlorothiazide and Metformin on Aquaresis and Nephroprotection by a Vasopressin V2 Receptor Antagonist in ADPKD: A Randomized Crossover Trial.

BACKGROUND AND OBJECTIVES: The vasopressin V2 receptor antagonist tolvaptan is the only drug that has been proven to be nephroprotective in autosomal dominant polycystic kidney disease (ADPKD). Tolvaptan also causes polyuria, limiting tolerability. We hypothesized that cotreatment with hydrochlorothiazide or metformin may ameliorate this side effect. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We performed a clinical study and an animal study. In a randomized, controlled, double-blind, crossover trial, we included 13 tolvaptan-treated patients with ADPKD. Patients were treated for three 2-week periods with hydrochlorothiazide, metformin, or placebo in random order. Primary outcome was change in 24-hour urine volume. We also measured GFR and a range of metabolic and kidney injury markers. RESULTS: Patients (age 45±8 years, 54% women, measured GFR of 55±11 ml/min per 1.73 m2) had a baseline urine volume on tolvaptan of 6.9±1.4 L/24 h. Urine volume decreased to 5.1 L/24 h (P<0.001) with hydrochlorothiazide and to 5.4 L/24 h (P<0.001) on metformin. During hydrochlorothiazide treatment, plasma copeptin (surrogate for vasopressin) decreased, quality of life improved, and several markers of kidney damage and glucose metabolism improved. Metformin did not induce changes in these markers or in quality of life. Given these results, the effect of adding hydrochlorothiazide to tolvaptan was investigated on long-term kidney outcome in an animal experiment. Water intake in tolvaptan-hydrochlorothiazide cotreated mice was 35% lower than in mice treated with tolvaptan only. Combination treatment was superior to "no treatment" on markers of disease progression (kidney weight, P=0.003 and cystic index, P=0.04) and superior or equal to tolvaptan alone. CONCLUSIONS: Both metformin and hydrochlorothiazide reduced tolvaptan-caused polyuria in a short-term study. Hydrochlorothiazide also reduced polyuria in a long-term animal model without negatively affecting nephroprotection. PODCAST: This article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2022_03_21_CJN11260821.mp3.

Metformin improves relevant disease parameters in an autosomal dominant polycystic kidney disease mouse model.

Autosomal dominant polycystic kidney disease (ADPKD), caused by mutations in the polycystin 1 (PKD1) or polycystin 2 genes, presents with progressive development of kidney cysts and eventual end-stage kidney disease with limited treatment options. Previous work has shown that metformin reduces cyst growth in rapid ADPKD mouse models via inhibition of cystic fibrosis transmembrane conductance regulator-mediated fluid secretion, mammalian target of rapamycin, 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 knockin point mutation in the Pkd1 gene. This mutation causes ADPKD in humans. Pkd1RC/RC male and female mice, which have a slow progression to end-stage kidney disease, received metformin (300 mg/kg/day in drinking water vs. water alone) from 3 to 9 or 12 mo of age. As previously reported, Pkd1RC/RC females had a more severe disease phenotype as compared with males. Metformin treatment reduced the ratio of total kidney weight-to-body weight relative to age-matched and sex-matched untreated controls at both 9 and 12 mo and reduced the cystic index in females at 9 mo. Metformin also increased glomerular filtration rate, 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 versus 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.NEW & NOTEWORTHY Metformin treatment improved ADPKD disease severity in a relevant, slowly progressive ADPKD mouse model that recapitulates a PKD-associated PKD1 mutation. Relative to controls, metformin reduced kidney weight/body weight, cystic index and BUN levels, while improving GFR, blood pressure and anemia. Metformin also reduced key inflammatory and injury markers, along with cell proliferation markers. These findings suggest several beneficial effects of metformin in this ADPKD mouse model, which may help inform new ADPKD therapies in patients.

Association of Baseline Urinary Metabolic Biomarkers with ADPKD Severity in TAME-PKD Clinical Trial Participants.

