Solute Transport, Energy Consumption, and Production in the Kidney

Na ⁺ Transport and O ₂ Consumption

The energy utilized in the kidney is primarily required for the active reabsorption of Na ⁺ from the glomerular filtrate. This seems rational considering that the amount of Na ⁺ reabsorbed by the kidney is much higher than that of HCO ₃ ⁻ (4,000 mmole/day), Ca ²⁺ (210 mmole/day), other electrolytes, and organic substances. The active Na ⁺ transport also energizes the reabsorption of water and other solutes by the osmotic gradient generated by Na ⁺ transport, and by the electrochemical gradient of Na ⁺ across the plasma membrane.

The data shown in Figures 6.1 and 6.2 indicate another important point. Extrapolation of the regression line to the Y ordinate indicates the basal oxygen consumption ( Figure 6.1 ). This value is identical to the energy used for cellular functions other than Na ⁺ transport, and was estimated to be between 3 to 18%, while other studies indicated a higher value for this basal O ₂ consumption in the kidney.

With regard to energy production, the kidney generates approximately 95% of the ATP produced via aerobic mechanism, while in some nephron segments anaerobic metabolism also occurs efficiently. This is reasonable because of the highly efficient ATP production by mitochondrial oxidative phosphorylation compared to anaerobic glycolysis. Thirty-six moles of ATP are generated by the mitochondrial oxidative phosphorylation of one mole of glucose, whereas only two moles of ATP are produced via glycolysis in the absence of O ₂ . Thus, historically, the relationship between Na transport and O ₂ consumption (QO ₂ ) in renal tissues has been extensively investigated.

Several investigators have examined Na ⁺ /O ₂ stoichiomety in the kidney. Thurau demonstrated a linear relationship between Na ⁺ transport and QO ₂ with a ratio of 28 Na ⁺ /O ₂ in the whole dog kidney ( Figure 6.1 ). This stoichiometry is equal to a ratio of 4.6 for Na ⁺ /ATP, which indicates more efficient transport in the kidney compared to that in the frog skin and toad bladder. The discrepancy has been studied by many investigators and is attributed to the following.

First, there are alternative pathways for Na ⁺ transport in the nephron. In the leaky epithelia, such as proximal tubule (PT), paracellular Na ⁺ transport occurs, while only transepithelial transport is possible in the tight epithelia in cortical collecting duct (CCD) cells. Second, coupling of Na ⁺ transport to that of other solutes occurs, such as bicarbonate transport in PT, and Na ⁺ -K ⁺ -2Cl ⁻ transport via NKCC2 in the thick ascending limb of Henle (TAL). These two mechanisms produce more efficient Na ⁺ transport in certain nephron segments compared to that observed in frog skin and toad bladder, where only cellular transport of Na ⁺ driven by Na ⁺ ,K ⁺ -ATPase occurs. The different Na ⁺ /O ₂ /ATP stoichiometry among individual nephron segments will be described below.

Proximal Tubule

In the PT, the Na ⁺ /QO ₂ was reported to be 24 to 30, corresponding to a Na ⁺ /ATP ratio of 4 to 5. In the early portion of the PT (S1), preferential absorption of bicarbonate occurs, resulting in a rise in the luminal Cl ⁻ concentration, defined as axial anionic asymmetry. In the basolateral membrane of proximal tubular cells, a Na ⁺ -bicarbonate co-transporter (NBC) extrudes Na ⁺ with HCO ₃ ⁻ with a stoichiometry of 1: 2~3. The electrochemical gradient of HCO ₃ ⁻ across the plasma membrane in the PT cells, which is generated by the coordinated function of carbonic anhydrase and H ⁺ -ATPase or Na ⁺ -H ⁺ exchanger, drives Na ⁺ efflux via NBC. Because PT is more permeable to Cl ⁻ than to HCO ₃ ⁻ , a driving force develops for isotonic fluid transport. It was suggested that 30% to 50% of Na ⁺ transport in the PT is passive, and not directly related to ATP consumption. In addition, in the early part of the PT, glucose, amino acids, and phosphate are actively reabsorbed by the Na ⁺ -co-transport mechanism, thereby rapidly reducing their concentration in the lumen. This luminal hypotonicity also contributes to the solvent drag which involves Na ⁺ . Thus, in the PT, more than 3 Na ⁺ could be transported via the hydrolysis of one mole of ATP.

