INTRACELLULAR Na⁺ ACCUMULATION AND HEPATOCYTE INJURY DURING COLD STORAGE

Abstract

Background. 

The mechanisms responsible for liver damage during cold storage are still not completely understood. We have investigated the role played by alterations of Na⁺ homeostasis in cell injury during cold hypoxia.

Methods. 

The changes in Na⁺ distribution were investigated in isolated rat hepatocytes stored at 4°C under hypoxic conditions.

Results. 

Hepatocyte cold stored up to 72 hr in Krebs-Henseleit-Hepes buffer showed a progressive increase in intracellular Na⁺ content that preceded the loss of cell viability. Na⁺ accumulation and cell death were prevented using Na⁺-free, acidic (pH 6.5) or glycine-supplemented storage media. The Na⁺ ionophore monensin reverted the cytoprotection exerted by glycine and by the acidic medium, but not that given by Na⁺-free Krebs-Henseleit-Hepes. A low Na⁺ content was also important for the cytoprotection observed using University of Wischonsin solution.

Conclusions. 

Na⁺ overload might contribute to liver graft injury occurring during cold storage.

Poor organ preservation still represents an important cause of primary graft nonfunction during liver transplantation ^{(1, 2)}. In recent years, many studies have focused on the possible mechanisms involved in causing liver graft failure. Little is known about the mechanisms responsible for hepatic injury during cold preservation. It has been postulated that the impairment of Na⁺/K⁺ and Ca⁺⁺ translocases due to hypotermia and the lowering of intracellular energy stores induce liver swelling and the activation of nonlysosomal proteases leading to hepatic cell damage ⁽²⁾. Furthermore, a recent report has stressed the contribution of oxidative stress in causing the injury of isolated cold stored hepatocytes and endothelial cells ⁽³⁾.

Studies using isolated rat hepatocytes exposed to oxidative stress, mitochondrial inhibitors, or hypoxia have demonstrated that ATP depletion is followed by an increase in intracellular Na⁺ levels ^{(4, 5)}. In these conditions Na⁺ accumulation results from the activation of Na⁺/H⁺ exchanger and of Na⁺/HCO₃⁻ cotransporter in response to intracellular acidification ⁽⁵⁾. In addition, the lowering of cellular ATP also contributed to the rise of intracellular Na⁺ by interfering with Na⁺ efflux throughout Na⁺/K⁺ ATPase ⁽⁵⁾. As a result of Na⁺ increase, Ca⁺⁺ accumulation and cell swelling occurred leading to the loss of plasma membrane integrity ⁽⁵⁾. Consistently, hepatocyte incubation in a Na⁺-free buffer or in an acidic medium (pH 6.5) greatly reduced Na⁺ overload and cell killing ^{(4, 5)}. Moreover, De Groot's group has recently reported that furosemide or bumetamide, two inhibitors of the Na⁺-K⁺-2Cl⁻ cotransporter decreased Na⁺ accumulation and tissue injury in isolated perfused livers exposed to warm ischemia ⁽⁶⁾. Intracellular acidosis and depletion of ATP stores are common features also in cold preserved explanted livers ⁽¹⁾. Therefore, we have investigated the possibility that perturbations of Na⁺ homeostasis might contribute to liver cell damage during cold storage.

Isolated rat hepatocytes were prepared from male Wistar rats (180-250 g weight) (Nossan, Correzzana, Italy) by liver perfusion with collagenase (type I, Sigma Chemical Co., St Louis, MO) as previously described ⁽⁵⁾. Cell viability, estimated at the beginning of the experiments, ranged between 85 and 90%. The basic incubation medium consisted in a Krebs-Henseleit-Hepes (KHH)^* buffer containing 118 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L KH₂PO₄, 1.3 mmol/L CaCl₂, 25 mmol/L NaHCO₃⁻, and 20 mmol/L N-(2-hydroxyethyl)-piperazine-N′-(2-ethanesulfonic acid) (Hepes) at pH 7.4. In the acidic Krebs-Henseleit buffer, Hepes was replaced by 20 mmol/L 2-(N-morpholino)-ethanesulfonic acid (MES) and the pH was adjusted to 6.5 with HCl. For the experiments performed in the absence of Na⁺, NaCl, and NaHCO₃ were replaced by 118 mmol/L choline chloride and 25 mmol/L KHCO₃, respectively. Isolated rat hepatocytes were maintained up to 72 hr at 4°C in differently modified Krebs-Henseleit buffer pH 7.4 under a nitrogen atmosphere obtained by fluxing the incubation flasks with 95% N₂-5% CO₂. In some experiments hepatocytes were cold stored in University of Wischonsin (UW) solution (Du Pont, Wilmington, DE) under 100% N₂ atmosphere. Cell viability and intracellular Na⁺ content were measured at different time points immediately after rewarming at room temperature. Cell viability was estimated by microscope-counting the hepatocytes excluding Trypan blue or by measuring propidium iodide fluorescence. Briefly, aliquots (2 mL) of the cell suspension were incubated 5 min at room temperature with 100 μg/mL propidium iodide and fluorescence was measured at 520 nm (excitation) and 605 nm (emission) wavelength pair before and after cell permeabilization with digitonin (10 μg/mL). The fluorescence readings before and after digitonin addition were used for calculating the percentage of viable cells. In our work these procedure gave comparable results. Intracellular Na⁺ levels were measured in viable cells separated by centrifugation through 3 mL of Percoll (Pharmacia, Uppsala, Sweden) solution (d = 1.06) in 0.25 mol/L sucrose. After centrifugation, the Percoll solution was rapidly removed by aspiration, and the cell pellets were extracted with 0.5 mL of 0.8 N perchloric acid. Na⁺ was measured in aliquots of the protein-free acidic supernatants diluted 200 times with distilled water using a Varian AA-1475 atomic absorption spectrophotometer (Varian Instruments Division, Palo Alto, CA). The values were corrected for the protein content of each pellet and Na⁺ concentration was expressed as nmol/mg of proteins.