BACKGROUND: Recent work suggests that dysregulated cellular metabolism may play a key role in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD). The TAME-PKD clinical trial is testing the safety, tolerability, and efficacy of metformin, a regulator of cell metabolism, in patients with ADPKD. This study investigates the cross-sectional association of urinary metabolic biomarkers with ADPKD severity among TAME-PKD trial participants at baseline. METHODS: Concentrations of total protein, targeted metabolites (lactate, pyruvate, succinate, and cAMP), and key glycolytic enzymes (pyruvate kinase M2 [PKM2], lactate dehydrogenase A [LDHA], and pyruvate dehydrogenase kinase 1 [PDK1]) were measured by ELISA, enzymatic assays, and immunoblotting in baseline urine specimens of 95 TAME-PKD participants. These analytes, normalized by urinary creatinine or osmolality to estimate excretion, were correlated with patients' baseline height-adjusted total kidney volumes (htTKVs) by MRI and eGFR. Additional analyses were performed, adjusting for participants' age and sex, using multivariable linear regression. RESULTS: Greater htTKV correlated with lower eGFR (r=-0.39; P=0.0001). Urinary protein excretion modestly correlated with eGFR (negatively) and htTKV (positively). Urinary cAMP normalized to creatinine positively correlated with eGFR. Among glycolytic enzymes, PKM2 and LDHA excretion positively correlated with htTKV, whereas PKM2 excretion negatively correlated with eGFR. These associations remained significant after adjustments for age and sex. Moreover, in adjusted models, succinate excretion was positively associated with eGFR, and protein excretion was more strongly associated with both eGFR and htTKV in patients <43 years old. CONCLUSIONS: Proteinuria correlated with ADPKD severity, and urinary excretion of PKM2 and LDHA correlated with ADPKD severity at baseline in the TAME-PKD study population. These findings are the first to provide evidence in human urine samples that upregulated glycolytic flux is a feature of ADPKD severity. Future analysis may reveal if metformin treatment affects both disease progression and the various urinary metabolic biomarkers in patients throughout the study.

Primary results of the randomized trial of metformin administration in polycystic kidney disease (TAME PKD).

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/1.73m2 per 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.

Generation of patterned kidney organoids that recapitulate the adult kidney collecting duct system from expandable ureteric bud progenitors.

Current kidney organoids model development and diseases of the nephron but not the contiguous epithelial network of the kidney's collecting duct (CD) system. Here, we report the generation of an expandable, 3D branching ureteric bud (UB) organoid culture model that can be derived from primary UB progenitors from mouse and human fetal kidneys, or generated de novo from human pluripotent stem cells. In chemically-defined culture conditions, UB organoids generate CD organoids, with differentiated principal and intercalated cells adopting spatial assemblies reflective of the adult kidney's collecting system. Aggregating 3D-cultured nephron progenitor cells with UB organoids in vitro results in a reiterative process of branching morphogenesis and nephron induction, similar to kidney development. Applying an efficient gene editing strategy to remove RET activity, we demonstrate genetically modified UB organoids can model congenital anomalies of kidney and urinary tract. Taken together, these platforms will facilitate an enhanced understanding of development, regeneration and diseases of the mammalian collecting duct system.

Oral delivery of metformin by chitosan nanoparticles for polycystic kidney disease.

Nanoparticle drug delivery has many advantages over small molecule therapeutics, including reducing off-target side effects and increasing drug potency. However, many nanoparticles are administered parenterally, which is challenging for chronic diseases such as polycystic kidney disease (PKD), the most common hereditary disease worldwide in which patients need continuous treatment over decades. To address this clinical need, we present the development of nanoparticles synthesized from chitosan, a widely available polymer chosen for its ability to improve oral bioavailability. Specifically, we optimized the synthesis parameters of chitosan nanoparticles and demonstrate mucoadhesion and permeation across an intestinal barrier model in vitro. Furthermore, when administered orally to mice, ex vivo imaging of rhodamine-loaded chitosan nanoparticles showed significantly higher accumulation in the intestines compared to the free model drug, as well as 1.3 times higher serum area under the curve (AUC), demonstrating controlled release and improved serum delivery over 24 h. To test its utility for chronic diseases such as PKD, we loaded the candidate PKD drug, metformin, into chitosan nanoparticles, and upon oral administration to a PKD murine model (Pkd1fl/fl;Pax8-rtTA;Tet-O cre), a lower cyst burden was observed compared to free metformin, and was well tolerated upon repeated dosages. Blood urea nitrogen (BUN) and creatinine levels were similar to untreated mice, demonstrating kidney and biocompatibility health. Our study builds upon previous chitosan-based drug delivery approaches, and demonstrates a novel, oral nanoformulation for PKD.

AMPK is activated during lysosomal damage via a galectin-ubiquitin signal transduction system.

