Renal Calcium Metabolism

Serum Calcium

In adult humans, the calcium concentration of extracellular fluid averages 10 mg/dl (=5 mEq/l, 2.5 mM). Plasma calcium exists as three distinct chemical forms: protein-bound, complexed (but diffusible), and ionized. The relations between the various forms of calcium are shown schematically in Fig. 64.1 . Forty percent of the serum calcium is bound to plasma proteins, with albumin accounting for some 90% of this. Smaller percentages are bound, though with greater affinity, to ß-globulin, α ₂ -globulin, α ₁ -globulin, and γ-globulin. Ten percent of the serum calcium is complexed with small polyvalent anions. Calcium complexes are formed by ion pairing with phosphate and citrate and, to a lesser extent, with bicarbonate, and sulfate. The degree of complexation depends upon the concentrations of ionized calcium, the complexing anion, and the ambient pH. The different moieties of calcium are important because only diffusible calcium, i.e., the free plus complexed calcium, is filtered at the glomerulus and crosses cell membranes. A comprehensive discussion of the biologically relevant forms of calcium and their interrelations is available elsewhere.

The ultrafilterable, or ionized fractions, of calcium as summarized above are affected by changes in the total serum calcium concentration, blood pH, plasma protein concentration, and the concentration of complexing anions. Increases in total serum calcium concentrations are usually accompanied by concomitant elevations in the concentration of ultrafilterable calcium, at least up to a total concentration of about 4 mM. Above this concentration, the ultrafilterable fraction declines such that increases of ultrafilterable calcium no longer parallel elevations of total calcium concentration. Reduction in calcium ultrafiltration with hypercalcemia has been postulated to result from the formation of insoluble Ca ₃ (PO ₄ ) ₂ protein complexes. This idea is supported by the finding that ultrafilterable phosphate concentrations also decline. Conversely, hypocalcemia is generally associated with a fall in calcium ultrafiltration.

Changes in the concentration of serum proteins are usually accompanied by parallel changes in the total serum calcium concentration so that the ultrafilterable fraction remains constant. In severe hypoproteinemia, however, the ultrafilterable fraction increases.

The concentration of ionized Ca ²⁺ varies inversely with blood pH. Acidemia increases ionized Ca ²⁺ concentrations, whereas alkalemia causes decreases. Increases in the serum concentration of complexing anions, such as phosphate, citrate, sulfate, or bicarbonate, reduce the ionized Ca ²⁺ concentration by sequestering Ca ²⁺ .

The amount of calcium in the extracellular fluid represents a dynamic balance between intestinal absorption, renal reabsorption, and osseous resorption. Symptoms of hypocalcemia vary in relation to the ionized serum calcium concentration. Mild reductions of calcium are associated with paresthesias and muscle cramps; more severe decreases of calcium may induce seizures. Increases of plasma calcium, on the other hand, have been implicated in attenuation of the renal effects of parathyroid hormone (PTH), the antidiuretic action of vasopressin, and reduced renal concentrating capacity. A schematic representation of calcium balance, for an adult human, is shown in Fig. 64.2 . Assuming a daily dietary calcium intake of 1,000 mg, net intestinal absorption amounts to about 200 mg, with the remaining 800 mg excreted in the feces. In balance, net intestinal absorption is matched by urinary excretion, while calcium accretion and loss from bone are equal. Thus, approximately 200 mg of calcium are excreted daily. In adults, net calcium balance is effectively zero, suggesting that in the absence of a calcium challenge such as lactation, the kidneys represent the dominant regulatory site of calcium metabolism.

The renal responses to alterations of extracellular calcium are transduced by the calcium-sensing receptor (CaSR), which is expressed prominently at the sites of PTH and vasopressin action in the kidney.

General Considerations, Calcium Clearance

Maintenance of extracellular calcium balance requires the daily urinary excretion of 200 mg of calcium. The amount of calcium excreted equals the difference between that filtered and reabsorbed:

calcium excreted=filtered load – calcium reabsorbed

or,

where U _{Ca ²⁺} is the concentration of calcium in the urine, V is the rate of urine flow, GFR is the glomerular filtration rate, $P_{C{a}^{2+}}^{uf}$ is the concentration of ultrafilterable calcium in plasma (filtered load being the production of GFR and plasma ultrafiltrable calcium), and T _{Ca ²⁺} is the rate of net Ca ²⁺ reabsorption.

