Metabolic Energetics and Drug Metabolism in the Kidneys PDF

Metabolic Energetics and Drug Metabolism in the Kidneys 1 Metabolic Energetics and Drug Metabolism in the Kidneys Lawrence H. Lash, Ph.D. Professor, Department of Pharmacology Office: 7312 Scott Hall T: 1-313-577-0475; E-mail: [email protected] Learning Objectives: 1. Rec...

Metabolic Energetics and Drug Metabolism in the Kidneys 1 Metabolic Energetics and Drug Metabolism in the Kidneys Lawrence H. Lash, Ph.D. Professor, Department of Pharmacology Office: 7312 Scott Hall T: 1-313-577-0475; E-mail: [email protected] Learning Objectives: 1. Recognize the importance of energy metabolism as a critical underlying process for sustaining kidney function. 2. Apply knowledge of substrate supply and regulation of respiratory rates in the kidneys to determining how nutritional status can influence kidney function. 3. Apply knowledge of regulation of redox processes in mitochondrial and kidney function to explain the responses of mitochondria to nutritional and other environmental factors and to identify potential therapeutic targets in various forms of kidney injury and disease. 4. Apply knowledge of the mechanisms by which mitochondrial biogenesis and function are regulated to explain the responses of mitochondria to nutritional and other environmental factors and to identify potential therapeutic targets in various forms of kidney injury and disease. 5. Apply knowledge of the responses of mitochondria during aging to understand kidney functional changes in aging.  Metabolic Energetics and Drug Metabolism in the Kidneys 2 Lecture Outline I. Introduction. A. Why is it important to understand energetics in the kidneys to understand normal kidney function? B. Why is it important to understand drug metabolism in the kidneys to understand normal kidney function? C. Why is it important to understand drug transport in the kidneys to understand normal kidney function? II. Substrate supplies and respiratory rates in different kidney regions. III. Drug metabolism and metabolic heterogeneity in different nephron cell types. IV. Redox balance and renal mitochondrial function. V. Mitochondrial biogenesis as a pharmacological target. A. Mitochondrial turnover, fission and fusion; biogenesis B. Mechanistic (Mammalian) Target of Rapomycin (mTOR) and Adenosine Monophosphate-Activated Kinase (AMPK) C. Sirtuins VI. Aging and kidney function. VII. Summary & Conclusions.  Metabolic Energetics and Drug Metabolism in the Kidneys 3 I. Introduction. A. Why is it important to understand energetics in the kidneys to understand normal kidney function? The kidneys are second only to the heart in terms of O2 consumption. Relative to other organs, the kidneys receive a very high blood flow based on relative organ weight, although O2 extraction by the kidneys is relatively low. However, the kidneys are particularly susceptible to hypoxic injury; role of renal hypoxia in the development and progression of both acute and chronic renal disease is of interest. About 95% of the ATP in the kidneys is supplied by oxidative metabolism, and thus renal mitochondria play an integral role in maintaining energy-requiring processes. Specific dependence of several important renal functions on ATP, and on the interrelationship between these functions and ATP production in the kidneys: Renal Na+ pump (i.e., the Na++K+-ATPase on the basolateral plasma membrane. Parallel alterations observed between the Q02 (respiration rate) and Na+ transport: rate of ATP production by the mitochondria is tightly coupled to Figure 1. Relationship between renal ATP the rate of ATP consumption by consumption and ATP synthesis in the Na+ pump. Thus, the Na+ mitochondria. pump acts as a ‘pacemaker’ for cellular respiration. Tissue-specific synthesis of key proteins: e.g., erythropoietin, Vitamin D receptor. The kidneys can be a gluconeogenic tissue.  