Lysosomal damage activates AMPK, a regulator of macroautophagy/autophagy and metabolism, and elicits a strong ubiquitination response. Here we show that the cytosolic lectin LGALS9 detects lysosomal membrane breach by binding to lumenal glycoepitopes, and directs both the ubiquitination response and AMPK activation. Proteomic analyses have revealed increased LGALS9 association with lysosomes, and concomitant changes in LGALS9 interactions with its newly identified partners that control ubiquitination-deubiquitination processes. An LGALS9-inetractor, deubiquitinase USP9X, dissociates from damaged lysosomes upon recognition of lumenal glycans by LGALS9. USP9X's departure from lysosomes promotes K63 ubiquitination and stimulation of MAP3K7/TAK1, an upstream kinase and activator of AMPK hitherto orphaned for a precise physiological function. Ubiquitin-activated MAP3K7/TAK1 controls AMPK specifically during lysosomal injury, caused by a spectrum of membrane-damaging or -permeabilizing agents, including silica crystals, the intracellular pathogen Mycobacterium tuberculosis, TNFSF10/TRAIL signaling, and the anti-diabetes drugs metformin. The LGALS9-ubiquitin system activating AMPK represents a novel signal transduction system contributing to various physiological outputs that are under the control of AMPK, including autophagy, MTOR, lysosomal maintenance and biogenesis, immunity, defense against microbes, and metabolic reprograming. ABBREVIATIONS: AMPK: AMP-activated protein kinase; APEX2: engineered ascorbate peroxidase 2; ATG13: autophagy related 13; ATG16L1: autophagy related 16 like 1; BMMs: bone marrow-derived macrophages; CAMKK2: calcium/calmodulin dependent protein kinase kinase 2; DUB: deubiquitinase; GPN: glycyl-L-phenylalanine 2-naphthylamide; LLOMe: L-leucyl-L-leucine methyl ester; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAP3K7/TAK1: mitogen-activated protein kinase kinase kinase 7; MERIT: membrane repair, removal and replacement; MTOR: mechanistic target of rapamycin kinase; STK11/LKB1: serine/threonine kinase 11; TNFSF10/TRAIL: TNF superfamily member 10; USP9X: ubiquitin specific peptidase 9 X-linked.

Activation of AMP-activated protein kinase during sepsis/inflammation improves survival by preserving cellular metabolic fitness.

The purpose was to determine the role of AMPK activation in the renal metabolic response to sepsis, the development of sepsis-induced acute kidney injury (AKI) and on survival. In a prospective experimental study, 167 10- to 12-week-old C57BL/6 mice underwent cecal ligation and puncture (CLP) and human proximal tubule epithelial cells (TEC; HK2) were exposed to inflammatory mix (IM), a combination of lipopolysaccharide (LPS) and high mobility group box 1 (HMGB1). Renal/TEC metabolic fitness was assessed by monitoring the expression of drivers of oxidative phosphorylation (OXPHOS), the rates of utilization of OXPHOS/glycolysis in response to metabolic stress, and mitochondrial function by measuring O2 consumption rates (OCR) and the membrane potential (Δψm ). Sepsis/IM resulted in AKI, increased mortality, and in renal AMPK activation 6-24 hours after CLP/IM. Pharmacologic activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) or metformin during sepsis improved the survival, while AMPK inhibition with Compound C increased mortality, impaired mitochondrial respiration, decreased OCR, and disrupted TEC metabolic fitness. AMPK-driven protection was associated with increased Sirt 3 expression and restoration of metabolic fitness. Renal AMPK activation in response to sepsis/IM is an adaptive mechanism that protects TEC, organs, and the host by preserving mitochondrial function and metabolic fitness likely through Sirt3 signaling.

AMPK, a Regulator of Metabolism and Autophagy, Is Activated by Lysosomal Damage via a Novel Galectin-Directed Ubiquitin Signal Transduction System.

AMPK is a central regulator of metabolism and autophagy. Here we show how lysosomal damage activates AMPK. This occurs via a hitherto unrecognized signal transduction system whereby cytoplasmic sentinel lectins detect membrane damage leading to ubiquitination responses. Absence of Galectin 9 (Gal9) or loss of its capacity to recognize lumenal glycans exposed during lysosomal membrane damage abrogate such ubiquitination responses. Proteomic analyses with APEX2-Gal9 have revealed global changes within the Gal9 interactome during lysosomal damage. Gal9 association with lysosomal glycoproteins increases whereas interactions with a newly identified Gal9 partner, deubiquitinase USP9X, diminishes upon lysosomal injury. In response to damage, Gal9 displaces USP9X from complexes with TAK1 and promotes K63 ubiquitination of TAK1 thus activating AMPK on damaged lysosomes. This triggers autophagy and contributes to autophagic control of membrane-damaging microbe Mycobacterium tuberculosis. Thus, galectin and ubiquitin systems converge to activate AMPK and autophagy during endomembrane homeostasis.

