Renal Cell Apoptosis and New Treatment Options in Sepsis-Induced Acute Kidney Injury

Abstract

Sepsis is a common and important cause of mortality in critically ill patients. Acute kidney injury (AKI) is one of the most important factors determining morbidity and mortality in the prognosis of sepsis. Recent studies have indicated that the pathogenetic mechanism in septic AKI is totally different from that in non-septic AKI. Our understanding of sepsis-associated AKI pathophysiology is shifting from renal vasoconstriction, ischemia, and acute tubular necrosis to heterogeneous vasodilation, hyperemia, and acute tubular apoptosis. Especially, apoptosis is gradually gaining importance in the understanding of the development of renal injury. The frequency of renal tubular apoptosis on biopsies of septic patients has been pointed out in recently published studies. Apoptosis can be triggered by ischemia, exogen toxins, or endogen mediators. It has been shown in some animal models that hyperglycemia, which is common in critically ill patients, causes apoptosis in renal tubular cells. New treatment options have emerged in the light of recent findings. Ghrelin that inhibits pro-inflammatory cytokines, caspase inhibitors that block the apoptotic pathway, and suppression of anti-inflammatory reactions are under study. Among the existing methods of treatment, usage of arginine, which is a vasopressor agent, ventilation with a low tidal volume, and hemofiltration methods cleaning toxic mediators from the circulation should be considered in the first place. Hyperglycemia treatment is of major importance, since, besides its anti-inflammatory effect, it has a protective role on the kidney. Regarding pathogenesis, rates of morbidity and mortality are aimed to be reduced through the new agents of therapy that have been studied on.

• sepsis
• apoptosis
• renal failure
• new treatment options

INTRODUCTION

Apoptosis is a term in ancient Greek used for leaves that yellow and fall off the trees. Defined as a programmed cell death, apoptosis is the process of regular removal of unwanted cells through a molecular program requiring active energy. Apoptosis is necessary for the organism to control the number of cells and the tissue volume and to protect itself from the unamenable cells threatening the homeostasis.^1,2 Cells undergoing apoptosis contract rapidly, lose their intracellular connections, and display dense chromatin intensification. As a result of apoptosis, nuclear fragmentation, cytoplasmic granulation, cellular fragmentation, and small apoptotic particles occur as well. These apoptotic particles are removed rapidly by the neighboring cells and macrophages. It is difficult to determine the apoptosis in the preparations, which contain millions of cells, because the morphologic changes occurring in apoptosis take place and are completed in less than an hour.³

Many morphological changes seen in apoptotic cells are caused by the cysteine proteases active in these cells. These enzymes, which are called death enzymes, are generally similar to each other and belong to a big protein family called caspases.⁴ A dozen of caspases and more than 100 caspase substrates have been defined in humans, at least a third of which playing a role in the apoptosis process.⁵ Caspases are accepted as the central killers of the apoptosis process, because inhibition of caspases through mutation or using small molecules that bear inhibitor characteristics could slow down or completely stop the apoptosis process.^5,6 Although it is accepted that the apoptosis process cannot be stopped once it has started, the pharmacological inhibition of caspases generally, if not always, saves the cells from apoptosis.⁷

It has been shown that apoptosis occurs in renal diseases. Apoptosis can be triggered by ischemia, exogen toxins, or endogen mediators. Apoptosis plays a role not only in starting the damage in the kidney but also in maintaining it. Mononuclear cellular infiltration, which consists basically of monocytes/macrophages and T cells, is characteristic in most of the renal diseases. Monocytes/macrophages secrete tumor necrosis factor-alpha (TNF-α), Fas ligand, free oxygen radicals, and nitric oxide. In this way, inflammatory cells act as factors increasing the apoptosis.⁸

The role of renal cellular apoptosis in septic acute kidney injury (AKI) has been studied by using immune-mediated stimulants such as TNF. When high doses of TNF are added to renal proximal cell cultures, DNA fragmentation and expressions of Fas messenger RNA and Fas related death zone protein increase.⁹ TNF and lipopolysaccharide (LPS) affect the apoptotic cell death in glomerular endothelial cells, depending on time and concentration.¹⁰ This apoptotic activation is thought to occur very early in septic kidneys.¹¹

