| Summary: | 博士 === 國立臺灣大學 === 臨床醫學研究所 === 95 === Renal failure is a common complication seen in clinical practice. Renal failure usually results in electrolyte imbalance, fluid overload and uremia which cause further complications leading to higher morbidity or mortality. Renal failure is divided into acute reanl failure (ARF) and chronic renal failure (CRF) according to the developmentory rate. The incidence of ARF is 1~15% in cardiovascular surgery, 19 % in moderate sepsis, 23 % in severe sepsis, and 51% in septic shock when blood cultures are positive. In these events ischemia plays an important role in the pathogenesis of shock-related acute renal injury. However, the development of ARF is sometimes the result of synergistic effect of ischemia with other intra-renal response subsequent to disease process, such as inflammation in septic ARF, thus further complicates the diagnosis and treatment of ischemic ARF. CRF is also an important disease in Taiwan. The prevalence and incidence of end stage renal disease (most coming from CRF) under dialysis is 1706 and 375/million respectively which are the top two in the world. Diabetes nephropathy and chronic glomerulonephritis are the two major diseases that lead to CRF therefore end stage renal disease. Renal failure not only hurt the personal health but also put a great impact on the family and society.
The kallikrein-kinin system is an important cytokine/paracrine system in kidney. This system includes kallikrein, kininogen, kinin, and bradykinin B2 and B1 receptors. Kallikrein cleaves kininogen substrate to release vasoactive kinin peptide via limited proteolysis. Binding of intact kinin to the B2 receptor activates secondary messengers, such as nitric oxide (NO)/cGMP and prostacyclin, and triggers many biological effects, such as vasodilation, inflammation, natriuresis, and edema formation. In kidney, bradykinin could increase renal perfusion which may improve the renal blood supply after acute ischemic renal injury or attenuate ischemia in chronic renal disease (CKD), which is found in many CKD includning chronic glomerulonephritis. The anti-hypertensive effect may be helpful in attenuating the disease progression in CKD. Besides, the cellular proliferative effect may also help the cell survival and renal repair process after reanl injury either in acute kidney injury (AKI) or CKD. However, the inflammatory effect may be detrimental to AKI and CKD, since inflammation is a very important factor in reanl damage.
We expect that kallikrein-bradykinin system will exert their action and influence the disease process in AKI and CKD. For the study of AKI, we apply acute ischemic reperfusion (I/R) renal injury as animal model and study the role of this system in I/R injury. For CKD, we conduct clinical study to explore the role of kallikrein-bradykinin system since there is lack of good animal model of major CKD, diabetes nephropahty and chronic glomerulonephritis. It is also difficult to obtain renal tissue or to measure the bradykinin level in urine. So, we first study the relationship between urinary kallikrein and renal function or the status of reanl injury to find out any clue of the role of kallikrein-bradykinin system in CKD.
The kallikrein-kinin system contributes to the protection of ischemic heart. Schoelkens et al. were the first to report the cardioprotective effects of bradykinin. Kallikrein gene delivery has also been observed to attenuate ischemic stroke and acute myocardial ischemia/reperfusion (I/R) injury. Conversely, several studies have shown bradykinin plays a deleterious role in brain, heart and other organs, such as lung, liver and intestine after I/R injury. Yet there is still no study evaluating the role of kallikrein-bradykinin system in I/R renal injury.
After the ischemic with/without perfusion injury, there will be effacement of the brush boder and necrosis/apoptosis of proximal tubular cells. The dead cells will slough from tubular lumen which will combine with luminal protein and form cast in the lumen. The basement membrane is denuded. Patchy necrosis is seen in outer medulla, especially in the S3 segment of proximal tubule. If the ischemia is more severe, tubular necrosis will extend to proximal tubule in the cortex. There is edema and inflammatory infiltration in the interstitium. However, the glomerulus is unaffected. Due to the ischemic insult, the endothelium is injured which impair the release the endothelium-derived nitric oxide. In contrast, the vasoconstrictors, such as endothelin-1, are released. Both mechanisms impair the renal reperfusion resulting in persistent ischemia in reperfusion phase, further extend renal injury. Bradykinin may improve the renal perfusion in the reperfusion phase of I/R injury. This effect may be beneficial in I/R renal injury.