Metabolic Energetics and Drug Metabolism in the Kidneys 4 B. Why is it important to understand drug metabolism in the kidneys to understand normal kidney function? Many drugs, especially anionic and cationic drugs, undergo renal excretion. The kidneys, particularly the proximal tubules, possess significant drug metabolism activities, similar to hepatocytes: CYP enzymes, FMOs, GSTs, UGTs, and SULTs. Significant species-dependent differences in renal drug metabolism; implication is that experimental data in other mammalian species (e.g., rats and mice) need to be carefully extrapolated to humans. Translational relevance: Renal drug metabolism is a critical factor in drug disposition and may impact efficacy and toxicity. Genetic polymorphisms in drug-metabolizing enzymes à Individual differences. C. Why is it important to understand drug transport in the kidneys to understand normal kidney function? The kidneys, in particular the proximal tubular cells, possess a large array of plasma membrane transporters that can mediate uptake, intracellular accumulation, or excretion of both drugs and many endogenous chemicals. Figure 2. Transporters for organic anions and cations in the human renal proximal tubular cell. Schematic of major basolateral and brush-border (apical) plasma membrane transporters for organic anions and cations in human proximal tubular cells. Abbreviations: MATE, multidrug and toxic compound extrusion; MRP, multidrug resistance protein; OA–, organic anion; OAT, organic anion transporter; OATP, organic anion transporting polypeptide; OC+, organic cation; OCT, organic cation transporter; OCTN, organic cation and carnitine transporter; P-gp, P-glycoprotein; URAT, uric acid transporter.  Metabolic Energetics and Drug Metabolism in the Kidneys 5 3 key processes involved in renal handling of drugs and metabolites: Glomerular filtration Reabsorption Secretion Plasma membrane transport processes are primary determinants of reabsorption and secretion. Translational relevance: Competition between drugs for specific renal transporters can lead to important drug-drug interactions that impact drug efficacy or drug toxicity. Competition between drugs and endogenous metabolites (e.g., uric acid) can lead to acute kidney injury (AKI). II. Substrate supplies and respiratory rates in different kidney regions. Contrary to expectation, glucose is a rather poor fuel of respiration in kidney cortex; preferred fuels are short- and long-chain fatty acids, endogenous lipids, ketone bodies, lactate, and some amino-acids. Primary fuel for respiration in outer stripe of the outer medulla (OSOM), inner stripe of outer medulla (ISOM), and medulla is very different from that in cortex. Renal carbohydrate metabolism: simultaneous presence of glycolytic and gluconeogenic pathways. The proximal convoluted tubule (PCT) does not metabolize glucose to any appreciable extent. The thick ascending limb of Henle’s loop (TALH) and the distal convoluted tubule (DCT), on the other hand, have a high capacity for glucose (and lactate) oxidation. The collecting-tubule system exhibits the highest capacities of aerobic glycolysis (lactate formation in the presence of oxygen) but simultaneously performs oxidative metabolism. Summary points: Heterogeneity of metabolic activity along the nephron. Glycolysis is linked closely to free-water clearance and possibly to sodium, potassium, and hydrogen ion transport.  Metabolic Energetics and Drug Metabolism in the Kidneys 6 Glucose oxidation, while not the major source of renal energy, is crucial in sodium, potassium, and phosphate reabsorption. Gluconeogenesis recovers carbon compounds generated during the process of renal ammoniagenesis. The complex network of biosynthetic and catabolic pathways of glucose metabolism may have evolved in the kidney to protect the organism against wide variations in glucose demand which would otherwise be unavoidable during the course of rapidly fluctuating renal electrolyte loads. III. Drug metabolism and metabolic heterogeneity in different nephron cell types. Cytoplasmic and mitochondrial enzymes of gluconeogenesis found exclusively in the PCT and proximal straight tubule (PST), with lower activities in the latter portion. Even under maximal stimulation, no gluconeogenic enzymes are found in the distal nephron segments. Na,K-ATPase: When normalized to tubule length, activity is highest in the DCT, TALH, and the PCT. The lowest activity was measured in the thin limb, and intermediate levels of activity were present in the collecting duct and PST. Heterogeneity of mitochondrial energy production in the kidney: As might be expected on the basis