The functional state of the kallikrein-kinin and renin-angiotensin-aldosterone systems in patients with localized kidney cancer

Introduction

The formation of a malignant neoplasm in a kidney affects its functional status. The association between renal function and carcinogenesis can be explained by several mechanisms. Tumor formation destroys renal tissue through direct invasion into the parenchyma or mechanical change of renal architecture caused by the compression of the renal parenchyma, collector tubules, renal tubules, and nephrons. Besides, cancer can secrete biologically active substances that can mediate the negative effect on the functional condition of the organ [1][2].

The development of tumors in the renal parenchyma primarily affects structural-functional renal units (nephrons). A significant role in the formation of nephropathy is played by the kallikrein-kinin system (KKS) [3]. It is a functional intermediate between the systems of blood coagulation and fibrinolysis. It contributes to the activation of the systems of complement and renin-angiotensin KKS affects microcirculation, renal excretion of electrolytes and water, contributes to the release of biologically active substances, participates in the regulation of the systemic and local renal circulation, etc. A sharp increase in the levels of kallikrein or insufficient levels of blood inhibitory potential can lead to the vicious loop that either maintains or enhances pathological reactions at the systemic and local levels. A disbalance between the activity of proteolytic enzymes and inhibitors of proteolysis greatly determines the final effect of KKS, its adaptive, and pathogenetic role in patients with various tubulointerstitial renal diseases [4][5][6][7]. Along with that, the role of changes in the KKS for formation of renal functional disorders in patients with renal tumor lesions is understudied.

Currently, it is believed that the renin-angiotensin-aldosterone system (RAAS), which is directly involved in the regulation of glomerular filtration, plays a significant role in the formation of functional renal disorders of various genesis. Renin induces a cascade of reactions that lead to the expression of angiotensin, which acts as a vasoconstrictor, reduces the volume of blood in the nephron glomerular barrier, and stimulates the synthesis of aldosterone. The secretion of renin is primarily controlled by the endocrine feedback mechanism with a direct activating effect of kallikrein [5][8]. Angiotensin-converting enzymes (ACE) transform angiotensin-I into angiotensin-II with a simultaneous degradation of bradykinin that exerts vasodilating and hypotensive effects. Aldosterone is synthesized in the adrenal gland glomerular cortex and participates in the regulation of К⁺, Na⁺, Н⁺, and NН₄⁺ ions [9]. The role of cortisol is acknowledged in patients with malignant neoplasms. It activates endogenous protective mechanisms, and at the same time, it restrains these mechanisms to prevent the excessive reaction that will lead to the damage or death of cells [10].

Some studies showed that the development of structural-functional disorders of the organs is inevitably associated with complicated and, probably, diverse changes in various RAAS components. At the same time, the results of numerous experimental and clinical studies demonstrate that aldosterone, angiotensin-I, and renin can be significant independent factors of the development and progression of renal disorders [11][12][13]. It should be highlighted that RAAS and KKS perform in close “cooperation” balancing each other by mutual enzymes (plasma kallikrein and kininases) [14].

The study aimed to evaluate the changes in the components of the KKS and RAAS in patients with localized kidney cancer.

Materials and methods

The study was approved by the Ethical Committee of the Rostov Cancer Research Institute, transformed into the National Medical Research Center for Oncology since 2020 (Protocol No. 24 dated 11/27/2016). The criteria of inclusion in the study were age > 18 years old, normal blood creatinine and/or glomerular filtration rate indicators, and lack of decompensated comorbid pathology.

The main group included 45 patients (25 men and 20 women) with diagnosed renal carcinoma at the stage T₁N₀M₀ that were examined and treated within 2016 – 2018. The mean age of patients in the group was 55.6 ± 7.8 years old. The control group included 13 relatively healthy male and female volunteers without oncological diseases. The mean age of patients in this group was 54.2 ± 6.9 years old. The studied groups were comparable by age, sex, and clinically significant comorbid somatic diseases in the anamnesis. All patients and relatively healthy volunteers signed a form of informed consent for participation in the study and processing of personal data.