After ischemia cellular ATP is degraded into ADP and AMP. The AMP is further degraded into hypoxanthine and the accumulation of hypoxanthine will stimulate the generation of reactive oxygen species (ROS). During the reperfusion phase, xanthine oxidase is activated and transforms hypoxanthine into xanthine with the by-product formation of hydrogen peroxide and superoxide. ROS can induce apoptosis by damaging DNA, oxidizing membrane lipids, and/or directly activating the expression of the genes/proteins responsible for apoptosis. The generated ROS also act synergistically with calcium to activate mitochondrial phospholipase A2 enhancing further inflammatory response and renal damage in renal I/R injury. In mitochondria, the generated ROS opens the permeability transition pore (PTP) which induces swelling of mitochondria, collapse of mitochondrial membrane potential, and uncoupling of mitochondrial oxidative phosphorylation which produces more ROS. Cytochrome c is therefore released into cytosole leading to cell necrosis and apoptosis. The overproduction of ROS and accumulation of mitochondrial Ca2+ have been proposed to be the main triggers of the PTP opening.
Calcium also plays an important role in ischemic reperfusion (I/R) renal injury. Ischemia causes the loss of ATP which will result in the impairment of Na+-K+ pump in cell membrane leading to loss of membrane potential and opening of voltage-gated Ca2+ channel, therefore influx of extracellular Ca2+ and elevation of intracellular Ca2+. Besides, ATP depletion also impairs the re-uptake of Ca2+ by endoplasmic reticulum, further enhances the intracellular Ca2+ accumulation. Elevation of intracellular Ca2+ will activate protease, phospholipase and degradation of cytoskeleton leading to cell death. Mitochondria takes intracellular excess Ca2+ and initiate the opening of PTP leading to mitochondrial damage and related apoptotic pathway. Blocking the Ca2+ channel or using Ca2+ chelator attenuates I/R renal injury. Bradykinin is known to enhance the intracellular free Ca2+ accumulation, mitochondria Ca2+ uptake and mitochondrial ROS generation in cell culture. So it may enhance the Ca2+ accumulation and ROS generation in kidney during I/R renal injury leading to more cell damage.
Therefore, we hypothesize that kallikrein-bradykinin system could play a role in I/R renal injury, either beneficial or detrimental. We conducted animal and cell culture studies to evaluate the role of kallikrein-bradykinin system in acute I/R renal injury. We activated the tissue kallikrein-kinin system by administration of rat tissue kallikrein protein in a rat model of renal I/R injury and examined the effects on renal function, pathology, ROS generation, apoptosis and inflammatory response. In cell culture studies, we use ATP depletion model in renal proximal tubular cells to simulate ischemia. The effect of bradykinin on cellular death, ROS generation, mitochondrial damage, and apoptotic pathway activation induced by ATP depletion was examined. Possible signaling pathway was also approached. The effect of bradykinin on cell death was further examined in hypoxic study. These results provided insights into the role of the kallikrein-kinin system in acute I/R renal injury.
Forty eight hours after I/R renal injury, the serum creatinine, blood urea nitrogen and excretion fraction of sodium increased in control rats. Administration of kallikrein protein further increased the impairment of renal function and this effect was blocked by co-treatment with bradykinin B2 receptor (B2R) antagonist (HOE140), but not bradykinin B1 receptor (B1R) antagonist (Lys-(des-Arg9- Leu8-bradykinin). Supplement with phosphate buffered saline could not reverse the detrimental effect of kallikrein. Administration with B2R or B1R antagonist only did not attenuate I/R renal injury. We also measured the renal artery blood flow one hour after I/R injury. Renal blood flow did decrease after I/R injury in control rats, but there was no difference between rats whether treated with kallikrein or not. The blood pressure 1 hour after reperfusion was also not different between each group. These results indicate that the activation of B2R is detrimental to I/R renal injury and this effect is not through enhanced natriuresis by bradykinin activation, change of renal blood flow or systemic blood pressure.