Thick Ascending Limb of Henle

In the TAL, the most efficient sodium transport occurs, with the Na ⁺ -K ⁺ -2Cl ⁻ co-transporter (NKCC2) playing an important role. The transport by NKCC2 is electrically neutral, and the K ⁺ reabsorbed by NKCC2 in the TAL is leaked back into the tubular lumen via the ROMK channel in the apical membrane. This K ⁺ leakage results in a positive electrical potential difference in the lumen, which drives paracellular transport of Na ⁺ from the lumen to the plasma ( Figure 6.3 ). Although no direct measurement has been made of the Na ⁺ /O ₂ /ATP stoichiometry in the TAL, the results obtained in the rectal gland and tracheal epithelium indicated that the Na ⁺ /ATP ratio could theoretically be up to 6 in the TAL.

CCD

In the CCD, the efficiency of Na ⁺ transport is the lowest among the nephron segments. In this segment, the junction between the epithelia is very tight, and paracellular transport is minimal. In addition, Na ⁺ entry from the luminal side into CCD cells is mediated only by ENaC, which is not associated with any coupled transport other than that of Na ⁺ . Thus, the Na ⁺ :ATP ratio in the CCD is estimated to be 3.

P-type-ATPases

P-type-ATPases ( Figure 6.4 ), including Na ⁺ ,K ⁺ -ATPase, H ⁺ ,K ⁺ -ATPase, and Ca ²⁺ -ATPase, share several common features: (1) they possess a seven amino acid motif with aspartate to which ATP binds; (2) they are transiently phosphorylated during the cation transport cycle (the term P-type derives from this transient phosphorylation); and (3) they catalyze cation transport between E ₁ and E ₂ conformations (P-type-ATPase was previously called E ₁ -E ₂ -ATPase). The transport activity of P-type-ATPases is commonly inhibited by vanadate.

Na ⁺ ,K ⁺ -ATPase

Na ⁺ ,K ⁺ -ATPase extrudes 3 Na ⁺ and takes up 2 K ⁺ across the plasma membranes through the hydrolysis of one ATP molecule in the presence of Mg ²⁺ . Na ⁺ ,K ⁺ -ATPase generates an inwardly-directed Na ⁺ gradient and inside a negative electrical gradient. Na ⁺ ,K ⁺ -ATPase accounts for approximately half of the total Na ⁺ reabsorption in the kidney.

Na ⁺ ,K ⁺ -ATPase is a heterodimeric integral membrane protein, with a minimal composition of α- and β-subunits. The α-subunit possesses ten membrane-spanning domains with a molecular mass of approximately 100 kDa. The β-subunit is a glycosylated type II membrane protein with a molecular weight of approximately 55 kDa. In mammalian genomes, four α-subunits and at least three β-subunits of Na ⁺ ,K ⁺ -ATPase have been identified. In addition, a γ-subunit, a member of the FXYD family of type II transmembrane proteins, constitutes an Na ⁺ ,K ⁺ -ATPase. In the kidney, two γ-subunit isoforms are expressed. The combination of each isoform comprises a number of Na ⁺ ,K ⁺ -ATPase isozymes that are expressed in a tissue- and cell-specific manner to evolve distinct properties to respond to cellular requirements. In the kidney, α1β1 is predominantly expressed.

The α-subunit is the catalytic subunit of the Na ⁺ ,K ⁺ -ATPase, and α1, α2, and α3 isoforms differ in their affinities for ATP, Na ⁺ and K ⁺ . β-subunits are suggested to facilitate the correct membrane integration and packing of the α-subunit, and β-subunits also participate in the determination of the intrinsic transport properties of Na ⁺ ,K ⁺ -ATPase. The γ-subunit was shown to be a specific regulator of renal α1β1 isozymes. A putative dominant-negative mutation in the gene encoding the γ-subunit (FXYD2) which leads to defective routing of the protein in a family with dominant renal hypomagnesaemia indicates the physiological importance of the γ-subunit. The overall structure of the α-subunit of Na ⁺ ,K ⁺ -ATPase determined by electron crystallography using two-dimensional crystals is similar to the X-ray structure of Ca ²⁺ -ATPase.

