Angiotensin-converting enzymes (ACE and ACE2) are downregulated in renal tumors.

The angiotensin-converting enzymes (ACE and ACE2) are highly expressed in renal tubules and play an important role in the regulation of renal function by the intrarenal renin-angiotensin system (iRAS). Dysregulation of these cell-surface peptidases has been associated with renal injury. Most of these studies, however, have focused on non-neoplastic kidney diseases. In the present study, ACE and ACE2 activity and protein and mRNA expression were analysed in a subset of clear-cell (CCRCC) and chromophobe (ChRCC) renal cell carcinomas, and in renal oncocytoma (RO). Enzyme activity was measured by spectrofluorometric (ACE2) and spectrophotometric assays (ACE), and protein and mRNA expression were determined by immunohistochemistry and qRT-PCR assays, respectively. The enzyme activities and immunohistochemistry showed that both enzymes are mainly downregulated in these neoplasms. qRT-PCR studies in CCRCC showed no positive correlation between ACE and ACE2 activity/protein expression and mRNA levels, whereas downregulation of ACE2 mRNA levels was observed in tumors from the distal nephron (ChRCC and RO). These findings suggest a metabolic imbalance in iRAS and a role of this system in renal neoplastic diseases, and point to ACE and ACE2 as potential prognostic/diagnostic markers.

Expression of cannabinoid receptors in human kidney.

The presence of CB1 and CB2 cannabinoid receptors and their physiological role in the kidney has been described in animal models but not in humans. Our aim in this study was to evaluate the presence of these receptors in human kidney, adult and fetal. For this purpose, RT-PCR, western-blot and immunohisto-chemical assays were performed. RT-PCR confirmed the presence of CB1 receptor mRNA receptor and the absence of the CB2 receptor mRNA in adult and fetal kidney. Western-blot and immunohistochemical assays revealed the presence of the CB1 cannabinoid receptor protein, which displayed a similar distribution in fetal and adult kidneys. Proximal and distal convoluted tubule cells and intercalated cells in the collecting ducts showed marked positivity. Conversely, the CB2 cannabinoid receptor protein was consistently negative in all cases. Our data suggest a possible implication of the endocannabinoid system in the physiology and development of the human kidney.

Changes in cell-surface peptidase activity in papillary renal cell carcinoma.

BACKGROUND: Renal cancer is one of the ten most common malignant tumours in humans and its histological classification, best clinical management and treatment strategies are continuously debated. Roughly 10% of renal carcinomas are papillary renal cell carcinomas (RCCs), a histologically well characterized tumour subtype that is linked to alterations on chromosomes 7 and 17. Peptidases are proteolytic enzymes known to be involved in oncological processes, although their precise role in renal cancer is poorly understood. MATERIALS AND METHODS: Eighteen papillary RCCs were selected for the study. Tumour and normal tissue samples were frozen for enzymatic analysis. The catalytic activity for a pool of peptidases (EC numbers: 3.4.11.14; 3.4.11.2; 3.4.11.6; 3.4.11.21; 3.4.11.7; 3.4.14.5; 3.4.24.11; 3.4.21.26; 3.4.19.3) was measured fluorometrically. RESULTS: Statistically significant decreases were observed in the following cell surface activities: EC.3.4.11.2 (six-fold decrease in tumour vs. non-tumour); 3.4.11.6 (five-fold decrease); 3.4.11.7 (eight-fold decrease); 3.4.24.11 (four-fold decrease). No significant alterations were observed in the soluble activities. CONCLUSION: These data confirm the involvement of cell-surface peptidases in the mechanisms underlying RCC aetiogenesis and suggest that the peptidase activity profile in the RCC may be a diagnostic/prognostic marker.

Expression and activity profiles of DPP IV/CD26 and NEP/CD10 glycoproteins in the human renal cancer are tumor-type dependent.

