Catecholamines production by kidney tissue and mesangial cell culture is differentially modulated by diabetes / A produção de catecolaminas pelo tecido renal e a cultura de células mesangiais é modulada diferencialmente pelo diabetes

Abstract Introduction: According to the International Diabetes Federation, the number of people with diabetes mellitus may reach 700 million in 2045. Catecholamines are involved in the regulation of several kidney functions. This study investigates the effects of hyperglycemia on catecholamines' metabolism in kidney tissue from control, diabetic, and insulin-treated diabetic rats, both in vivo and in vitro. Methods: Male Wistar-Hannover rats were randomized into: control, diabetic, and insulin-treated diabetic groups. Diabetes was induced by a single injection of streptozotocin, and diabetic treated group also received insulin. After 60 days, blood and kidney tissue from all groups were collected for catecholamines' quantification and mesangial cells culture. Results: diabetic rats had lower body weight, hyperglycemia, and increase water intake and diuresis. Additionally, diabetes promoted a sharp decrease in creatinine clearance compared to control group. Regarding the whole kidney extracts, both diabetic groups (treated and non-treated) had significant reduction in norepinephrine concentration. In mesangial cell culture, catecholamines' concentration were lower in the culture medium than in the intracellular compartment for all groups. Norepinephrine, epinephrine, and dopamine medium levels were increased in the diabetic group. Conclusion: The major finding of the present study was that 8 weeks of diabetes induction altered the kidney catecholaminergic system in a very specific manner, once the production of catecholamines in the excised kidney tissue from diabetic rats was differentially modulated as compared with the production and secretion by cultured mesangial cells.

Resumo Introdução: Segundo a Federação Internacional de Diabetes, o número de pessoas com diabetes mellitus pode chegar a 700 milhões em 2045. As catecolaminas estão envolvidas na regulação de várias funções renais. Este estudo investiga os efeitos da hiperglicemia no metabolismo das catecolaminas no tecido renal de ratos controle, diabéticos e diabéticos tratados com insulina, tanto in vivo como in vitro. Métodos: Os ratos Wistar-Hannover machos foram randomizados em: grupos controle, diabéticos e diabéticos tratados com insulina. O diabetes foi induzido por uma única injeção de estreptozotocina, e o grupo diabético tratado também recebeu insulina. Após 60 dias, sangue e tecido renal dos grupos foram coletados para quantificação de catecolaminas e cultura de células mesangiais. Resultados: ratos diabéticos apresentaram peso corporal mais baixo, hiperglicemia, e aumento da ingestão de água e diurese. Ademais, o diabetes promoveu uma redução acentuada na depuração de creatinina comparado com o grupo controle. Quanto aos extratos de rim total, ambos os grupos diabéticos (tratados/não tratados) tiveram redução significativa na concentração de noradrenalina. Na cultura de células mesangiais, a concentração de catecolaminas foi menor no meio de cultura do que no compartimento intracelular para todos os grupos. Níveis médios de noradrenalina, adrenalina e dopamina estavam aumentados no grupo diabético. Conclusão: O principal achado deste estudo foi que 8 semanas de indução de diabetes alteraram o sistema catecolaminérgico renal de maneira muito específica, já que a produção de catecolaminas no tecido renal excisado de ratos diabéticos foi modulada diferencialmente comparada com produção e secreção por células mesangiais cultivadas.

Catecholamines production by kidney tissue and mesangial cell culture is differentially modulated by diabetes.

INTRODUCTION: According to the International Diabetes Federation, the number of people with diabetes mellitus may reach 700 million in 2045. Catecholamines are involved in the regulation of several kidney functions. This study investigates the effects of hyperglycemia on catecholamines' metabolism in kidney tissue from control, diabetic, and insulin-treated diabetic rats, both in vivo and in vitro. METHODS: Male Wistar-Hannover rats were randomized into: control, diabetic, and insulin-treated diabetic groups. Diabetes was induced by a single injection of streptozotocin, and diabetic treated group also received insulin. After 60 days, blood and kidney tissue from all groups were collected for catecholamines' quantification and mesangial cells culture. RESULTS: diabetic rats had lower body weight, hyperglycemia, and increase water intake and diuresis. Additionally, diabetes promoted a sharp decrease in creatinine clearance compared to control group. Regarding the whole kidney extracts, both diabetic groups (treated and non-treated) had significant reduction in norepinephrine concentration. In mesangial cell culture, catecholamines' concentration were lower in the culture medium than in the intracellular compartment for all groups. Norepinephrine, epinephrine, and dopamine medium levels were increased in the diabetic group. CONCLUSION: The major finding of the present study was that 8 weeks of diabetes induction altered the kidney catecholaminergic system in a very specific manner, once the production of catecholamines in the excised kidney tissue from diabetic rats was differentially modulated as compared with the production and secretion by cultured mesangial cells.

Plasma proteomics for the assessment of acute renal transplant rejection.