The pump activity of Na ⁺ ,K ⁺ -ATPase has been investigated using direct measurement of hydrolysis activity, and axial heterogeneity in the nephron segments was demonstrated ( Figure 6.4 ). Na ⁺ ,K ⁺ -ATPase hydrolysis activity was high in the TAL, distal convoluted tubule (DCT), and proximal convoluted tubule (PCT), and low in the pars recta (PST) and collecting tubule (CT). Ouabain binding studies show the highest density of Na ⁺ ,K ⁺ -ATPase (20–30 fmol/mm length of tubule) in the DCT and the MTAL, intermediate density (10 fmol/ mm) in the PCT and connecting tubule (CNT), and lowest density (2–7 fmol/mm) in the PST, CTAL, and CT. The pump activity was proportional to the number of catalytic units. α1β1 has a maximum turnover rate of 7,700/min. The measurement of Na ⁺ ,K ⁺ -ATPase hydrolytic activity at V _max , and initial rates of ouabain-sensitive Rb uptake indicated that in intact cells the pump works at approximately 20–30% of its V _max . Western blotting analysis, RT-PCR using microdissected nephron segments, in situ hybridization, and immunohistochemical analysis demonstrated similar intranephron heterogenetic localization of Na ⁺ ,K ⁺ -ATPase consistent with those observed in the studies on pump activity.

Regulation of Na ⁺ ,K ⁺ -ATPase by dexamethasone, deoxycorticosterone, intracellular Na concentration, cAMP, potassium depletion, aldosterone and vasopressin was demonstrated. Many of these modulations influence the cell surface expression of Na ⁺ ,K ⁺ -ATPase, while MEK1/2 inhibitors changed the intrinsic activity rather than cell surface expression.

Ca ^{2 +} -ATPase

Renal calcium transport is comprised of two processes: a paracellular, passive process that predominates in most nephron segments; and a transcellular, energy-dependent step in the DCT. Transcellular calcium transport involves: (1) entry into the DCT cell via a Ca ²⁺ channel (ECaC) across the luminal membrane; (2) intracellular Ca movement facilitated by the presence of the vitamin D-dependent calcium-binding protein (calbindin D); and (3) extrusion by the Ca ²⁺ -ATPase located at the basolateral membrane. The extrusion of Ca ²⁺ , the final step in the transcellular transport of Ca ²⁺ , is mediated by the plasma membrane Ca ²⁺ -ATPase (PMCA), which is a P-type-ATPase.

PMCA is a monomeric protein consisting of approximately 1,220 amino acids with a molecular mass of 140 kDa. The sequence contains the calmodulin-binding domain, and two domains resembling calmodulin, one of which may play a role in the binding of Ca ²⁺ . There are at least four isoforms of PMCA, and isoforms 1 and 4 are more widely distributed than 2 and 3. The activity of renal PMCA showed two saturable components: a high-affinity component with a Km of 33 μM ATP, and a low-affinity component with a Km of 0.63 mM ATP. PMCA is regulated by calmodulin, estrogen and dihydrotestosterone, protein kinase C or A, extracellular ATP, and pathophysiologic states such as hypercalciuria. RT-PCR, immunohistochemical analysis, and Western blot analysis demonstrated high expression of PMCA in the DCT. PMCA was also detected in Madin–Darby canine kidney (MDCK) cells. Doucet and co-workers examined sodium azide-insensitive plasma membrane Ca ²⁺ -ATPase activity. The Ca ²⁺ -ATPase was maximally activated by Ca ²⁺ concentrations with an apparent Km of 0.3–0.4 μM. Ca ²⁺ -ATPase activity was found in all segments of the nephron: activity was highest in the DCT and CCT, intermediate in the PCT and MTAL, and lowest in the PST, CTAL and MCT.

In addition to PMCA, there exists another distinct Ca ²⁺ -ATPase, the sarco/endoplasmic reticulum Ca ²⁺ -ATPase (SERCA), which also belongs to the P-type-ATPase. The SERCA family includes three gene products, SERCA1 (ATP2A1), SERCA2 (ATP2A2), and SERCA 3 (ATP2A3), which function in the removal of free cytosolic Ca ²⁺ into the sarco/endoplasmic reticulum. Although thapsigarigin is known to be a specific inhibitor of the endoplasmic reticulum Ca ²⁺ -pump, no study has been reported on the relative energy consumption rate of SERCA in the kidney.