We further examined the degree of tubular necrosis and inflammatory response after I/R injury. Similar phenomenon was seen. Activation of B2R enhanced the tubular necrosis, macrophage/neutrophil infiltration, the gene/protein expression of MCP-1 and TNF-alpha in renal tissue. To explore the possible mechanism, we measured the oxidative stress between different groups. Using real-time chemiluminescence recording to measure superoxide production, we found that superoxide generation began in the beginning of ischemia and increased progressively. The initiation of blood re-flow in beginning of reperfusion stimulated a surge of superoxide production and this production sustained till 3.5 hours after I/R procedure. Activation of B2R aggravated the ROS generation in both ischemic and reperfusion phases. Measurement of tissue hydrogen peroxide, free malondialdehyde, reduced and oxidized glutathione also demonstrated the enhancement of oxidative stress by activation of B2R. The renal apoptotic cells, stained by TUNEL and anti-caspase-cleaved fragment (p85) of human poly (ADP-ribose) polymerase (PARP) antibody, were examined 4 hours after I/R injury from the same tissue from ROS measurement. The kallikrein treated rats, which had more ROS generation, also had higher degree of tubular cell apoptosis. Therefore, the in vivo study indicates that early activation of B2R aggravates I/R renal injury through enhancement of ROS generation in rats. This finding is compatible with the findings of Chien et al. (2001), in that the amount of ROS was positively correlated to the duration of ischemia and the degree of apoptosis.
The sources for ROS generation after I/R may come from inflammatory cells or the resident cells. In kidney Chien et al. (2001) demonstrated the major sources of ROS came from proximal tubular cells in early stage (within 4 h after ischemia) of renal I/R injury. The site of ROS generation was compatible with the site of tubular necrosis induced by I/R injury. It is probable the augmentation of ROS generation by bradykinin receptor activation also happened in proximal tubular cells after I/R injury. In cell culture, bradykinin increases mitochondria ROS generation in cardiomyocyte or vascular smooth muscle cell. Therefore, we hypothesize that bradykinin could enhance ROS generation, mitochondrial injury and cellular death induced by ischemic injury in renal tubular cells culture model.
Cell culture studies were conducted to uncover the underlying mechanism of the detrimental effect of bradykinin system in ischemic injury. Using substrate deprivation and treatment with antimycin A, cellular ATP was depleted to simulate ischemia in proximal tubular cell line, NRK52E. We found that activation of B2R aggravated the cellular death induced by ATP depletion, which were revealed by more LDH release, more subG0/G1 cells, more apoptotic body and Annexin V biding assay in bradykinin treated cells. Using sub G0/G1 evaluation, we further confirmed that bradykinin also enhanced the apoptosis induced by hypoxic stimuation for 9 hours. Using fluorescence dye and flow cytometry, we proved that activation of B2R enhanced the ROS generation (including superoxide and hydrogen peroxide) and loss of mitochondrial membrane potential induced by ATP depletion. Loss of mitochondrial membrane potential causes mitochondrial damage, therefore related apoptotic pathway activation. Our results revealed that there was more cytochrome c released into cytoplasma in bradykinin treated cells. The activated form of caspase 9 and PARP was more in bradykinin treated cells than in control cells. Renal tubular cell was known to be the major source of ROS in I/R renal injury and prolong ischemia was associated with more ROS generation and tubular damage. Our studies demonstrated that activation of B2R enhanced ROS generation induced by ATP depletion in renal tubular cells. With more ROS, more mitochondrial damage and cell death were observed in bradykinin treated cells when compared to cells subjected to ATP depletion only. This in vitro study complements the in vivo evidence that the enhancement of ischemic reperfusion renal injury by early B2R activation was through the enhancement of ROS generation.
In addition to ROS, calcium also facilitate the opening of mitochondrial membrane permeability transition pore and plays an important role in ischemic/reperfusion injury in vivo and in vitro. Bradykinin is known to enhance intracellular Ca2+ accumulation in cell culture. In this study, we demonstrated activation of B2R also enhanced the intracellular and mitochondrial Ca2+ accumulation induced by ATP depletion. Even in cells already subjected to ATP depletion for 1.5 hours, bradykinin also could increase intracellular Ca2+ concentration. More intracellular and mitochondrial Ca2+ act synergistically with ROS to open the mitochondrial membrane permeability transition pore leading to greater loss of mitochondrial membrane potential, therefore more severe mitochondrial swelling, and rupture of the mitochondrial membrane. Accompanying the mitochondrial damage, cytochrome c is released into cytosole, which cleaves procaspase 9 into the active form of caspase 9, thus conferring the apoptotic death signal. These more apoptotic events are seen in bradykinin treated cells. These results indicate the important role of Ca2+ in bradykinin mediated detrimental role in cellular death induced by ATP depletion.