Statistical analysis for multiple comparisons was performed by one-way analyses of variance test with Bonferroni's corrections for multiple comparisons. The distribution normality of the groups considered was preliminary evaluated by the Shapiro-Wilk test.

Cold storage of isolated rat hepatocytes suspended in KHH medium led to a progressive increase of intracellular Na⁺(Fig. 1A) Na⁺ accumulation was evident already after 12 hr of incubation at 4°C and preceded hepatocyte death. Cell viability started to decline after 24 hr of storage in cold KHH and was less than 20% after 72 hr (Fig. 1B). Hepatocyte cold-storage in Na⁺-free KHH buffer afforded an almost complete protection against Na⁺ accumulation and cell killing (Fig. 1). Lowering the pH of the incubation medium to 6.5 also effectively reduced Na⁺ overload and preserved hepatocyte viability (Fig. 1). The addition of 2 mmol/L glycine also afforded a good protection against the effects of cold hypoxia (Fig. 1). The cytoprotective action of the acidic medium and of glycine was, however, lost by promoting Na⁺ increase by the addition of Na⁺/H⁺ ionophore monensin (10 μmol/L) (Fig. 2). However, monensin was not cytotoxic when added to cells suspended in Na⁺-free KHH medium (Fig. 2), indicating that alterations of intracellular Na⁺ homeostasis are critical for hepatocyte survival during cold storage. A low Na⁺ content (25 mmol/L) and the substitution of extracellular Cl⁻ with the impermeant anion lactobionate (4-O-β-galactopyranosyl-D-gluconic acid) are the main features of UW solution that is currently used for the cold preservation of human liver grafts. We have observed that intracellular Na⁺ content and cell viability of isolated hepatocyte stored up to 72 hr at 4°C in UW solution were comparable to those of cells maintained in Na⁺-free Krebs-Henseleit-Hepes buffer (Fig. 1). The addition of monensin also did not interfere with the cytoprotective effect of UW solution (Fig. 2). To evaluate the relative contribution of low extracellular Na⁺ and of lactobionate in protecting against cold storage injury, isolated hepatocytes were stored up to 48 hr at 4°C in a modified Krebs-Henseleit-Hepes containing either sodium or potassium lactobionate (115 mmol/L) as the major osmolite. Figure 3 shows that there was no differences in Na⁺ accumulation and in cell death between hepatocytes cold stored 24 hr in the presence of either Na-lactobionate or K-lactobionate. However, after 48 hr of cold preservation hepatocytes maintained in K-lactobionate containing buffer showed a better viability and lower Na⁺ content than those kept in Na-lactobionate containing buffer (Fig. 3).

Figure 1: Changes in intracellular Na⁺ concentrations (A) and in hepatocyte viability (B) during cold storage under different conditions. Isolated rat hepatocytes (10⁶ cells/ml) were maintained up to 72 hr at 4°C under a nitrogen atmosphere in, respectively, KHH buffer pH 7.4 (•), UW solution (▪), a Na⁺-free KHH buffer (▴), KHH buffer containing 2 mmol/L glycine (▾), and Krebs-Henseleit-MES buffer pH 6.5 (⋄). The results are means of five different experiments ± S.D. Statistical significance: *P<0.01 versus cells maintained in modified KHH buffer or UW solution.