Unlike phosphate or magnesium, which exhibit saturable reabsorptive kinetics (i.e., a tubule reabsorption maximum, T ^m ), most studies suggest that calcium does not display such behavior. However, some evidence for a calcium $T^m{(T}_{C{a}^{2+}}^m)$ has been advanced. The $T_{C{a}^{2+}}^m$ is conventionally determined by measuring calcium excretion as a function of the plasma calcium, or ultrafilterable calcium, concentration. Two explanations have been proposed for the apparent failure of calcium excretion to saturate at elevated serum calcium concentrations. It has been argued that, in vivo , only a limited excursion of ultrafilterable plasma calcium concentrations can be tolerated and that within this range the filtered load of calcium may be less than the $T_{C{a}^{2+}}^m$ . It has also been postulated that since the bulk of calcium absorption is passive and may be accomplished by a combination of diffusion and solvent drag, no $T_{C{a}^{2+}}^m$ exists.

Calcium is reabsorbed throughout its passage along the nephron. As detailed below and shown schematically in Fig. 64.3 , 60–70% of the filtered calcium is reabsorbed by proximal tubules, an additional 20% by thick ascending limbs, and 5–10% by distal tubules. Final adjustments of calcium excretion are achieved in collecting ducts, where transport may be absorptive or secretory. The net result of these processes is that only 0.5–1.5% of the filtered calcium is normally excreted in the voided urine.

The interested reader is referred to several excellent monographs for more comprehensive treatments of calcium homeostasis and clearance micropuncture and isolated tubule studies of renal calcium transport.

Glomerular Filtration

The renal disposition of calcium begins with its ultrafiltration across the capillaries of the glomerulus. The ultrafilterable forms of calcium include ionized Ca ²⁺ and calcium complexed to small anions. Calcium bound to plasma proteins is not filtered. The calcium concentration in the ultrafiltrate, measured by directly sampling tubular fluid from Bowman’s space is 1.31 mM, or 63% of that in plasma. This value compares favorably to that of ultrafiltrate prepared with artificial membranes.

Glomerular ultrafiltration is a passive process, driven by the net hydraulic and osmotic pressure gradient across the capillary membranes. The glomerulus is generally not considered a site of regulation of calcium absorption or homeostasis. However, it should be borne in mind that PTH depresses the glomerular ultrafiltration coefficient K _f , which may thereby diminish single nephron filtration rates and contribute to the hormone’s calcium-sparing action (cf. Hypercalcemia ). mRNA transcripts for the type 1 PTH receptor (PTH1R) and type 2 PTH receptor (PTH2R) are expressed in glomeruli.

Proximal Convoluted Tubule

Following its ultrafiltration across the glomerular capillaries, calcium is reabsorbed throughout the nephron. As shown in Fig. 64.3 , the majority of the filtered calcium is recovered by the proximal tubules. The proximal nephron of both superficial and juxtamedullary tubules consists of three ultrastructurally distinct segments: S ₁ , S ₂ , and S ₃ . Operationally, however, calcium transport has been characterized in the microscopically recognized proximal convoluted tubules, corresponding primarily to the S ₁ segment, and to a lesser extent in superficial proximal straight tubules ( pars recta ), S ₂ segments.

Sixty to Seventy percent of the filtered calcium is absorbed by the end of the proximal convoluted tubule. Using in vivo micropuncture techniques, Lassiter and colleagues, and subsequently others, demonstrated that two-thirds of the filtered calcium is reabsorbed by the end of the accessible proximal convolution in the rat. Somewhat less complete absorption, 37–45%, obtains in the rabbit. Remarkably, in the hamster, which exhibits the most robust calcium-sparing effect of PTH, calcium absorption by proximal tubules is negligible. This observation underscores the primacy of distal segments in regulating final calcium excretion.

In general, random collections of proximal tubule fluid samples under control conditions result in tubular fluid to glomerular filtrate calcium ratios, (TF/GF) _{Ca ²⁺} between 1.0 and 1.2. Based on these findings it is commonly held that the concentration of calcium within the proximal tubular fluid remains essentially identical to that of glomerular filtrate or ultrafilterable plasma (UF) calcium, i.e., that calcium transport along the length of the proximal convoluted tubule proceeds essentially as an isoosmotic process. These observations, in turn, have been interpreted as indicating that the bulk of calcium absorption is energetically passive. However, a detailed examination of calcium absorption along the length of the accessible superficial proximal tubule revealed that (TF/GF) _{Ca ²⁺} increases from a value close to unity at the earliest micropuncture locations to a value of about 1.2, still relatively early in the nephron, and stayed constant for the remaining accessible portions of the proximal nephron. The cause of the elevated (TF/GF) _{Ca ²⁺} ratios is unclear but may have its origin in molecular sieving of calcium at the luminal pole of the tight junctions as calcium absorption lags behind that of water. Alternatively, the presence of nontransported, anionically complexed calcium within the lumen might contribute to or account for the elevated values of (TF/GF) _{Ca ²⁺} . Inasmuch as the proximal tubule transepithelial voltage is about −2.5 mV, these findings are consistent with the idea that a component of proximal calcium transport may be energetically active since it proceeds against an electrochemical gradient.