of the differences in the transport activity along the nephron, the distribution of mitochondria also varies. The density of mitochondrial volume (mitochondrial volume per unit volume of cytoplasm) decreases from 33% to 22% along the proximal tubule (PCT to PST). Along the distal portion of the nephron it decreases from 44%, its highest level (found in the medullary TALH), to 31 % in the DCT. The density in the thin limbs is only 6-8%, and the collecting tubule has about 20 and 10% in the cortical and medullary segments, respectively. Using the amount of mitochondrial cristae membrane surface and calculating the number of membrane respiratory chain units and their consumption of O2, an upper limit for the rate of ATP formation in the different segments of the nephron can be estimated. Proximal segments synthesized 14.2 µmol ATP/min, the thick limb and distal segments together produced 5.9 µmol/min, and the contribution from the remaining portions of the nephron was less than 1 µmol/min. The sum for the entire kidney was 21.0 µmol/min, which compares favorably with the rate of 18-36 µmol/min estimated from the rates of O2 consumption of the kidney as a whole.  Metabolic Energetics and Drug Metabolism in the Kidneys 7 Figure 3. Renal regional, cellular, and subcellular distribution of key drug-metabolizing enzymes. The proximal tubules are the predominant site within nephrons for most drug metabolism. Includes all the major enzymes of Phase I and Phase II drug metabolism. Selective localization of some drug-metabolism enzymes in other nephron segments. Metabolic Energetics and Drug Metabolism in the Kidneys 8 Important role of drug-metabolism enzymes in generation of metabolites critical for roles of kidneys in regulation of fluid balance and blood pressure: HETEs and EETs derived from action of CYPs on arachidonic acid (AA) released by cytoplasmic phospholipase A2 (cPLA2) action on plasma membrane lipids. Some species-dependent differences in renal CYPs. Roles in blood pressure illustrated in Figures 5 and 6.  Metabolic Energetics and Drug Metabolism in the Kidneys 9 Point to Remember: CYPs are not only important in drug metabolism but in metabolism of endogenous chemicals as well.  Metabolic Energetics and Drug Metabolism in the Kidneys 10 Summary of selected metabolic and functional differences in key nephron segments:  Metabolic Energetics and Drug Metabolism in the Kidneys 11 IV. Redox balance and renal mitochondrial function. Figure 7. Central role of renal mitochondria in oxidative stress and the antioxidant defense system. [from Bhargava and Schnellmann (2017) Mitochondrial energetics in the kidney. Nature Rev. Nephrol. 13, 629-646.] Renal mitochondria can be both targets for and sources of reactive oxygen species (ROS). Insults (e.g., toxic chemicals, reactive metabolites of drugs) can increase the production of ROS in the cytoplasm and mitochondria. NADPH oxidase 2 (NOX2) and NOX4 can also contribute to the production of ROS. ROS à breaks in mitochondrial DNA (mtDNA), damage to lipids and proteins. Damaged mtDNA à aberrant mitochondrial proteins;inhibits mitochondrial protein synthesis.  Metabolic Energetics and Drug Metabolism in the Kidneys 12 Damaged lipids and proteins à impaired mitochondrial function à further increases in mitochondrial ROS. ROS also activate nuclear factor erythroid 2-related factor 2 (NRF2), which translocates to the nucleus and binds to antioxidant-responsive elements (AREs) to activate the transcription of genes encoding antioxidant enzymes, such as mitochondrial superoxide dismutase 2 (SOD2), glutathione peroxidase (GPX) and catalase. SOD2 reduces superoxide anions to hydrogen peroxide (H2O2) and oxygen (O2). Catalase, found in peroxisomes, and GPX, located in the cytoplasm and mitochondria, reduce H2O2 to water. GPX also oxidizes glutathione (GSH), resulting in glutathione disulfide (GSSG) as a byproduct of reducing hydrogen peroxide to water. GSSG in mitochondria (mGSSG) is converted back to GSH by glutathione reductase (GR) in a process that requires the presence of NADPH. The activity of the mitochondrial uncoupling protein 2 (UCP2) is increased, dissipating the proton motive force and decreasing ROS production. mGSH, mitochondrial GSH. The electron transport chain