Ca
2+ overload is characteristic of tissues with lethally injured cells, since the breakdown of the plasma membrane barrier to Ca
2+ causes a large increase in cytosolic Ca
2+, which is sequestered in part by the mitochondria. Specifically, building on the hypothesis that homeostatic mechanisms controlling cellular Ca
2+ are disturbed in AKI, it has been shown that radiocontrast-induced AKI (
51,
52) and cadaveric kidney transplant dysfunction (
53,
54), for example, can be attenuated by administration of chemically dissimilar Ca
2+ channel blockers. These are two clinical conditions in which intense renal vasoconstriction is demonstrable, a situation where delivery of oxygen and nutrients to renal tubules is compromised. The administration of Ca
2+ channel blockers reduces the intensity of renal vasoconstriction and provides better delivery of nutrients to renal tissues. With ischemia, the poor nutrient flow to renal tubules also results in tubule Ca
2+ overload, which can be lessened by the Ca
2+ channel blockers. Although Ca
2+ channel blockers have been shown to be efficacious in these two aforementioned clinical conditions, a full understanding of the mechanisms by which cytosolic or tissue Ca
2+ increases in underperfused situations and how this increase may contribute to organ injury is the focus of much recent research. It is important, therefore, to understand the normal cellular Ca
2+ regulation before discussing the newer insights that have been gained using experimental approaches to further improve our understanding of the pathogenesis of AKI.
Normal Regulation of Cell Ca2+
Three major cellular Ca
2+ pools exist: (a) a pool bound to plasma membranes, (b) a pool bound to or sequestered within intracellular organelles, and (c) a pool both free and bound within the cytoplasm (
55).
Although 60% to 70% of all Ca
2+ in renal epithelial cells is located in the mitochondria, cytosolic free ionized Ca
2+ is the most critical with regard to regulation of intracellular events. Cytosolic free Ca
2+ (Ca
2+)i is normally kept at about 100 nM, which is 1/10,000 of the extracellular level (
56). Ca
2+ efflux is mediated on basolateral membranes by both Ca
2+ ATPase, which is adenosine triphosphate (ATP) dependent, and a Na
+/Ca
2+ exchanger on the basolateral membrane, which is ATP independent (
57). Normally, the cell membrane is impermeable to Ca
2+ and maintains a steep Ca
2+ gradient between the cytosol and the extracellular space. However, when cytosolic Ca
2+ increases in response to increased cellular membrane permeability or decreased Ca
2+ efflux or both, the mitochondria and endoplasmic reticulum actively increase their Ca
2+ uptake. Mitochondrial uptake and retention of Ca
2+ become substantial only when cytosolic levels exceed 400 to 500 nM, as occurs with cell injury (
56). Mitochondrial uptake is regulated by a Ca
2+ uniporter in the mitochondrial inner membrane. During cell injury, active mitochondrial sequestration appears to be quantitatively the most important process for buffering elevations in cytosolic Ca
2+.
Tubular Effects of Ca2+ Accumulation
In vivo studies of intact kidney cannot discriminate between protective effects at the vascular sites compared to tubular sites or a combination thereof. As the proximal tubule is the main site of injury in I/R models in vivo and the human allograft with AKI (
58), the study of isolated proximal tubules during conditions of oxygen deprivation either in suspension or in primary culture has provided insight into the pathophysiology of proximal tubular injury. Numerous studies in both freshly isolated rabbit and rat proximal tubules as well as various models of proximal and distal tubules in culture have demonstrated an increase in cytosolic Ca
2+ in these renal epithelial cells during chemical anoxia, hypoxia, and Ca
2+ ionophore treatment (
59,
60,
61,
62,
63,
64,
65,
66,
67). When exposed to anoxia in vitro, proximal and distal tubules in culture rapidly exhibit cell death after reoxygenation (
68). However, if Ca
2+ is removed from the bathing medium during the first 2 hours of reoxygenation and then replaced, cell viability is greatly enhanced (
68). Ca
2+ channel blockers have also been shown to delay the onset of anoxic cell death in primary cultures of rabbit proximal tubules and cortical collecting tubules, suggesting that Ca
2+-mediated hypoxic cell death is not limited to the proximal tubules (
69).
Ca
2+ channel blockers have no effects on the rate of Ca
2+ influx into normoxic proximal tubules. However, during hypoxia or anoxia in vitro, Ca
2+ influx rate into tubules is increased above normal levels, and Ca
2+ channel blockers reduce this rate to or toward normal (
70). This is an important observation because (Ca
2+)i could increase as the result of normal influx rates in the presence of reduced efflux rates secondary to decreased ATP-dependent Ca
2+ ATPase or decreased Na
+/Ca
2+ antiporter activity. The efficacy of Ca
2+ channel blockers to prevent the increased Ca
2+ influx rate during hypoxia and not during normoxia suggests a hypoxia-induced alteration in membrane permeability to Ca
2+ that is sensitive to Ca
2+ channel blockers. This permeability pathway appears to be sensitive, in part, to the decrease in ATP that occurs during hypoxia. For example, reduced ATP levels in rat proximal tubules with a phosphate-free incubation medium result in increased Ca
2+ influx rate (
71). This ATP-dependent change in Ca
2+ permeability has not been examined in detail; however, acidosis prevents the increased Ca
2+ influx rate in tubules and delays the onset of cell injury, as assessed by lactate dehydrogenase (LDH) release even though ATP remains at low levels (
72). Cellular protection is also observed with an acidotic perfusate in the isolated perfused kidney (
73). Intracellular acidosis is more likely to develop in complete anoxia than in hypoxia, and this may explain the only very short-lived increase in Ca
2+ influx rate (
70) as well as the absence of appreciable tissue Ca
2+ overload during anoxia, as assessed by atomic absorption spectroscopy (
74).