Proximal Straight Tubule

Because proximal straight tubules ( pars rectae ) do not extend to the surface of the kidney and are not amenable to conventional micropuncture techniques, calcium transport by this segment has been less thoroughly studied than in proximal convoluted tubules. Indirect estimates of calcium reabsorption derived from the difference between the calcium concentration of fluid samples obtained from late proximal convoluted tubules and those from the tip of the loop of Henle suggested that 10% of the filtered calcium is recovered by proximal straight tubules. This approach necessitates obtaining proximal samples from a superficial nephron, while the samples from the loop of Henle are taken from the tip of a juxtamedullary nephron. Nonetheless, direct in vitro microperfusion studies of proximal straight tubules substantiated the conclusion that proximal straight tubules absorb 10% of the filtered calcium.

Mechanisms of Proximal Tubule Calcium Transport

Calcium absorption by proximal tubules may be mediated by a combination of passive and active transport mechanisms. The transepithelial absorption of calcium can be conceptually described by the following relation:

calcium absorption=passive transport+active transport

where passive absorption is the sum of diffusion and solvent drag. These relations can be expressed formally as:

where, P _{Ca ²⁺} is the apparent calcium permeability, ΔC _{Ca ²⁺} is the transepithelial calcium concentration difference ([lumen-to-bath] – [bath-to-lumen]), z _i is the valence, R the gas constant, T the absolute temperature, C _{Ca ²⁺} is the average transmural calcium concentration across the tubule ([lumen-to-bath+bath-to-lumen]/2), ΔΨ is the transepithelial voltage, σCa ²⁺ is the reflection coefficient for calcium, J _v is the net fluid absorption, and ${J\{}_{{\mathrm{Ca}}^{2+}}^{\mathrm{active}}\}$ is the metabolically active, transcellular calcium transport.

Most evidence suggests that proximal tubule calcium transport is passive, i.e., energetically independent, and occurs primarily by diffusion and solvent drag. These mechanisms imply that calcium absorption proceeds primarily by the paracellular route ( Fig. 64.4 ) through the lateral intercellular space between adjoining cells. By contrast, active transport is a two-step process, wherein calcium enters the cell across apical plasma membranes and is then extruded across basolateral plasma membranes. Basolateral efflux occurs against a steep electrochemical gradient and is powdered by the hydrolysis of ATP by the Na-K-ATPase. A general schematic representation of these processes in proximal tubules is shown in Fig. 64.4 .

It should be noted that, although small by comparison with paracellular calcium absorption, active cellular absorption by proximal tubules amounts to some 20 µmol/min, which, in fact, is approximately twice that of the distal nephron, where calcium absorption is entirely cellular.

The mechanism of calcium transport by the S ₂ segment of the proximal straight tubule resembles that of the proximal convoluted tubule. Studies using isolated perfused rabbit S ₂ proximal tubules, under experimental conditions designed to minimize net fluid movement and the electrochemical gradient for Ca ²⁺ , generally are consistent with the idea that passive driving forces are the major determinant of calcium absorption. However, evidence for a significant amount of active calcium transport has been reported. Sacks and Bourdeau showed that when passive driving forces across isolated S ₂ segments of rabbit proximal straight tubules were experimentally manipulated, the direction and rate of net calcium flux were predicted by the magnitude of the imposed electrochemical gradient. Thus, passive diffusion appears to be the major mechanism of transport in proximal straight tubules.

The presence of active, transcellular calcium absorption by proximal tubules, no matter how slight its magnitude, necessitates specific transport proteins in apical plasma membranes to admit calcium and others in basolateral membranes to mediate its extrusion. As far as is presently known, cellular calcium entry is mediated by calcium channels. Support for the presence of such channels takes the form of electrophysiological and pharmacological evidence. The molecular identity of such proximal tubule calcium channels is unknown but appears not to be TrpV5 or TrpV6. Basolateral calcium efflux in energetically dependent. Two proteins capable of mediating such transport are the plasma membrane Ca ²⁺ -ATPase (PMCA) or the NCX Na ⁺ /Ca ²⁺ exchanger. Proximal tubule cells express PMCA1 and PMCA4 isoforms, which may serve as the primary mechanism of cellular Ca ²⁺ efflux. Proximal tubules also express the NCX1 Na ⁺ /Ca ²⁺ exchanger. The relative contribution of NCX1 and PMCA1/4 to cellular calcium absorption is not known.