Figure 2: Effect of Na⁺/H⁺ ionophore monensin on Na⁺ content (A) and cell viability (B) of isolated rat hepatocytes maintained 24 hr at 4°C under a nitrogen atmosphere. Hepatocytes were suspended in, respectively, Krebs-Henseleit-MES buffer pH 6.5, Na⁺-free KHH buffer, KHH buffer containing 2 mmol/L glycine (Gly), and UW solution in the presence (stripped bars) or in the absence (open bars) of 10 μmol/L monensin. The results are means of three different experiments ± S.D. Statistical significance: *P<0.001 versus samples not containing monensin.

Figure 3: Relative contribution of Cl⁻ and Na⁺ substitution on hepatocyte preservation during cold storage. Isolated hepatocytes were cold stored in modified KHH buffers containing, respectively, 115 mmol/L Na-lactobionate (open bars) or K-lactobionate (stripped bars) and the changes intracellular Na⁺ content (A) and hepatocyte viability (B) were monitored after 24 and 48 hr of incubation at 4°C under a nitrogen atmosphere. The results are means of three different experiments ± S.D. Statistical significance: *P<0.01 versus cell preparations incubated with K-lactobionate.

Gizewski et al. ⁽⁷⁾ have previously reported that short-term (30 min) cold incubation of cultured hepatocytes and liver endothelial cells in UW solution or Krebs-Henseleit buffer caused a transient lowering of intracellular Na⁺ and Cl⁻ content. In our work, the Na⁺ levels of hepatocytes exposed to prolonged hypothermia in Na⁺-free KHH buffer or UW solution were not significantly lower (89 ± 22 and 95 ± 15 nmol/mg protein, respectively) than those of freshly isolated cells (115 ± 12 nmol/mg protein). However, a progressive increase in the Na⁺ content was appreciable in hepatocytes maintained in complete KHH buffer. Fiegen et al. ⁽⁶⁾ have reported that the inhibition of Na⁺-K⁺-Cl⁻ cotransporter by bumetamide reduced by about 30% Na⁺ accumulation during warm hypoxia of isolated perfused livers. Although with the inhibition of plasma membrane Na⁺/K⁺ ATPase by low temperature ⁽¹⁾ and the activation of Na⁺-K⁺-Cl⁻ cotransporter might contribute to Na⁺ influx during cold hypoxia, we have observed that cell incubation in an acidic medium greatly reduced Na⁺ accumulation. Conversely, promoting the exchange of Na⁺ for H⁺ by the addition of monensin reverted the effect of the acidic medium. Thus, influx of Na⁺ throughout the acid buffering systems in response to intracellular acidification likely represents the main cause for Na⁺ accumulation during cold hypoxia.

It has been reported that the substitution in UW solution of extracellular Cl⁻ with the impermeant anion lactobionate is critical for liver preservation, although replacing K⁺ with Na⁺ do not affect liver enzyme release and the transplant outcome in different animal models ⁽¹⁾. We have observed that Na⁺ accumulation is greatly reduced during cold storage in lactobionate-containing buffers. An interference with anion fluxes through hepatocyte plasma membranes might account for the effect of lactobionate, because the substitution of chloride with cell membrane impermeant cation gluconate, but not with the permeant ion nitrate, decreases Na⁺ influx during hepatocyte warm hypoxia ⁽⁸⁾. However, during prolonged hypothermia the use of lactobionate as a main cation did not afford a complete protection against Na⁺ accumulation. Thus, the low Na⁺ content should be regarded as an additional important factor for the cytoprotective action of UW solution.

Hepatocyte swelling can be observed as a result of Na⁺ increase during metabolic inhibition ⁽⁴⁾. Furthermore, cold preservation affects the capacity of hepatocytes to regulate cell volume in response to osmotic loads ⁽⁹⁾. It is therefore possible that Na⁺-mediated cytotoxicity during cold storage might involve osmotic stress. Nonetheless, the involvement of Ca⁺⁺-dependent mechanisms, including the activation of Ca⁺⁺-activated proteases and phospholypases can not be excluded, considering that the reversed activation of Na⁺/Ca⁺⁺ exchanger is an important cause for the increase of hepatocyte cytosolic Ca⁺⁺⁽¹⁰⁾.

Glycine has been shown to improve cold storage and reperfusion injury of rat livers ⁽¹¹⁾. Such a beneficial effects has been ascribed to the ability of glycine to prevent the injury of liver sinusoidal endothelial cell during reperfusion ⁽¹¹⁾. However, the effect of glycine on Na⁺ accumulation might also contribute in maintaining cell viability during cold hypoxia. The action of glycine on Na⁺ influx appears to be related to the block of specific Cl⁻ channels, because the glycine-receptor antagonist, strychnine, and the incubation in a Cl⁻-free medium similarly prevented Na⁺ overload and hepatocyte killing during warm hypoxia or KCN poisoning ⁽⁸⁾.

In conclusion the results presented demonstrate that Na⁺ overload might contribute to liver graft injury by hypothermia and suggest that preservation of Na⁺ homeostasis during cold storage might improve graft preservation.