All patients had components of KKS and RAAS assessed along with general medical examination. The indicators of kallikrein were established by a kinetic method after blood plasma and urine chromatography using DEAE-Sephadex А-50 (Amersham Biosciences Corp., Sweden). Further, its activity was assessed in the reaction with ethyl N2-benzoyl-L-arginine (BAEE) as a substrate (Sigma-Aldrich Pty Ltd., USA). The levels of prekallikrein were assessed using the same unadsorbed fraction of blood plasma and urine with further activation of prekallikrein with trypsin and evaluation of its level in the reaction with BAEE. The activity of kallikrein and the level of prekallikrein were expressed in milliunits per 1 ml of blood plasma (mU/ml), wherein “mU” is the content of enzyme that hydrolyzes 1 µM of BAEE per minute at 25°С [15]. Total activity of trypsin-like proteinases was established with a kinetic method in the same conditions without fractioning with BEAA in comparison with the control reaction with reagents. The activity of trypsin proteinases was expressed in milliesterase units (mEU) of BAEE hydrolyzed per 1 minute. The activity of kinin-degrading enzyme carboxypeptidase N (kininase 1) was assessed by a kinetic method in the whole blood plasma and urine by the rate of hydrolysis of hippuryl-L-lysin in the reaction in the presence of cobalt ions for 20 minutes at 37°С. It was expressed in µM/ml of the studied fluid per 1 minute (blood plasma, urine) [15]. Kinetic measurements in the reactions were performed with double beam spectrophotometer “HITACHI U-2900 IIO UV Solutions” (Hitachi Ltd., Japan) in thermostatted cells. The evaluation of the levels of α-1-proteinase inhibitor and α-2-macroglobulin in the blood plasma of patients was made using enzyme-linked immunosorbent assay using standard test kits. The levels of angiotensin-1, renin, and cortisol were studied by the method of radioimmune analysis.

Statistical analysis. Statistical analysis was conducted using the software StatSoft Statistica 8.0 (StatSoft Inc., IBM Corp., USA) and Student-Fisher tests. Statistical hypotheses were considered significant at р < 0.05.

Results

The levels of the components of the KKS (trypsin proteinases and plasma inhibitors of proteolysis) in the blood plasma in patients with localized renal cell carcinoma before anti-tumor therapy and healthy volunteers are presented in Table 1.

Patients with renal cell carcinoma (RCC) had significant differences in the composition of the blood plasma in comparison with healthy volunteers. Thus, a two-fold decrease of prekallikrein in patients was associated with a 2.3-fold increase in the activation of kallikrein in comparison with healthy volunteers (p < 0.05). It was accompanied by 1.4-fold increased activity of carboxypeptidase that acted as an ACE (p < 0.05).

Because kallikrein is an acute-phase glycoprotein that mediates the regulation of urine filtration via bradykinin, the authors studied its activity concerning other proteinases of trypsin type. It can be seen that patients with localized RCC have a synchronous increase in the total activity of trypsin proteinases by 2.6 times and other trypsin proteinases (TP) in the blood plasma by 3 times in comparison with normal values of these parameters (p < 0.05). On average, the level of active kallikrein in the total activity of trypsin proteinases was 9%.

The most significant plasma inhibitors of proteolytic enzymes of the blood include α-1-proteinase inhibitor (α-1PI) and α-2-macroglobulins (α-2M) that are characterized by interchangeability and inhibition of the enzymatic activity. The obtained data shows that patients with localized RCC in the blood plasma had decreased activity of α-1-PI and α-2M by 2.3 and 1.9 times, respectively, in comparison with normal values (p < 0.05). The formation of proteinase-inhibitory disbalance with the activation of proteolytic processes was confirmed by a higher interaction coefficient of kallikrein/α-2М and low α-1PI/α-2М in comparison with normal parameters (p < 0.05).

The study of changes in the urine indicators was similar to the changes observed in the blood (Table 2).

The baseline level of kallikrein in the urine of patients with RCC was 3.8 times higher and prekallikrein was 1.6 times higher in comparison with a group of healthy volunteers (p < 0.05). Absolute values of trypsin proteases were significantly higher in patients with RCC and the indicators of proteolysis inhibitors were lower in comparison with normal parameters. The total activity of trypsin proteases was 2.4 times higher than in donors and the levels of trypsin proteinases were higher by 2 times (p < 0.05). Besides, these patients had higher interaction coefficients TP/α-1PI and K/α-2М.