It has been shown in some in experimental models of acute ischemic and toxic renal injury, such as glycerol-induced AKI (Gly-AKI), ischemia/reperfusion-induced AKI (I/R-AKI) and LPS-induced AKI (LPS-I-AKI) that human renal tubular cells are killed by apoptosis as well as by necrosis. Hotchkiss and his friends proved that not only necrosis but also apoptosis plays an important role in sepsis and septic shock.¹² It has been claimed in a recent study that apoptosis is the most important factor in septic AKI. In this study, postmortem renal biopsies are applied to eight traumatic patients, nine non-septic AKI patients, and 19 patients who died of septic shock. It is reported that the histopathological findings observed in biopsies taken from septic AKI patients are acute tubular injuries or necrosis in different degrees, leukocyte infiltration in glomeruli, interstitial capillaries, and tubular lumen, and apoptosis in tubular and rarely in glomerular cells. Thrombotic findings were rarely seen. Acute tubular apoptosis was not observed in non-septic AKI patients. While this study is the only controlled study made after 1980 which evaluates biopsies taken from humans, it has some drawbacks as well. As biopsies were taken from the traumatic patients just at the site of the accident, it’s probable that not enough time passed for the development of histopathological findings in the kidney. Another drawback of the study is that since the biopsies are taken postmortem, the reversible changes expected to occur in recovering patients could not be observed.¹³

The importance of treatment with caspase inhibitors in apoptotic–inflammatory AKI is increasing every day. Indeed, caspase inhibitors ameliorate ischemia-reperfusion injury in different organs, including the kidney. Like caspase inhibition, treatment options blocking the apoptotic pathway are also promising.¹⁴ It was found in a rat model of Gly-AKI that caspases caused inflammation, apoptosis, vasoconstriction, and tubular necrosis. Early caspase inhibition attenuated these processes and improved the renal functions significantly.¹⁵ In another study, murine models of septic AKI and I/R-AKI were compared to understand biochemical, histologic, and cytokine changes. Apoptosis was assessed, and the effect of caspase-3 inhibition on renal functions was also examined. The effect of interleukin-10 blocking was also compared. It was found that, in septic kidneys, acute tubular necrosis or inflammation was minimal but tubular cell apoptosis was prominent and caspase-3 activity was positively correlated with renal dysfunction. A decrease in apoptosis by caspase-3 inhibitors resulted in attenuation of renal dysfunction. Septic models were associated with an increase in interleukin-10 and also showed massive immune cell apoptosis with increased regulatory T cells. Blocking interleukin-10 rescued septic mice from the development of AKI, whereas it had no effect in ischemia/reperfusion injury.¹⁶ An important reason that the immunity is repressed in septic patients is the lymphocyte apoptosis. Septic patients are generally lymphopenic. In addition, a decrease in B and CD4 lymphocyte subgroups is also observed in these patients. Anergy and decrease in T cell response seen in most of the septic patients is an excessive counter-response aiming to balance the pro-inflammatory response that had initially emerged. This situation could lead to the development of an organ dysfunction that can occur afterwards. Various researchers suggested that prevention of the emerging immunosuppression might have a role in the treatment of sepsis. It has been indicated that prevention of lymphocyte apoptosis decreased the mortality rate in the sepsis that occurs after the cecal ligation in subject animals. In another small study in which interferon gamma therapy is used, a relatively better rate of survival is obtained.^17–19 These findings suggest that suppression of the anti-inflammatory reaction may be an important treatment option in sepsis.

A recent study showed that extracorporeal therapy with polymyxin B reduced the pro-apoptotic activity in the plasma of septic patients whose renal cells were cultured. It seems likely that plasma separation techniques can prove to be beneficial in renal injury by removing the pro-apoptotic factors and cytokines.²⁰

Ghrelin shows a protective effect on kidneys by inhibiting pro-inflammatory cytokines, particularly TNF-α, in the circulation and in kidneys. Ghrelin may thus hold promise for managing endotoxemia-induced AKI. Although further evidence confirming this beneficial effect is definitely needed, studies with this agent in septic AKI seem justified.²¹

Vasopressor agents applied in septic AKI are also of importance. It has been shown in an animal model, which is in a septic shock, that usage of low doses of arginine vasopressin causes less tubular apoptosis, systemic inflammation, and renal damage when compared to noradrenalin.²²

It’s been indicated in experimental studies with ischemia-reperfusion models that erythropoietin decreases apoptotic cell death and induces tubular proliferation.²³ It has also been shown in a retrospective clinical study that erythropoietin decelerates the pace of developing a chronic renal dysfunction in pre-dialysis patients.²⁴ However, high doses of erythropoietin might lead to renal vasoconstriction, hypertension, and increased risk of thrombosis. Recently derivated erythropoietins with no hematopoietic activity might prove to be safer in these patients.