BACKGROUND: Cell-surface glycoproteins play critical roles in cell-to-cell recognition, signal transduction and regulation, thus being crucial in cell proliferation and cancer etiogenesis and development. DPP IV and NEP are ubiquitous glycopeptidases closely linked to tumor pathogenesis and development, and they are used as markers in some cancers. In the present study, the activity and protein and mRNA expression of these glycoproteins were analysed in a subset of clear-cell (CCRCC) and chromophobe (ChRCC) renal cell carcinomas, and in renal oncocytomas (RO). METHODS: Peptidase activities were measured by conventional enzymatic assays with fluorogen-derived substrates. Gene expression was quantitatively determined by qRT-PCR and membrane-bound protein expression and distribution analysis was performed by specific immunostaining. RESULTS: The activity of both glycoproteins was sharply decreased in the three histological types of renal tumors. Protein and mRNA expression was strongly downregulated in tumors from distal nephron (ChRCC and RO). Moreover, soluble DPP IV activity positively correlated with the aggressiveness of CCRCCs (higher activities in high grade tumors). CONCLUSIONS: These results support the pivotal role for DPP IV and NEP in the malignant transformation pathways and point to these peptidases as potential diagnostic markers.

Increased prolyl endopeptidase activity in human neoplasia.

Prolyl endopeptidase (EC 3.4.21.26) (PEP) is a serine peptidase that converts several biologically active peptides. This enzyme has been linked to several neurological, digestive, cardiovascular and infectous disorders. However, little is known about its involvement in neoplastic processes. This study analyzes fluorimetrically cytosolic and membrane-bound PEP activity in a large series (n=122) of normal and neoplastic tissues from the kidney, colon, oral cavity, larynx, thyroid gland and testis. Cytosolic PEP activity significantly increased in clear cell renal cell carcinoma, urothelial carcinoma of the renal pelvis and head and neck squamous cell carcinoma. Both cytosolic and membrane-bound PEP activity were also increased in colorectal adenomatous polyps. These data suggest the involvement of PEP in some mechanisms that underlie neoplastic processes.

Increased APN/CD13 and acid aminopeptidase activities in head and neck squamous cell carcinoma.

BACKGROUND: Involvement of peptidases in carcinogenic processes of several tumor types has been investigated in recent years. Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers worldwide and accounts for more than 90% of all head and neck cancers. Increased understanding of its pathophysiology has led to implication of several proteinases, specially matrix metalloproteinases, in its genesis, growth, and dissemination. However, very little is known about involvement of peptidases in this neoplasm. METHODS: Seventeen HNSCC tissue samples were selected for the study. Tumor and normal tissue samples were frozen for enzymatic study. The catalytic activity for a pool of peptidases (PSA, APN/CD13, APB, APA, Asp-AP, CAP, DPPIV/CD26, NEP/CD10, and PGI) was measured fluorometrically. RESULTS: The activity of 2 cell surface aminopeptidases (APN/CD13 and APA) and a cytosolic aminopeptidase (Asp-AP) was significantly increased in HNSCC tissues. CONCLUSIONS: These data show the involvement of cell surface and cytosolic peptidases in the mechanisms underlying HNSCC.

Acid, basic, and neutral peptidases present different profiles in chromophobe renal cell carcinoma and in oncocytoma.

Renal cell carcinomas (RCCs) are neoplasias with high prevalence and mortality. We previously reported that several peptidases may be involved in the pathophysiology of clear cell renal cell carcinoma (CCRCC). Now, to gain insight into the reasons that lead the various RCC types to behave very differently with regard to aggressiveness and response to anticancer treatments, we analyzed subsets of chromophobe renal cell carcinoma (ChRCC), and renal oncocytoma (RO), a benign tumor; as well as different grades and stages of CCRCCs. Particulate APN, APB, and APA activities were decreased in both ChRCC and RO (tumor vs. nontumor tissues). Interestingly, activities were downregulated in a tumor-type specific way and the intensities of the decreases were stronger in the benign tumor than in the malignant type. Moreover, when two key histopathological parameters for tumor prognosis (high vs. low stage and grade) were analyzed, increases of activity were also observed in several of these cell surface peptidases (APN, APB). Some soluble activities (APB, Asp-AP) were also downregulated in the RCCs. With respect to genetic expression, PSA and APN were in a positive correlation related to their activities in both ChRCC and RO; but not APB, Asp-AP, APA, and PGI. These results may suggest an involvement of several peptidases in the pathophysiology of renal cancer, since they presented different patterns of activity and expression in tumors with different behaviors.

Cystinyl aminopeptidase activity is decreased in renal cell carcinomas.