The results of the study on the indicators of RAAS and cortisol in patients with localized RCC before the anti-tumor therapy and healthy volunteers are presented in Table 3.

The results showed that all patients included in the study had decreased cortisol/renin ratio by 1.3 times and increased interaction coefficients renin/AT-1 by 2.9 times, cortisol/angiotensin-I – by 2.3 times, cortisol/angiotensin-I – by 2.3 times, and aldosterone/angiotensin-I – by 1.2 times in comparison with normal values of these indicators (p < 0.05).

Discussion

This was the first complex study of the components of KSS and RAAS in patients with localized RCC.

The obtained results revealed significant differences in the KKS between healthy volunteers and patients with localized RCC before anti-tumor therapy.

It is known that kallikrein is a proteolytic enzyme. It is a serine proteinase of trypsin type with a priority in the protein metabolism and the lowest energy requirements during proteolysis [5][7][16][17]. The study of baseline levels of prekallikrein and kallikrein in the blood serum showed that they were significantly higher than the normal values. It can be explained by the compensatory nature of these changes because, in the conditions of renal dysfunction, an increase in the activity of kallikrein provides the required blood flow rate and excretion of sodium in the viable nephrons. Various publications showed that a significant increase in kallikrein and/or a decrease in the inhibitory potential of the blood can lead to the development of pathologic reactions at the local and systemic levels in patients with various tubulointerstitial renal diseases [4][5][6][7].

Currently, some studies demonstrate the general mechanism of humoral regulation of proteolytic systems with proteinase inhibitors [4, 5, 18]. An increase in the total activity of trypsin in the blood plasma in comparison with normal values was registered in the present study. It agrees with updated available data on the interaction of all trypsin proteinases with the possibility of mutual activation [5].

It is known that the insufficiency of α-1-PI provokes the cytolytic effect of proteinases with unregulated proteolysis and damage of nephrons [4][5][16][18]. The obtained study results agree with the published data of some researchers that revealed a consistency in the activation of trypsin type proteinases in patients with oncological pathology. At the same time, the activation of proteolysis and changes in the proteinase inhibitory equilibrium of the blood are the factors that contribute to the damage of capillary endothelium, internal leaf of the Bowman’s capsule, and basal membrane [18][19]. Because of these disorders, the barrier function is not acting for the blood proteins. Besides, a lysing effect of proteinases inhibits tubular reabsorption and the damaged tubular structures do not prevent proteins from penetrating secondary urine, which was confirmed by the present study.

The study results also showed that patients with localized RCC in the blood have increased activity of kallikrein and trypsin proteinases with sufficient deficit of their inhibitors (α-1-PI and α-2-M). The authors revealed an increased interaction coefficient kallikrein/α-2М and a decreased interaction coefficient α-1PI/α-2М in comparison with a group of healthy volunteers. α-2-M is characterized by the polyfunctionality of its action. As an inhibitor, it neutralizes the activated proteinases in the blood in the lack of reserves of other plasma inhibitors and blocks the proteolytic activity of proteinases of all classes. A decrease in the activity of α-2-M completely destabilizes physiological balance and shifts the priority from proteolysis.

The results of the study suggest that these changes are associated with the development of a malignant lesion in the kidney.

Such functional disorders as a disbalance in the activity of proteolytic enzymes and inhibitors of proteolysis determine the final effect of the KKS and its role in the formation of nephropathy. This predisposes the prognosis and course of the disease at these stages of anti-tumor therapy. In other words, the functional status of the renal parenchyma and detection of early signs of metabolic renal dysfunction can be important factors from the point of view of possible renal failure development after radical surgical treatment [20].

The study results showed that all patients with RCC had the levels of renin, angiotensin-I, aldosterone, and cortisol in the blood were significantly elevated in comparison with normal values. It could be a compensatory reaction in response to the stress associated with the development of the oncological process.

The revealed disbalance in the ratios of the components of the RAAS and cortisol indicates the disturbances in the regulation adaptation mechanisms in patients with renal cell carcinoma.

Conclusion

Localized RCC is associated with the formation of tubular interstitial dysfunction, the shift of proteinase inhibitory balance to the activation of proteolysis, dysregulation of mechanical adaptation, and disorders in the local metabolism in the kidneys.