H ⁺ ,K ⁺ -ATPase

H ⁺ ,K ⁺ -ATPase was originally characterized in a study of gastric mucosa. The gastric H ⁺ ,K ⁺ -ATPase is located in the apical membrane of stomach parietal cells, and mediates electroneutral exchange of K ⁺ and H ⁺ . Gastric H ⁺ ,K ⁺ -ATPase activity is independent of extracellular sodium, and is inhibited by vanadate.

Molecular cloning identified two types of H ⁺ ,K ⁺ -ATPase: gastric and colonic type H ⁺ ,K ⁺ -ATPase. H ⁺ ,K ⁺ -ATPase is comprised of α- and β-subunits. The catalytic α-subunit of gastric H ⁺ ,K ⁺ -ATPase shows structural similarity to that of Na ⁺ ,K ⁺ -ATPase, and the greatest homology occurs in the phosphorylation site region, and domains presumably involved in nucleotide binding and energy transduction. The α-subunit of colonic H ⁺ ,K ⁺ -ATPase exhibits 63% amino acid identity to that of the gastric H ⁺ ,K ⁺ -ATPase. The β-subunit of H ⁺ ,K ⁺ -ATPase shows 41% amino acid sequence identity to the β2-subunit of Na ⁺ ,K ⁺ -ATPase in the rat.

In the kidney, the existence of both gastric H ⁺ ,K ⁺ -ATPase and colonic H ⁺ ,K ⁺ -ATPase was demonstrated. Gastric H ⁺ ,K ⁺ -ATPase is expressed constitutively along the length of the collecting duct, is responsible for H ⁺ secretion and K ⁺ reabsorption under normal conditions, and may be stimulated with acid–base perturbations and/or K ⁺ depletion. The level of expression of colonic H ⁺ ,K ⁺ -ATPase is much lower in the kidney than in the distal colon.

Using in vitro microperfusion, Wingo and colleagues provided evidence of the existence of omeprazole-sensitive acidification and a K ⁺ -absorptive mechanism in OMCD in rabbits. By enzymatic analysis, Doucet and Garg quantified the K ⁺ -stimulated, Na-insensitive ATPase activity in the nephron segments. K ⁺ -stimulated ATPase activity was identified in the CNT, CCT, and MCT, although the activities were very low compared to those of other P-type-ATPases in the kidney. The renal K ⁺ -ATPase had a high affinity for K ⁺ (Km of approximately 0.2~0.4 mM) and was inhibited by vanadate, omeprazole, and SCH 28080, specific inhibitors of gastric H ⁺ ,K ⁺ -ATPase, but was insensitive to ouabain. A correlation between the magnitude of enzymatic activity and the percentage of intercalated cells in a given segment suggested that K ⁺ -ATPase activity originates in intercalated cells.

Immunohistochemical analysis revealed H ⁺ ,K ⁺ -ATPase in intercalated cells in the CCD and OMCD. In all segments studied, except for the CCD, the percentage of H ⁺ ,K ⁺ -ATPase-immunoreactive cells corresponded to the percentage of intercalated cells. The RT-PCR technique demonstrated the gastric H ⁺ ,K ⁺ -ATPase α-subunit in the CCD and IMCD, and a specific hybridization signal for the gastric H ⁺ ,K ⁺ -ATPase α-subunit cDNA was demonstrated. The colonic H ⁺ , K ⁺ -ATPase α-subunit is specifically expressed in the CCD and OMCD in K ⁺ -depleted rats. An increase in the H ⁺ ,K ⁺ -ATPase activity, and enhanced expression of gastric H ⁺ ,K ⁺ -ATPase α-subunit and colonic H ⁺ ,K ⁺ -ATPase in K ⁺ depletion, suggests the physiological adaptation of renal H ⁺ ,K ⁺ -ATPase.