Hyperglycemia is frequent in intensive care units and in critically ill patients and has negative effects on morbidity and mortality. In healthy individuals, the plasma glucose is kept within a narrow range by using insulin and counter-regulatory hormones (glcocagone, epinephrine, cortisole, and growth hormone) which balance the glucose metabolism between the liver and peripheral tissues. In cases of stress, however, counter-regulatory hormones increase and this, while increasing the glucose production, decreases the usage of glucose by peripheral tissues, thus causing hyperglycemia.^25,26 Apart from stress hyperglycemia, factors such as usage of steroids and vasopressor agents, immobilization and enteral and parenteral nutritions or fluid infusions containing excessive amounts of glucose also contribute to the hyperglycemia in intensive care patients. Hyperglycemia has also some negative effects such as fluid imbalance, immunosuppression, increase in inflammation, leukocyte dysfunctions, tendency to thrombosis, and endothelial dysfunction.²⁶ With a tight glucose control, a remarkable decrease in mortality and morbidity has been achieved; and in patients requiring intensive care for more than 5 days, there has been a decrease in the time they stay in the intensive care and in the development of renal failure. Also, a 46% regression in sepsis and a less critical disease polyneuropathy has been observed.²⁷ Yet, tight glucose control bears a risk of hyperglycemia. NICE-SUGAR (Normoglycemia in Intensive Care Evaluation- Survival Using Glucose Algorithm Regulation) study, comparing the intensive insulin therapy with the traditional insulin therapy in intensive care patients, is one of the most comprehensive study on this subject.²⁸ In this study, a significant increase in mortality has been observed in the group in which intensive insulin therapy has been applied when compared to the traditional therapy group. Considering, too, the risk of hypoglycemia in general meaning, glucose level should be kept below 150 mg/dL. When a glycemic control strategy is started, a nutrition protocol in which, preferably, the enteral way is used should be adopted. The risk of hypoglycemia should be reduced by providing a continuous glucose support.²⁹

Apart from reducing the glycemia, insulin therapy is claimed to have some other independent effects. It is reported in some recent publications that it has a protective effect on kidneys. It is thought to prevent AKI in sepsis with its anti-inflammatory and—on tubular epithelial cells—anti-apoptotic effects.³⁰ Although the protective role of insulin on the kidney could not clearly be explained, some mechanisms regarding to it have been suggested. Its effects on the lipid metabolism have been studied. Hypocholesterolemia [low levels of low density lipoprotein (LDL) and high density lipoprotein (HDL)] and hypertriglyceridemia are seen in critically ill patients and intensive insulin therapy has been proved to improve this profile. Improvement of the lipid profile, especially increase in LDL could be considered as a mechanism of the protective effect on kidneys. This analysis does not substantiate a cause–effect relationship but at least suggests that the improved lipid profile and especially the elevation of the LDL levels may represent one potential mechanism for the renoprotective effect of intensive insulin therapy, consistent with previous observations in the animal model of renal ischemia-reperfusion.³¹

The damaging effect of hyperglycemia on the endothelial cells is described in ischemia/reperfusion mediated AKI models. Intercellular adhesion molecule-1 (ICAM-1) and E-selectin levels are high in AKI. It has been shown in some experiments done with animal models that antibodies, antisense oligonucleotides and E-selectin inhibition against ICAM-1 in ischemic damage, have protective effects on kidneys. Therefore, it is stated that the protective effect of intensive insulin therapy on the endothelium could be observed on kidneys, as well.³¹

Another interesting point in septic AKI pathophysiology is the communication among the organs. In acute respiratory distress syndrome (ARDS), applying low tidal volume ventilation reduces the renal damage. Observation of apoptosis when renal cells are incubated into rabbit plasmas in which damaging ventilation methods are used lead us to think that this relationship between the organs might be with Fas-ligand mediators. In ARDS patients ventilated with high tidal volume, a significant relationship between plasma Fas-ligand levels and serum creatinine has been found as well.^11,32

CONCLUSIONS

Sepsis related AKI continues to be a very serious health problem today, causing significant amounts of losses due to its high incidence and mortality rate. The main reason for this is that its pathogenesis has not been sufficiently understood. In recent studies, the view that the main pathogenetic factor in septic renal damage is not hemodynamic failures or ischemia as is thought, but it is inflammation and renal cellular apoptosis that act as the main agents gained priority. As a result, searches for the therapy shifted to that direction and agents aiming to suppress apoptosis and inflammation are currently being considered. In the light of these new findings, it is inevitable that the stereotyped treatment algorithms should change. Among the existing methods of treatment, usage of arginine, which is a vasopressor agent, hyperglycemia treatment, ventilation with a low tidal volume, and hemofiltration methods removing inflammatory cytokines from the blood should be considered in the first place. Ghrelin that inhibits pro-inflammatory cytokines, caspase inhibitors that block the apoptotic pathway, and suppression of anti-inflammatory reaction with interleukin-10 antagonists are treatment methods that are likely to be widely applicable in the future. As the mortality rate is high it would be appropriate to individualize the treatment and plan it according to the needs of the patient.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.