The involvement of peptidases in carcinogenetic processes of several tumor types has been researched in recent years. Although kidney is one of the major tissues known to express cystinyl-aminopeptidase (CAP), little is known about its role in renal neoplasia. This study analyzes fluorimetrically membrane-bound and soluble CAP activity in the three main renal cancers: clear cell (CCRCC), papillary (PRCC), and chromophobe (ChRCC) renal cell carcinomas. Overall, a marked decrease of membrane-bound CAP activity in all the three renal cell carcinomas was detected when compared with their respective surrounding non-tumor tissues. So, the tumor vs. non-tumor CAP ratios (units of peptidase per mg of protein) was as follows: 926+/-111 vs. 3778+/-276 for CCRCCs, 737+/-181 vs. 4351+/-950 for PRCCs, and 592+/-118 vs. 4905+/-935 for ChRCCs. In contrast, the soluble fraction of this enzyme displayed minor and non-significant changes when comparing tumor and non-tumor CAP activities in the whole series. After stratification by stage and grade, CCRCCs displayed significant differences: pT3 category had significantly higher levels of membrane-bound activity than pT1, and high grade cases (G3-4) had higher soluble CAP activity than low grade ones (G1-2). These data may open additional possibilities in the study of renal cell carcinoma with regard to the prognosis of patients.

Altered levels of acid, basic, and neutral peptidase activity and expression in human clear cell renal cell carcinoma.

Peptides play important roles in cell regulation and signaling in many tissues and are regulated by peptidases, most of which are highly expressed in the kidney. Several peptide convertases have a function in different tumor stages, and some have been clearly characterized as diagnostic and prognostic markers for solid tumors, including renal cancer; however, little is known about their in vivo role in kidney tumors. The present study compares the activity of a range of peptidases in human tumor samples and nontumor tissue obtained from clear cell renal cell carcinoma (CCRCC) patients. To cover the complete spectrum and subcellular distribution of peptide-converting activity, acid, neutral, basic, and omega activities were selected. CCRCC displays a selective and restricted pattern of peptidase activities. Puromycin-sensitive aminopeptidase activity in the tumor increases [tumor (t) = 10,775 vs. nontumor (n) = 7,635 units of peptidase (UP)/mg protein; P < 0.05], whereas aminopeptidase N decreases (t = 6,664 vs. n = 33,381 UP/mg protein; P < 0.001). Aminopeptidase B activity of the particulate fraction in tumors decreases (t = 2,399 vs. n = 13,536 UP/mg protein; P < 0.001) compared with nontumor tissues, and aspartyl-aminopeptidase activity decreases significantly in CCRCC (t = 137 vs. n = 223 UP/mg protein; P < 0.05). Soluble and particulate pyroglutamyl peptidase I activities, aminopeptidase A activity, and soluble aminopeptidase B activity do not vary in renal cancer. The relative expression for the aforementioned peptidases, assayed using quantitative RT-PCR, increases in CCRCC for aminopeptidases B (1.5-fold) and A (19-fold), aspartyl-aminopeptidase (3.9-fold), puromycin-sensitive aminopeptidase (2.5-fold), and pyroglutamyl peptidase I (7.6-fold). Only aminopeptidase N expression decreases in tumors (1.3-fold). This peptidase activity profile in the neoplastic kidney suggests a specific role for the studied convertases and the possible involvement of an intracrine renin-angiotensin system in the pathogenesis of CCRCC.

Subcellular distribution of membrane-bound aminopeptidases in the human and rat brain.

We evaluated the subcellular distribution of four membrane-bound aminopeptidases in the human and rat brain cortex. The particulate enzymes under study--puromycin-sensitive aminopeptidase (PSA), aminopeptidase N (APN), pyroglutamyl-peptidase I (PG I) and aspartyl-aminopeptidase (Asp-AP)--were fluorometrically measured using beta-naphthylamide derivatives. Membrane-bound aminopeptidase activity was found in all the studied subcellular fractions (myelinic, synaptosomal, mitochondrial, microsomal and nuclear fractions), although not homogenously. Human PSA showed highest activity in the microsomal fraction. APN was significantly higher in the nuclear fraction of both species, while PG I showed highest activity in the synaptosomal and myelinic fractions of the human and rat brain. The present results suggest that in addition to inactivating neuropeptides at the synaptic cleft, these enzymes may participate in other physiological processes. Moreover, these peptidases may play specific roles depending on their activity levels at the different subcellular structures where they are localized.