V-type ATPases

V-type (vacuolar) ATPases represent the second family of ATP-dependent ion pumps. Vacuolar H ⁺ -ATPase is primarily responsible for the acidification of intracellular compartments such as endosomes, lysosomes, Golgi apparatus, and clathrin-coated vesicles. H ⁺ -ATPase is also expressed in the plasma membrane, and functions in acid–base transport in epithelia. In the kidney, vacuolar H ⁺ -ATPase mediates H ⁺ secretion, mainly in the PT and CCD.

H ⁺ -ATPase is a multi-subunit complex composed of two functional domains. The V(1) domain is a 570 kDa peripheral complex composed of eight subunits of molecular mass 73–14 kDa (subunits A–H) that is responsible for ATP hydrolysis. The V(o) domain is a 260 kDa integral complex composed of five subunits of molecular mass 100–17 kDa (subunits a, d, c, c’, and c”) that is responsible for proton translocation.

H ⁺ -ATPase is insensitive to vanadate or ouabain, but inhibited by bafilomycin A, N,N’-dicyclohexylcarbodiimide (DCCD: Ki= 50 μM) and N-ethylmaleimide (NEM: Ki= 20 μM). Physiological experiments indicated the existence of H ⁺ -ATPase in the PT. DCCD caused a fall in CO ₂ absorption by 15% under eucapnia, and by 30% during acute hypercapnia in the PT. In other experiments, the S ₃ segment was shown to possess plasma membrane H ⁺ -ATPase activity.

The relative contribution of H ⁺ -ATPase to ATP consumption by the kidney was examined by Noel et al. In dog proximal tubules incubated under control conditions, 81% of the respiration was directly related to oligomycin-sensitive ATP synthesis, and 29% of this amount was inhibited by bafilomycin A. In rabbit and hamster PT, the bafilomycin-sensitive ATP requirement involves only 5 and 10%, respectively, of the total ATP turnover. Thus, the metabolic cost of H ⁺ -ATPase in PT varies significantly among species.

Ait-Mohamed et al. examined the localization of NEM-sensitive ATPase in all the segments of the rat nephron; its activity was highest in the PCT; intermediate in the PST, TAL and CCT; and lowest in the OMCD. Immunocytochemical analysis demonstrated localization of rat H ⁺ -ATPase in the PCT, the initial part of the thin descending limb, TAL, DCT, and CT consistent with the aforementioned H ⁺ -ATPase activity.

Garg and co-workers examined the effect of acid-base balance and aldosterone on NEM-sensitive ATPase activity, and demonstrated the modulation of NEM-sensitive ATPase activity by metabolic acidosis and administration of aldosterone, and these effects were observed mainly in the CT.

The significance of H ⁺ -ATPase in final urinary acidification along the collecting system has been confirmed by hereditary defects in H ⁺ -ATPase. Mutations in the gene encoding the B1-subunit of H ⁺ -ATPase cause distal renal tubular acidosis with sensorineural deafness, and defects in the 116 kDa subunit ATP6N1B cause recessive distal renal tubular acidosis with preserved hearing.

ABC Superfamily

The third subgroup of primary active transporters is the ABC (ATP-binding cassette) transporter family. The prototype ABC transporter is the P-glycoprotein (P-gp) encoded by the MDR gene. P-gp was originally isolated as a drug extrusion pump in cancer cells which confers multiresistance to antineoplastic drugs. Later, P-gp was also shown to be expressed in normal tissues such as the kidney, intestine, and brain capillary cells, where it acts as a functional barrier to xenobiotics by extruding them from the tissues. Then, a subfamily of ABC transporters, the MRP (multidrug-resistance-associated protein) family, was identified, and the number of its isoforms is expanding rapidly.

The common molecular structure of ABC transporters is as follows; they possess two transmembrane (TM) domains, each with six TM segments and two nucleotide-binding domains, both of which can hydrolyze ATP. The stoichiometry of two ATPs hydrolyzed per molecule of drug transported was proposed.

Although the molecular properties of the members of ABC transporters, such as tissue distribution and substrate selectivity, have been extensively characterized, their significance in energy consumption in the kidney remains to be investigated, and to date no information is available.

Comparison of Ion Transporting ATPase Activities and QO ₂ along the Nephron

In the upper panel of Figure 6.5 a comparison of the ion transporting ATPase activities is shown. In the lower part, relative distribution of QO ₂ along the nephron segments is depicted.