In summary, proximal tubules exhibit high calcium permeability and low transepithelial electrical resistance. Most calcium absorption proceeds through passive mechanisms and traverses the paracellular pathway ( Fig. 64.4 ). The majority of passive absorption is diffusive, with an additional slight contribution by solvent drag. The active component constitutes 20% of the total calcium absorption and proceeds through a transcellular pathway that involve entry through apical membrane calcium channels and exit across basolateral membranes that is mediated by isoforms of the plasma membrane Ca ²⁺ -ATPase and/or the Na ⁺ /Ca ²⁺ exchanger.

Descending and Ascending Thin Limbs

Limited information is available on calcium transport and permeability of thin descending limbs of Henle’s loop. The few studies failed to uncover evidence for net calcium movement (absorptive or secretory) (summarized in ). When single rabbit descending thin limbs were perfused in vitro , (TF/UF) Ca ²⁺ and (TF/P) inulin increased proportionately. This would allow calcium to accumulate in the tubular fluid. Furthermore, these studies revealed a remarkably low permeability to calcium; about one-tenth that of proximal convoluted tubules.

Thin ascending limbs of Henle’s loop also exhibit low calcium permeability and are not a site of net calcium transport. The low permeability of thin ascending limbs is particularly striking in view of its comparatively high permeability to sodium and chloride. In single, microperfused thin ascending limbs of the hamster, removal of luminal calcium had no effect on the concentration of intracellular free calcium, consistent with the low permeability.

In summary, calcium transport by thin descending limbs and thin ascending limbs contributes little to the overall reabsorptive process because of the low Ca ²⁺ permeability and absence of active calcium transport by these segments.

Thick Ascending Limb

Approximately 20–25% of the filtered calcium is reabsorbed by thick ascending limbs of Henle’s loop. Thick ascending limbs are comprised of medullary and cortical portions. This division is relevant because appreciable functional and species differences attend calcium transport and its hormonal regulation by these nephron segments. Calcium is absorbed by both medullary and cortical portions of thick ascending limbs, though to differing degrees. It is unclear if the gradual changes in cellular architecture between medullary and cortical portions of the thick ascending limb are related to differences in the mechanism or magnitude of calcium transport. The distinguishing feature of calcium transport in thick ascending limbs is the presence of parallel paracellular and cellular transport pathways ( Fig. 64.5 ). Under resting conditions, calcium absorption is energetically passive and proceeds through the paracellular pathway driven primarily by the lumen electropositive transepithelial voltage resulting from the absorption of Na ⁺ . The greater the rate of Na ⁺ absorption, the larger the voltage, and the consequent rate of Ca ²⁺ absorption. Conversely, lower rates of Na ⁺ absorption, following the administration of loop-acting diuretics, for instance, the lower the extent of Ca ²⁺ absorption. Thus, in the absence of hormonal stimulation, Ca ²⁺ and Na ⁺ absorption parallel one another in thick ascending limbs as they do in proximal tubules.

The pattern of parallel Ca ²⁺ and Na ⁺ absorption is disrupted, however, by PTH and by calcitonin. Thick ascending limbs express the parathyroid hormone receptor and the calcitonin receptor. The distribution of these receptors, as deduced by the ability of their respective ligands to induce cAMP accumulation, varies importantly between medullary and cortical ascending limbs and across species. Table 64.1 summarizes representative findings for humans and the mouse. Direct studies of the effect of PTH and calcitonin on calcium transport by isolated cortical and medullary thick limbs established that these peptides stimulate active, transcellular calcium absorption. Thus, resting calcium absorption proceeds through the paracellular pathway through tight junctions, and active calcium absorption follows a transcellular route. Moreover, whereas passive calcium transport in thick limbs parallels Na ⁺ movement, active calcium cellular calcium transport is inversely related to cellular sodium absorption. The reason for this is that apical membrane calcium influx is voltage dependent. Cellular Na ⁺ entry depolarizes the cell, thereby reducing the driving force for Ca ²⁺ entry, and conversely decreased Na ⁺ influx hyperpolarizes the transmembrane voltage and increases Ca ²⁺ influx. These complex relations between voltage and paracellular vs. cellular calcium movement makes it difficult to assess the relative contribution of cellular and paracellular calcium transport to the net absorption, especially under physiological conditions or in clinical settings.

Notably, other peptide hormone receptors, including those for ADH and glucagon, are also expressed in thick ascending limbs. Their activation is not accompanied by changes of calcium absorption. This may be explained by the requirement for activation of both adenylyl cyclase and phospholipase C, which occurs with PTH and calcitonin but not ADH or glucagon, for stimulation of cellular calcium transport in cortical ascending limbs.