complexes I–V are indicated as I, II, III, IV and V, and can be significant sites of ROS formation. V. Mitochondrial biogenesis as a pharmacological target. A. Mitochondrial turnover, fission and fusion; biogenesis. Mitochondrial homeostasis requires a fine-tuned balance between mitochondrial dynamics and mitochondrial energetics to ensure proper functioning of mitochondria. Mitochondria can adapt to different metabolic conditions by the regulation of mechanistic (mammalian) target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) nutrient sensing pathways. External stimuli can augment mitochondrial processes, such as mitophagy, fission, and fusion, and mitochondrial biogenesis to modulate levels of ATP production. Fission: the splitting of mitochondria in two; fission isolates damaged mitochondria from the mitochondrial network; once isolated, damaged mitochondria are targeted for mitophagy. Fusion: the combining of two mitochondria; leads to the elongation of mitochondria under physiological conditions, helping to maintain oxidative phosphorylation. Mitophagy: a process by which mitochondria are degraded.  Metabolic Energetics and Drug Metabolism in the Kidneys 13 Mitochondrial biogenesis: production of new, functional mitochondria; serves to increase ATP production in response to increasing energy demands. Mitochondrial biogenesis is regulated by a range of transcriptional co-activators and co- repressors (Figure 8). Figure 8. Activation and regulation of mitochondrial biogenesis. [Source: Bhargava and Schnellmann (2017) Nature Rev. Nephrol. 13, 629-646.] Activation of peroxisome proliferator-activated receptor g-co-activator 1a (PGC- 1a) in the cytoplasm causes its translocation to the nucleus and the transcription of various genes, including that for mitochondrial transcription factor A (TFAM), whose protein products are needed for oxidative phosphorylation (Oxphos), the tricarboxylic acid (TCA) cycle, and mitochondrial biogenesis. Activation of G-protein coupled receptors (GPCRs) (e.g., b2-adrenergic receptors (b2AR) and 5-hydroxytryptamine receptor 1F (5-HT1F)) leads to dissociation of heterotrimeric G proteins composed of Ga, Gb, and Gg subunits and the subsequent activation of protein kinase A (PKA) and endothelial nitric oxide synthase (eNOS). Although the pathway from GPCRs to eNOS is still unclear (indicated by dashed line), eNOS stimulates soluble guanylate cyclase (sGC) to form cGMP, which in  Metabolic Energetics and Drug Metabolism in the Kidneys 14 turn activates PGC-1a. cGMP can be converted to GMP by cGMP-specific 3’,5’- cyclic phosphodiesterase (PDE5). Several chemicals can activate nuclear receptors such as peroxisome proliferator-activated receptors (PPARs) and estrogen-related receptors (ERRs) to induce mitochondrial biogenesis. Once activated, these nuclear receptors can act as transcriptional co-activators (labeled as nuclear receptor transcription factors (NRTFs)) with PGC-1a to stimulated mitochondrial biogenesis. Other transcription factors, including nuclear respiratory factor 1 (NRF1) and NRF2, can also directly bind to PGC-1a to induce mitochondrial biogenesis. Stimuli such as caloric restriction, can activate eNOS, increasing levels of cGMP, thereby activating PGC-1a. Activity of sirtuin 1 (SIRT1) is increased by high NAD+:NADH ratios, leading to activation of PGC-1a. High AMP:ATP ratios also activate AMP-activated protein kinase (AMPK), leading to activation of PGC-1a by phosphorylation. B. Mechanistic (Mammalian) Target of Rapomycin (mTOR) and Adenosine Monophosphate-Activated Kinase (AMPK). mTOR: serine-threonine kinase complexes that comprise several proteins; two complexes exist: mTORC1 and mTORC2. mTORC1 proteins regulate cell growth and proliferation and inhibits autophagy by stimulating anabolic (i.e. biosynthetic) processes. mTORC2 proteins are believed to regulate K+ and Na+ levels in the kidneys. mTORC1: Acts as a nutrient sensor by being activated by growth factors, nutrients such as amino acids and glucose, and oxidative stress, triggering pathways that lead to increased protein synthesis, nucleotide synthesis, lipid synthesis, and mitochondrial biogenesis via activation of PGC-1a. The mTOR pathway can be inhibited by hypoxia and AMPK. AMPK acts as a nutrient sensor in the kidneys and stimulates catabolic (i.e., degradative) processes. High cellular ratios of AMP:ATP in the presence of low O2 concentrations leads to activation of AMPK. AMPK phosphorylates several proteins, leading to production of antioxidant enzymes, induction of mitochondrial biogenesis, increase in glycolysis, fatty acid oxidation, and glucose transport. AMPK stimulates mitochondrial biogenesis by phosphorylating PGC-1a, thereby increasing its activity. AMPK stimulates the production of cellular energy and inhibits energy-consuming pathways by inhibiting mTORC1.  