Effects of imipramine treatment on delta-opioid receptors of the rat brain cortex and striatum.

Imipramine (CAS 113-52-0) is being utilized widely for the treatment of major depression. In recent years, there has been evidence of the involvement of the endogenous opioid system in major depression and its treatment. There is some evidence indicating that opioid receptors could be involved in the antidepressant mechanism of action. Regarding this topic, mood-related behavior of endogenous enkephalins seems to be mediated by delta-opioid receptors. In this work, the effects of subacute (5 day) and chronic (15 day) treatments of imipramine on the density and the affinity of the delta-receptors in the striatum and in the parietal and frontal cortices of the rat brain are described. Studied parameters (Bmax and Kd) were calculated by a saturation binding assay with the delta-opioid agonists [3H]-DPDPE (tyrosyl-2,6-3H(N)-(2-D-penicillamine-5-D-penicillamine)-enkephalin) as specific ligand and DSLET ([D-serine2]-D-leucine-enkephalin-threonine) as non-radioactive competing ligand. It was found that 15 days treatment significantly decreased the delta-opioid receptor density,without changing the affinity, in the frontal cortex of the rat brain. That decrease was confirmed by delta-opioid receptor immunostaining. These results suggest that delta-opioid receptors could play a role in the chronic action mechanism of imipramine.

Effects of morphine administration and its withdrawal on rat brain aminopeptidase activities.

The endogenous opioid neuropeptide system seems to be involved in the neural processes which underlie drug addiction. Several studies have reported that the administration of morphine induces changes in the levels and/or activity of endogenous opioid peptides (enkephalin, dynorphin) and their precursors in specific brain regions of the adult CNS. The aim of this work was to study the effects of chronic morphine exposure and its withdrawal on certain aminopeptidases capable of degrading opioid peptides in brain areas including the amygdala, hypothalamus, hippocampus, striatum and brain cortices. In animals treated with morphine, aminopeptidase N presented higher enzyme activity levels in the striatum, the hypothalamus and the amygdala compared to control animals, although statistically significant differences were observed only in the case of the striatum. In addition, the activity of soluble puromycin-sensitive aminopeptidase (PSA) was found to be higher in the frontal cortex of these rats. In contrast, rats experiencing withdrawal symptoms presented decreased levels of aminopeptidase activity in certain brain areas. Thus, the activity of aminopeptidase N in the hippocampus and soluble puromycin-sensitive aminopeptidase in the frontal cortex were found to be lower in rats experiencing naloxone precipitated withdrawal symptoms, compared to the corresponding controls. Finally, the activity of the three studied aminopeptidases in vitro was unaltered by incubation with morphine, suggesting that the observed effects are not due to a direct action of this opioid upon the aminopeptidases. The results of the present report indicate that aminopeptidases may play an important role in the processes of tolerance and withdrawal associated with morphine administration.

Aminopeptidases in visceral organs during alterations in body fluid volume and osmolality.

Enzymatic cleavage of some peptides in the local environment could be included among the mechanisms related to the regulation of hydrosaline balance. In order to examine this hypothesis, we measured representative aminopeptidase activities in visceral organs of rats after applying certain hydrosaline challenges. Decreased levels (about 30%) of particulate puromycin-insensitive-neutral aminopeptidase in the renal medulla and of soluble acid aminopeptidase in the lung were observed under hyperosmolality and hypovolemia. Decreased levels (more than 45%) of particulate type-I-pyroglutamyl aminopeptidase in the heart were observed under altered volemia. These results indicate that aminopeptidases at these anatomical locations might be involved in the regulation of body fluid volume and osmolality.

Aminopeptidases in visceral organs during alterations in body fluid volume and osmolality

Enzymatic cleavage of some peptides in the local environment could be included among the mechanisms related to the regulation of hydrosaline balance. In order to examine this hypothesis, we measured representative aminopeptidase activities in visceral organs of rats after applying certain hydrosaline challenges. Decreased levels (about 30%) of particulate puromycin-insensitive- neutral aminopeptidase in the renal medulla and of soluble acid aminopeptidase in the lung were observed under hyperosmolality and hypovolemia. Decreased levels (more than 45%) of particulate type-I-pyroglutamyl aminopeptidase in the heart were observed under altered volemia. These results indicate that aminopeptidases at these anatomical locations might be involved in the regulation of body fluid volume and osmolality.