There are three sources of calcium mobilization to elevate intracellular Ca2+: (1) Ca2+ release from internal stores, such as the endoplasmic reticulum; (2) influx through dihydropyridine sensitive (voltage-dependent) Ca2+ channels; (3) and dihydropyridine insensitive (receptor mediated) Ca2+ input from extracellular space. The release of Ca2+ from internal stores could be stimulated by increase of inositol 1,4,5-trisphosphate (IP3). Activation of bradykinin receptors is known to activate PLC resulting in phosphoinositide hydrolysis in the plasma membrane and generation of IP3 in different cell types. We applied PLC inhibitor (U73122) in cell culture studies and demonstrated that the enhancement of ATP depletion related intracellular and mitochondrial Ca2+ accumulation by bradykinin could be blocked by PLC inhibitor to the similar level as cells subjected to ATP depletion only. This indicates the possible role of PLC-IP3 signaling pathway in bradykinin enhanced Ca2+ accumulation induced by ATP depletion. The detrimental effect of bradykinin on generation of ROS, loss of mitochondrial membrane potential and apoptosis was also reversed by co-treatment with PLC inhibitor. These results indicate the detrimental effect of bradykinin on ATP depletion related cellular injury is through activation of PLC signaling pathway. However, bradykinin could also increase intracellular Ca2+ through the opening of voltage-dependent or receptor mediate Ca2+ channel. The possible involvement of other two pathways in bradykinin enhanced calcium accumulation and ROS generation induced by ATP depletion needs further studies to clarify. Our studies revealed that PLC inhibitor could almost blunted the dentrimental effect of bradykinin on ATP depletion related cell injury, so the voltage-dependent or receptor mediate Ca2+ channel may play a minor role or should be activated through PLC pathway activation.
We do not know whether bradykinin receptor activation could enhance ROS generation and apoptosis induced by ATP depletion independent of mitochondrial injury. Endoplasmic reticulum (ER) stress is known to induce apoptosis in ischemic reperfusion injury. Bradykinin could enhance ROS generation, intracellular Ca2+ accumulation, release of Ca2+ from ER in cells subjected to ATP depletion. All these events could result in ER stress. Once ER stress is present, ER stress related apoptotic pathway, such as caspase 12 activation, c-Jun NH2-terminal kinase pathway activation and growth arrest and DNA damage–inducible gene 153 expression will be activated. Therefore, bradykinin may enhance cell death through enhancement of ER stress induced by ATP depletion. But this hypothesis needs further studies to clarify.
One of the other possible mechanisms in which activation of bradykinin B2 receptor aggravates I/R renal injury is through the activation of phospholipase A2 (PLA2). Several studies have shown that cytosolic, mitochondrial and microsomal subcellular fractions of PLA2 is activated after I/R injury. PLA2 could also be activated by increases in intracellular or extracellular Ca2+ elicited by the inflammatory mediator bradykinin. ROS has also been observed to act synergistically with calcium to activate mitochondrial PLA2 in isolated kidney and in vivo I/R renal injury. Therefore, it is probable that activation of PLA2 induced by ischemic reperfusion renal injury is further enhanced by activation of B2R. The activated PLA2 is critical to the production of proinflammatory lipid mediators, as the initiation of the inflammatory response. Inflammation is considered to be the major pathophysiological pathway of acute renal failure resulting from ischemia. More activation of PLA2 may induce more tissue injury in I/R renal injury with bradykinin system activation.
In contrast to the protective role of bradykinin system in heart and brain in some studies, our study did not reveal a beneficial role of early activation of bradykinin system in I/R renal injury. The reason for this discrepancy is unknown. One possible explanation is that bradykinin may play different role in different stage of I/R injury. In beneficial effect study of brain the activation of bradykinin indeed was initiated many hours after ischemia since Xia et al.(2004) administrated kallikrein gene after the initiation of reperfusion. The studies of detrimental effect of bradykinin in other organs were usually performed using early blocking of B2R strategy. In early stage bradykinin might exert the inflammatory response while in late stage bradykinin may facilitate cell proliferation. These effects put different impact on I/R injury. The other explanation is that heart and brain have collateral circulation that bradykinin could enhance collateral circulation through vasodilatory effect once one supply vessel is occluded. But there is only one artery to supply blood in kidney. Therefore, bradykinin may only exert the inflammatory effect in early stage of I/R renal injury.