Mitochondrial Oxidative Phosphorylation

Mitochondrial oxidative phosphorylation ( Figures 6.6, 6.7 ) is comprised of the following three steps: (1) production of reduced equivalents, i.e., NADH and FADH ₂ , mostly by the TCA cycle in the matrix of mitochondria; (2) electron transfer via the mitochondrial respiratory chain in the inner membrane of mitochondria, associated with proton extrusion across the inner membrane of mitochondria; and (3) ATP production by F ₀ F ₁ -ATPase using the proton gradient generated ( Figures 6.6 and 6.7 ). The mitochondrial respiratory chain catalyzes electron transfer via four large multimeric integral membrane protein complexes (complex I to IV), ATP synthase (alternatively called complex V), and two relatively small hydrophobic proteins, i.e., ubiquinone (Q: Coenzyme Q) and cytochrome c.

Tricarboxylic Acid Cycle

The tricarboxylic acid (TCA) ( Figure 6.8 ) cycle is present in all mammalian cells, except those lacking mitochondria such as mature red blood cells. The TCA cycle oxidizes acetyl CoA derived from carbohydrates, fatty acids, amino acids, and ketone bodies, and produces NADH and FADH ₂ . In addition, the TCA cycle provides intermediates that are utilized for the formation of glucose, lipids, and amino acids. Thus, the TCA cycle is central for metabolism, and is regulated to meet a variety of cellular metabolic demands.

Le Hir et al. assayed three TCA cycle enzymes, i.e., oxoglutarate dehydrogenase, citrate synthase and isocitrate dehydrogenase, in dissected rat nephron segments. The activities of the enzymes were higher in distal segments (TAL and DCT) than in the PT. The distal versus proximal ratios of activities were about 1.5, 2.5, and 2 for oxoglutarate dehydrogenase, citrate synthase, and isocitrate dehydrogenase, respectively. Oxoglutarate dehydrogenase showed the lowest activity along the entire nephron segments, and appeared to catalyze the rate-limiting step of the TCA cycle. Marver et al. determined citrate synthase levels in isolated rabbit nephron segments. The order of relative citrate synthase activities in normal rabbit nephron segments (per kg of dry tissue) was as follows: DCT> PCT> CTAL> CCD> PST. The activity in CCD was regulated by aldosterone.

β-Oxidation of Fatty Acids

Fatty acid is a major energy fuel in the kidney. β-Oxidation of short-, medium- and long- chain fatty acids ( Figure 6.8 ) occurs in mitochondria, and that of very long-chain fatty acids in peroxisomes. The 3-hydroxyacyl-CoA dehydrogenase activity, which mainly represents the mitochondrial β-oxidation pathway, is similarly distributed in all cortical proximal and distal segments, and is much lower in glomeruli and collecting ducts. The peroxisomal fatty acyl-CoA oxidase is restricted to the PT, with a capacity comparable to that in liver cells.

Ketone Body Metabolism

The kidney, as well as the muscle and brain, utilizes ketone bodies as metabolic fuel, while the liver cells do not. Acetoacetate and β-hydroxybutyrate are converted to acetyl CoA in the mitochondrial matrix. In this process, three enzymes, i.e., D-3-hydroxybutyrate dehydrogenase, 3-ketoacyl CoA transferase (3-oxoacid CoA- transferase), and acetoacetyl CoA thiorase, are involved.

Guder and co-workers measured 3-oxoacid CoA- transferase, and D-3-hydroxybutyrate dehydrogenase in mouse nephron. The activities of these enzymes were high in TAL and DCT, but decreased to nearly 20% in the CCD. In the PCT and PST, the 3-oxoacid CoA-transferase activity was almost equal, while the 3-hydroxybutyrate dehydrogenase activity was five-fold higher in PST than in PCT. In glomeruli and thin descending limbs of Henle’s loop, the enzymatic activities were markedly low. These results indicate that 3-hydroxybutyrate and acetoacetate can be metabolized in all of the mouse nephron segments with different capacities. The enzymatic activity for ketone body oxidation mirrors the distribution of mitochondria along the nephron segment.