Calcitriol directly sensitizes renal tubular cells to ATP-depletion- and iron-mediated attack

Abstract

Vitamin Ds have been reported to have diverse effects on cell homeostasis, leading to suggestions that they have therapeutic applications extending beyond their traditional actions on the Ca2+/parathyroid/bone axis. As some of these potential indications carry an inherent risk of acute renal failure (ARF; eg, cancer chemotherapy and organ transplantation), the goal of this study was to assess whether vitamin Ds directly affect renal tubule injury responses. Cultured human proximal tubular (HK-2) cells were exposed to physiological or pharmacological doses of either calcitriol (D3) or a synthetic vitamin D2 analogue (19-nor) for 3 to 48 hours. Their impact on cell integrity (percent lactate dehydrogenase (LDH) release and tetrazolium dye MTT uptake) under basal conditions and during superimposed injuries (ATP depletion/Ca2+ ionophore or iron-mediated oxidant stress) were determined. As vitamin Ds can be anti-proliferative, cell outgrowth ([3H]thymidine uptake and crystal violet staining) was also tested. Finally, the action of D3 on in vivo ARF (glycerol-induced myoglobinuria) and isolated proximal tubule injury responses were assessed. D3 induced a rapid, dose-dependent increase in HK-2 susceptibility to both ATP-depletion/Ca2+-ionophore- and Fe-mediated attack without independently affecting cell integrity or proliferative responses. In contrast, D2 negatively affected only Fe toxicity and only after relatively prolonged exposure (48 hours). D3 dramatically potentiated in vivo ARF (two- to threefold increase in azotemia), suggesting potential in vivo relevance of the above HK-2 cell results. Proximal tubules, isolated from these glycerol-exposed mice, suggested that D3 can worsen tubule injury despite a parodoxic suppression of H2O2 production. In contrast, D3 had a mild negative impact on cellular energetics (depressed ATP/ADP ratios), and it accentuated plasma membrane phospholipid breakdown. The latter was observed in both glycerol-treated and control tubules, suggesting a primary role in the injury- potentiation effect of D3. Vitamins D(s) may directly, and differentially, increase proximal tubule cell susceptibility to superimposed attack. This property should be considered as new uses for these agents are defined.

Figure 1.

Calcitriol effects on HK-2 cell MTT uptake under basal conditions and after 4 hours of ATP depletion/Ca²⁺ ionophore exposure. CAD, calcium ionophore plus antimycin plus 2-deoxyglucose. Calcitriol exposures were 0, 50, and 250 pg/ml for 18 hours before the CAD challenge. Under basal conditions, calcitriol had no effect on MTT uptake; however, it induced slight, but highly significant, reductions in MTT uptake after CAD exposure (consistent with an increase in CAD-mediated cytotoxicity).

Figure 2.

Calcitriol effects on ATP depletion/Ca²⁺ ionophore injury (CAD for 16 hours), as assessed by percent LDH release. Calcitriol exposure (250 pg/ml for 18 hours) did not independently alter percent LDH release. However, in the presence of the CAD challenge, it doubled LDH release, consistent with an exacerbation of CAD-mediated cell death.

Figure 3.

ATP depletion/Ca²⁺ ionophore (CAD × 16 hours) injury: effects of 30-hour calcitriol exposure. In contrast to the Figure 2 ▶ (18-hour) experiments, in these experiments cells were exposed to 0, 50, or 250 pg/ml calcitriol for 30 hours, and then cellular susceptibility to injury was assessed. Calcitriol induced dose-dependent increases in CAD-mediated LDH release without independently affecting LDH release.

Figure 4.

Effects of 19-nor on ATP depletion/Ca²⁺ ionophore (CAD for 4 hours) mediated injury, as assessed by MTT assay. These experiments recapitulated those shown in Figure 1 ▶ , except for substituting bio-equivalent doses of 19-nor for calcitriol. Unlike calcitriol, 19-nor had no impact on CAD-mediated reductions in MTT uptake.

Figure 5.

Effects of 19-nor on ATP depletion/Ca²⁺ ionophore (CAD for 16 hours) mediated injury, as assessed by percent LDH release. Exposing HK-2 cells to either 200 or 1000 pg/ml doses of 19-nor for either 18 or 48 hours failed to alter CAD-mediated injury.

Figure 6.

Calcitriol and 19-nor effects on 16 hours of iron-mediated HK-2 cell injury. Calcitriol exposure for 18 hours induced modest, but highly significant, dose-dependent increments in percent LDH release in response to Fe-mediated cell injury (left panel). In contrast, 18-hour 19-nor exposures had no injury-potentiating effect (right panel). Neither agent altered LDH release independent of the Fe challenge.

Figure 7.

Iron-mediated HK-2 cell injury (for 16 hours): effects of 48-hour calcitriol or 19-nor exposure. These experiments were analogous to those in Figure 6 ▶ , except that the cells were exposed to calcitriol or 19-nor for 48 hours (rather than 18 hours) before Fe addition. The results differed from those presented in Figure 6 ▶ in two respects: 1) 19-nor, as well as calcitriol, exacerbated Fe-mediated injury; and 2) the lower vitamin D dosages (50 pg/ml calcitriol; 200 pg/ml 19-nor) exerted greater injury-promoting effects than did the higher calcitriol/19-nor doses (200 and 1000 pg/ml, respectively).

Figure 8.

HK-2 cell proliferation, as assessed by [³H]thymidine incorporation, in the presence of calcitriol or 19-nor. HK-2 cells were cultured for 16 or 48 hours with either calcitriol (0, 50, or 250 pg/ml; left panel) or with 19-nor (0, 200, or 1000 pg/ml), and then [³H]thymidine incorporation was assessed. Calcitriol had no effect on [³H]thymidine uptake under any of the test conditions, and 19-nor slightly suppressed [³H]thymidine uptake, but only with the 1000 pg/ml dose. This was only a transient effect, as after more prolonged (48 hours) exposure, 19-nor had no effect.

Figure 9.

Effects of single-dose calcitriol administration on normal mice and on mice subjected to concomitant glycerol administration. When administered to control (C) mice, calcitriol induced no increments in BUN or plasma creatinine concentrations. However, when administered at the time of glycerol administration, calcitriol caused two- to threefold BUN/creatinine increments (assessed 18 hours after glycerol with or without calcitriol therapy).

Figure 10.

Total plasma calcium concentrations in normal mice (left) and in glycerol-treated mice (right) (18 hours with or without calcitriol administration). Calcitriol induced modest, and essentially identical, degrees of hypercalcemia in normal and glycerol-treated mice. C, controls (ie, no calcitriol treatment).

Figure 11.

Effects of calcitriol administration on H₂O₂ production by isolated tubules and on cell energetics, assessed by tubule ATP/ADP ratios. Left panel: Tubules harvested from mice 2 hours after glycerol injection demonstrated brisk H₂O₂ production, compared with control tubules (harvested from normal mice). In vivo calcitriol (vit D) treatment at the time of glycerol injection markedly suppressed tubule H₂O₂ production. Right panel: Tubules harvested from glycerol-treated mice had significant depressions in ATP/ADP ratios (an indirect assessment of state 3 respiration). In vivo calcitriol (vit D) treatment caused a modest, but significant, accentuation of this ATP/ADP suppression.