Baseline Characteristics and Patient-Reported Outcomes of ADPKD Patients in the Multicenter TAME-PKD Clinical Trial.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) has been associated with metabolic disturbances characterized by downregulation of AMP-activated protein kinase (AMPK), a critical sensor of the cellular energy status. Therapeutic activation of AMPK by metformin could inhibit cyst enlargement by inhibition of both the mammalian target of rapamycin pathway and fluid secretion via the CFTR chloride channel. METHODS: We designed a phase-2, randomized, placebo-controlled, clinical trial to assess the safety, tolerability, and efficacy of metformin on total kidney volume in adults without diabetes (age 18-60 years) with ADPKD and eGFR of ≥50 ml/min per 1.73 m2. There were no eligibility criteria relating to kidney volume. In addition to demographics and clinical/family history, baseline parameters included eGFR, total kidney and liver volumes measured by MRI, and patient-reported outcomes were ascertained by the Medical Outcomes Study Short Form-36, the Gastrointestinal Safety Rating Scale, and the HALT-PKD pain questionnaire. RESULTS: We successfully randomized 97 participants recruited from two university-based clinical sites in Baltimore and Boston. The mean age of participants was 41.9 years, 72% were female, and 94% of participants were White. The majority of study participants had early stage disease, with a mean eGFR of 86.8±19.0 ml/min per 1.73 m2. Approximately half of the study participants (48%) were classified as high risk for progression (Mayo imaging classes 1C, 1D, or 1E). There was no correlation between kidney and/or liver size and health-related quality of life (HRQoL) or gastrointestinal symptom severity. CONCLUSIONS: We report successful recruitment in this ongoing, novel, clinical trial of metformin in ADPKD, with a study sample comprising patients with early stage disease and nearly a half of participants considered at high estimated risk for progression. Participants reported a low gastrointestinal symptom burden at baseline, and HRQoL similar to that of the general population, with no differences in symptoms or HRQoL related to organomegaly. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Metformin as a Novel Therapy for Autosomal Dominant Polycystic Kidney Disease (TAME), NCT02656017.

AMPK phosphorylation of the ß1Pix exchange factor regulates the assembly and function of an ENaC inhibitory complex in kidney epithelial cells.

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-ß1 ß1Pix, 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 ß1Pix 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 ß1Pix in vitro. Among several AMPK phosphorylation sites on ß1Pix detected by mass spectrometry, Ser71 was validated as functionally significant. Compared with wild-type ß1Pix, overexpression of a phosphorylation-deficient ß1Pix-S71A mutant attenuated ENaC inhibition and the AMPK-activated interaction of both ß1Pix and Nedd4-2 to 14-3-3 proteins in cortical collecting duct cells. Similarly, overexpression of a ß1Pix-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 ß1Pix is critical for the formation of a ß1Pix/Nedd4-2/14-3-3 complex. With expression of a general peptide inhibitor of 14-3-3-target protein interactions (R18), binding of both ß1Pix 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 ß1Pix at Ser71, which promotes 14-3-3 interactions with ß1Pix and Nedd4-2 to form a tripartite ENaC inhibitory complex, in the mechanism of ENaC regulation by AMPK.

Ex vivo kidney slice preparations as a model system to study signaling cascades in kidney epithelial cells.

Several model systems have been used to study signaling cascades in kidney epithelial cells, including kidney histology after systemic treatments, ex vivo isolated tubule perfusion, epithelial cell lines in culture, kidney micropuncture, and ex vivo kidney slices. We and others have found the ex vivo kidney slice method useful to study the signaling cascades involved in the regulation of kidney transport proteins. In this chapter we describe our adaptations to this classic method for the study of the regulation of kinases and endocytosis in rodent kidney epithelial cells. Briefly, slices are obtained by sectioning of freshly harvested rat or mouse kidneys using a Stadie-Riggs tissue slicer. Alternatively, a vibratome can be used to obtain slices at a more consistent and finer thickness. The harvested kidney and kidney slices are kept viable in either cell culture media or in buffers that mimic physiological conditions equilibrated with 5% CO2 at body temperature (37°C). These buffers keep the slices viable during hours for incubations in the presence/absence of different pharmacological agents. After the incubation period the slices can be used for biochemistry experiments by preparing tissue lysates or for histological evaluation after fixation. Moreover, the fixed slices can be used to evaluate changes in subcellular trafficking of epithelial proteins or endosomes via immunolabeling followed by confocal microscopy. The resulting micrographs can then be used for systematic quantification of protein- or compartment-specific changes in subcellular localization under each condition.