In clinical practice, plasma kallikrein-bradykinin system is activated during sepsis leading to systemic vasodilation and enhances inflammatory response. Besides, recent studies have revealed that inflammatory response, in addition to ischemia, also played a role in the pathogenesis of septic ARF. Tissue kallikrein is found to be released during intestinal inflammation in animal, human inflammatory bowel disease and mechanical ventilation related lung injury. Therefore, it is possible that renal tissue kallikrein-bradykinin system is also released and activated by the stimulation of inflammation in sepsis. Since activation of B2R will aggravates I/R renal injury, kallikrein-bradykinin system may play a synergistic action with ischemia on the development of septic ARF. To test this hypothesis, we need to examine whether renal kallikrein-bradykinin system is activated during sepsis and study the correlation between the development of ARF and the activity of kallikrein-bradykinin system.
We also try to study the role or kallikrein-bradykinin system in CKD. Changes in the urinary excretion of kallikrein are predictive of the synthesis and activation of kallikrein within the kidney. Evaluation of urinary kallikrein excretion in patients with kidney disease may be helpful to clarify the role of kallikrein-kinin system in kidney disease. We conducted the clinical study to evaluate the relationship between urinary kallikrein and renal function or proteinuria. Using multiple regression analysis, we found that the urinary kallikrein excretion was positively correlated to reanl function but not the degree of proteinuria. After 12 months of follow up, the renal function deteriorated while the urinary kallikrein excretion also decreasesd. Kallikrein is excreted from renal tubule. As the renal injury progress there will be loss of the nephron number. So the urinary kallikrein excretion decreases. Our results indicate that urinarly kallikrein excretion may be an indicator of reanl function change.
Interestingly, the urinary kallikrein concentration is positively correlated to urinary MCP-1 concentration. Our analysis revealed that the correlation was not related to their relationship to renal function independently. The higher urinary MCP-1 may indicate the higher acitivity of inflammation in kidney. The result indicates that urinary kallikrein is excreted when renal inflammatory damage is present. This finding is compatible to the previous studies that kallikrein is excreted in animal and human inflammatory intestinal disease and in mechanimal lung injury. But the correlation is only found in one time point in our study. Serial follow up of the relationship between urinary kallikrein and MCP-1 is necessary to clarifly the true relationship. If urinary kallikrein did correlate to the activity of renal damage, measuring urinary kallikrein serially may be helpful in realizing the activity of disease process. But the application in clinical practice need further studies to clarify the relationship between urinary kallikrein excretion and status of renal injury, especially the correlation to the renal pathological findings.
After release kallikrein may exert its action, cleave kininogen into bradykinin and initiate the inflammatory reaction. But bradykinin is also reported to enhance matrix degradation through inhibition of PAI-1 expression. Inflammation and fibrosis are two important processes in CKD. Therefore, kallikrein-bradykinin system may have effect on renal disease progression. But whether the released kallikrein is beneficial or detrimental to CKD is unknown. To study the role of kallikrein-bradykinin system in CKD, we will conduct animal model study, by activating or blocking bradykinin receptor, to see the effect of bradykinin system on renal inflammation and fibrosis. These studies will more specifically demonstrate the role of bradykinin system in CKD.
In conclusion, early activation bradykinin B2 receptor has a deleterious effect on I/R renal injury through enhancement of ROS generation in both ischemic and reperfusion phases. The in vitro study reveals that the detrimental effect of B2R activation in cell injury induced by ATP depletion or hypoxia is through accumulation of intracellular Ca2+, generation of ROS, and mitochondrial damage by PLC pathway activation. However, the clinical implication and other possible signaling pathway of which B2R activation enhances I/R renal injury needs further studies to clarify. In CKD, urinary kallikrein excretion is positively correlated to renal function and urinary inflammatory cytokines. This indiates that urinary kallikrein may be a potential marker of renal damage in CKD. But the role of kallikrein-bradykinin system in CKD is still unclear and is to be studied.
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