Metabolic Energetics and Drug Metabolism in the Kidneys 15 C. Sirtuins. Sirtuins 1-7 are a family of evolutionarily-conserved, NAD+-dependent deacetylases that regulate histone proteins at specific lysine residues, promoting post-translational modification that results in chromatin silencing and transcriptional repression. The dependence of sirtuins on the cellular levels of the coenzyme NAD+ links sirtuin activity to energy metabolism. SIRT1: SIRT1 has garnered the most interest because of its positive regulation by NAD+ and its ability to act as a positive transcriptional regulator of PGC-1a and other mitochondrial-associated genes through promoter deacetylation. Resveratrol, a stilbenoid found in red wine, has been extensively studied as a SIRT1 activator and stimulator of mitochondrial biogenesis. Resveratrol has been shown to enhance mitochondrial biogenesis and oxidative metabolism, and to be protective in animal models of cardiovascular disease, neurodegeneration, and metabolic syndrome.  Metabolic Energetics and Drug Metabolism in the Kidneys 16 Mammals express seven sirtuins (SIRT1-7) that are localized in different subcellular compartments. SIRT1 is in the nucleus and regulates both nucleosome histone acetylation and the activity of several transcriptional factors. SIRT1 inhibits TNFa-dependent transactivation of NF-kB, limiting the expression of several proinflammatory genes. After DNA damage and oxidative stress, SIRT1-dependent deacetylation of p53 as well as forkhead box type O transcription factors (FoxO), results in reduced cell apoptosis and senescence. SIRT1 also regulates the activity and expression of hypoxia-inducible factor-2a, which is responsible for the hypoxic induction of erythropoietin (EPO) by renal cells. From a metabolic point of view, SIRT1 binds to and represses genes regulated by PPAR-g after food withdrawal and also controls gluconeogenesis and mitochondria biogenesis by deacetylating/activating PGC-1a. SIRT3 and other mitochondrial sirtuins: SIRT3 is the major regulator of the whole organelle acetylome. For example, within the mitochondrial electron transport chain, SIRT3 directly binds to and regulates complex I, succinate dehydrogenase A of complex II, and ATP synthase (complex V), thus powerfully boosting ATP levels. SIRT3 plays a major role in the regulation of mitochondrial antioxidant pathways and detoxification through de-acetylation and activation of SOD2, activation of isocitrate dehydrogenase 2, deacetylation of acetyl-CoA synthase 2 and glutamate dehydrogenase, which fuels the urea cycle. Moreover, SIRT3 promotes beta-oxidation by driving long-chain acyl CoA dehydrogenase activity, and ketone body generation by promoting the deacetylation of 3-hydroxy-3- methylglutaryl CoA synthase 2. Mitochondrial SIRT4 is predominantly an ADP-ribosylase and has the opposite effects to SIRT3, in that it inactivates the enzymes involved in the urea cycle and b-oxidation but induces lipogenesis through malonyl CoA decarboxylase deacetylation. SIRT4 governs the cellular metabolic response to DNA damage via glutamine metabolism inhibition, suggesting a role as a tumor suppressor. SIRT5 was initially described as a mitochondrial deacetylase that regulates the urea cycle through the direct activation of carbamoyl phosphate synthetase 1. Subsequent studies showed that SIRT5 also exhibits demalonylase and desuccinylase activities, through which it controls ketogenesis. SIRT5 also induces energetic flux via glycolysis.  