On the basis of these observations, the role of Ca
2+ influx rate in mediating proximal tubule hypoxic injury was examined. By employing a combination of ethylene glycol tetraacetic acid (EGTA) and various Ca
2+ concentrations in the tubule bathing medium (Ca
2+-modified Krebs buffer), a delay in the onset of cell injury during hypoxia was seen when extracellular Ca
2+ concentration was <10
-5 M (
64).
Thus, Ca
2+ ions enter renal proximal tubules at a faster rate than normal during oxygen deprivation. The removal of extracellular Ca
2+ ions or administration of Ca
2+ channel buffers reduces the injury associated with this increased influx rate of Ca
2+. Acidosis also reduces Ca
2+ influx rate (
72) and exerts cytoprotective effects (
71,
72,
73,
74). Finally, if Ca
2+ ions do enter hypoxic or anoxic cells, their deleterious effects can be mitigated by calmodulin inhibitors (
69). Together, these data strongly suggest that it is the increased cytosolic or intracellular burden of Ca
2+ that initiates the development of cell injury.
The level of the free cytosolic Ca
2+ increase during ATP depletion in proximal tubules has been studied. Previously it was difficult to determine peak cytosolic Ca
2+ levels using the high-affinity Ca
2+ fluorophore Fura-2. The (Ca
2+)i increases to >100
µM in ATP-depleted proximal tubules using the low-affinity Ca
2+ fluorophore Mag-Fura-2 (
75). Experiments were done in the presence of 2 mM glycine, which approximates the physiologic concentration in vivo. Ninety-one percent of the tubules studied in an individual experiment had a free cytosolic Ca
2+ that exceeded 10
µM. Thirty-five percent had levels >500
µM with no cell membrane damage. In this study, proximal tubules had a remarkable resistance to the deleterious effects of increased Ca
2+ during ATP depletion in the presence of glycine. In the isolated perfused rat kidney, intracellular Ca
2+ increases have also been measured using 19
F NMR and 5
F BAPTA. In these studies, there was a partially reversible increase from 256 to 660 nM of Ca
2+ (
76,
77).
The level of oxygen deprivation that is required to increase cytosolic Ca
2+ has also been studied. A rise in cytosolic Ca
2+ in anoxic but not hypoxic tubules was demonstrated (
78). In hypoxic perfusion, oxygen tension measured with a very sensitive electrode was 5 to 6 mm Hg. Complete anoxia was achieved with oxyrase in a nonperfused system. Ca
2+ did not increase during hypoxia, but there was an increase in Ca
2+ during anoxia. This increase paralleled the collapse in mitochondrial membrane potential as measured by rhodamine fluorescence. Because cell membrane damage occurred during both anoxia and hypoxia, it was concluded that an increase in cell Ca
2+ is not always necessary for cell injury.
However, despite these studies, a crucial question remained to implicate Ca
2+ as the primary factor in cell injury. Does the increase in cytosolic Ca
2+ precede the injury, or is it a postlethal event? To answer this question, a video imaging system was designed in which the rise in cytosolic Ca
2+ as well as cell membrane injury could be simultaneously measured in freshly isolated proximal tubules (
79). (Ca
2+)i in freshly isolated proximal tubules, as assessed with Fura-2, increased significantly after 2 minutes of hypoxia and continued to increase progressively with continued hypoxia (
67). This increase in (Ca
2+)i precedes the uptake by nuclei of the membrane-impermeable dye propidium iodide (PI) (
67). PI staining is reduced when hypoxic rat proximal tubules are incubated either in a Ca
2+-free medium or with the intracellular Ca
2+ chelator BAPTA (
67). This study strongly supports the hypothesis that a cause-and-effect relationship exists between the elevation in (Ca
2+)i and the development of hypoxic membrane damage. Furthermore, this early rise in (Ca
2+)i after 5 to 10 minutes of hypoxia is reversible, since return to a well-oxygenated medium results in a prompt (1 minute) return of (Ca
2+)i to baseline level. If membrane injury had been the cause of the increase in (Ca
2+) i, a return to basal levels would not have occurred with reoxygenation.
In support of a pathogenic role of Ca
2+ in cell injury, it has been demonstrated that voltage-dependent Ca
2+ channels are involved in cellular and mitochondrial accumulation of Ca
2+ that follows ATP depletion and that voltage-dependent Ca
2+ channels play an important role in regulating mitochondrial permeability transition, cytochrome c release, caspase activation, and apoptosis (
80). In this study, in a rat renal proximal tubular cell line treated with antimycin A, ATP depletion-induced apoptosis was preceded by increased [Ca(
2+)]i and mitochondrial Ca
2+ before activation of mitochondrial signaling. Antagonizing L-type Ca(
2+) channels with azelnidipine administration ameliorated cellular and mitochondrial Ca(
2+) accumulation, mitochondrial permeability transition, cytochrome c release, caspase-9 activation, and resultant apoptosis.