Metabolic Energetics and Drug Metabolism in the Kidneys 17 VI. Aging and kidney function. Figure 10. Changes occurring in the aging kidney. The aging kidney loses nephrons and mass; in turn, the remaining nephrons compensate for this loss by undergoing hypertrophic adaptation. The figure illustrates driving mechanisms for the aging-associated macroscopic and microscopic findings and the regenerative failure seen with older age. The histologic features interstitial fibrosis and tubular atrophy (IF/TA), glomerulosclerosis (GS), and microvascular rarefaction are partly interdependent. The importance of cellular senescence for tubular changes has been nicely demonstrated, but a contribution of cellular senescence to microvascular changes through endothelial or vascular senescence is also conceivable. Pericyte loss and an imbalance of pro- and antiangiogenic factors further contribute to the vascular phenotype with age. The age-related reduction in renal microvasculature may be an independent cause of nephron loss but could be also secondary to GS. Podocyte hypertrophy and the associated dysfunction are driving GS. SASP, senescence-associated secretory phenotype. [Source: Schmitt, R., and Melk, A. (2017) Molecular mechanisms of renal aging. Kidney Int. 92, 569-579.]  Metabolic Energetics and Drug Metabolism in the Kidneys 18 Figure 11. Molecular pathways present in renal epithelial cells. Autophagy is important for the aging phenotype. With age, the capacity to activate autophagy is reduced, and downregulation of this essential catabolic process results in impaired degradation of intracellular material. Positive effects of caloric restriction (CR) and the mammalian target of rapamycin (mTOR) inhibitor rapamycin are described in the text. Cellular senescence resulting from cumulative damage can be derived from signaling through telomere-p53 or the p16INK4a pathway eventually resulting hypophosphorylated retinoblastoma (Rb). Permanently arrested senescent cells are still viable and metabolically active. The presence of the associated cell’s secretory phenotype called SASP (senescence-associated secretory phenotype) converts them into potentially destructive cells. The inflammatory factors of the SASP act in paracrine fashion initially by recruiting immune cells, which destroy the microenvironment, and secondly by altering the function of neighboring cells. The SASP also induces functional changes in the secreting cell itself through autocrine action. ARF, alternate reading frame protein; ATM, ataxia telangiectasia–mutated; CDK, cyclin-dependent kinase; IL, interleukin; ROS, reactive oxygen species; TNF-a, tumor necrosis factor-a. [Source: Schmitt, R., and Melk, A. (2017) Molecular mechanisms of renal aging. Kidney Int. 92, 569-579.]  Metabolic Energetics and Drug Metabolism in the Kidneys 19 VII. Summary & Conclusions. Mitochondrial function is critical for maintenance of kidney function: o (Na++K+)-ATPase (i.e., Na+-pump) activity o Glomerular filtration o Protein synthesis (e.g., EPO, vitamin D receptor) o Gluconeogenesis in proximal tubules Renal mitochondria can adapt to energy consumption / energy demands. Renal drug metabolism is a critical factor in drug disposition and may impact efficacy and toxicity. The kidneys, in particular the proximal tubular cells, possess a large array of energy-dependent, plasma membrane transporters that can mediate uptake, intracellular accumulation, or excretion of both drugs and many endogenous chemicals; potential site of drug-drug interactions. Metabolic and energetic heterogeneity in the nephron: o The proximal tubules are the predominant site within nephrons for most drug metabolism. o Includes all the major enzymes of Phase I and Phase II drug metabolism. o Selective localization of some drug-metabolism enzymes in other nephron segments. o Glucose metabolism and mitochondrial function differs amongst cell types of nephron. CYP-dependent metabolism of arachidonic acid important in renal regulation of blood pressure and fluid homeostasis. Redox balance an important modulator of mitochondrial function. Regulation of mitochondrial biogenesis enables response to changes in energy needs of kidneys: o mTOR and AMPK o Sirtuins o PGC-1a Sirtuins as pharmacologic (therapeutic) targets to improve renal function and/or protect from tubular injury. Renal aging: o Nephron loss o Cellular senescence: Cell cycle changes o Cellular hypertrophy o Immunological and inflammatory responses