Renal transplant is the best treatment for patients with chronical kidney disease however acute graft rejection is the major impediment to success in renal transplantation leading to loss of the organ the first year after transplantation. The aim of this study was to identify plasma proteins that may be early biomarkers of acute rejection of renal allograft, developing a diagnostic model that avoids the loss of the transplanted organ. Shotgun proteomics (LC-MS/MS) method was used to analyze a set of thirty-one plasma samples, including 06 from patients with acute graft rejection after transplantation (rejection group/Rej-group) and twenty-five from renal transplant patients with stable renal graft function (control group/Ct-group). As results nineteen proteins were upregulated in the rejection group compared to the control group, and two proteins were downregulated; and three were present exclusively in the rejection group. After analysis, we selected four proteins that were related to the acute phase response and that were strongly associated with each other: they are alpha-1 antitrypsin (A1AT), alpha-2 antiplasmin (A2AP), serum amyloid A (SAA) and apolipoprotein CIII (APOC3). We think that simultaneous monitoring of SAA and APOC3 can provide insights into a broad profile of signaling proteins and is highly valuable for the early detection of a possible acute renal graft rejection. STATEMENT OF SIGNIFICANCE OF THE STUDY: In this study we did plasma shotgun patients with and without acute rejection of renal allograft. In a clinical setting an acute rejection is typically suspected upon an increase in plasma creatinine and renal biopsy. But these methods are late and unspecific; sometimes the rejection process is already advanced when there is an increase in serum creatinine. Therefore, it is necessary to find proteins that can predict the allograft rejection process. In our study were able to identify changes in the concentration of plasma protein belonging to a network of protein interaction processes the acute phase response. We believe, therefore, that development of a routine diagnosis of these proteins can detect early acute rejection of renal allograft process, thus preventing its loss.

Alternative pathways for angiotensin II production as an important determinant of kidney damage in endotoxemia.

Sepsis is an uncontrolled systemic inflammatory response against an infection and a major public health issue worldwide. This condition affects several organs, and, when caused by Gram-negative bacteria, kidneys are particularly damaged. Due to the importance of renin-angiotensin system (RAS) in regulating renal function, in the present study, we aimed to investigate the effects of endotoxemia over the renal RAS. Wistar rats were injected with Escherichia coli lipopolysaccharide (LPS) (4 mg/kg), mimicking the endotoxemia induced by Gram-negative bacteria. Three days after treatment, body mass, blood pressure, and plasma nitric oxide (NO) were reduced, indicating that endotoxemia triggered cardiovascular and metabolic consequences and that hypotension was maintained by NO-independent mechanisms. Regarding the effects in renal tissue, inducible NO synthase (iNOS) was diminished, but no changes in the renal level of NO were detected. RAS was also highly affected by endotoxemia, since renin, angiotensin-converting enzyme (ACE), and ACE2 activities were altered in renal tissue. Although these enzymes were modulated, only angiotensin (ANG) II was augmented in kidneys; ANG I and ANG 1-7 levels were not influenced by LPS. Cathepsin G and chymase activities were increased in the endotoxemia group, suggesting alternative pathways for ANG II formation. Taken together, our data suggest the activation of noncanonical pathways for ANG II production and the presence of renal vasoconstriction and tissue damage in our animal model. In summary, the systemic administration of LPS affects renal RAS, what may contribute for several deleterious effects of endotoxemia over kidneys.

Clinical relevance of diagnostic hysteroscopy with concurrent endometrial biopsy in the accurate assessment of intrauterine alterations.

PURPOSE: The aim of this retrospective observational study was to evaluate the reliability of diagnostic hysteroscopy, routinely performed along with endometrial biopsy, by analyzing and comparing both hysteroscopic and histopathological outcomes in asymptomatic infertile patients, previously to their IVF cycle. METHODS: The study included 84 consecutive infertile patients who underwent diagnostic hysteroscopy followed by endometrial biopsy. Four-micrometer sections were stained with hematoxylin and eosin and examined microscopically. The data evaluated the frequency and characteristics of endometrial abnormalities found in the biopsies of patients with normal hysteroscopy outcome. Descriptive data are presented as percentages, and the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of hysteroscopy for diagnosis of endometrial alterations were calculated on the basis of pathologic reports. RESULTS: The hysteroscopy evaluation showed 50.0 % of patients with a normal uterine cavity, 40.5 % with endometrial polyps, 6.0 % with endometrial hyperemia, and 3.5 % with other endometrial abnormalities. Among the 42 patients with a normal uterine cavity at hysteroscopic examination, 60.0 % also had a normal biopsy outcome, but in other 40.0 % of patients at least one histopathological abnormal aspect was diagnosed at biopsy. The sensitivity (67.3 %), specificity (80.6 %), PPV (85.4 %) and NPV (59.5 %) of diagnostic hysteroscopy were calculated on the basis of histopathological findings. CONCLUSIONS: Our results show that diagnostic hysteroscopy demonstrated intrauterine alterations in half of infertile patients; histopathological endometrial alterations suggest high rate of false-negative outcomes. Therefore, diagnostic hysteroscopy and concurrent endometrial biopsy should be used as complementary diagnostic and therapeutic approach, especially for patients with previous IVF failures.

O que influencia doação de óvulos quando não há compensação financeira? / What influences oocyte donation when there is no financial compensation?

Aim: To evaluate the influence of factors such as age, education level and previous treatment for infertility in the decision to donate or receive eggs. Methods: Patients visting our service for the first time answered the question: “Would you donate or receive eggs?”. We assessed whether the inclination to donate or receive was related to age, level of education and the previous unsuccessful treatment for infertility.Results: 313 patients were included and most (56.9%) said they would donate eggs while only 34.5% would receive a donation. When giving and receiving were evaluated jointly we observed a positive correlation between them (Pearson correlation: r = 0.537, p < 0.01). Patients that underwent previous treatments for infertility were significantly more prone to egg donation (63.4% yes vs. 36.6% no, p < 0.05 vs no previous treatment group), but not toreceive (41.8% yes vs. 58, 2% no). In high and low levels of education most patients were in favor of donation (55.4% and 61.3%, respectively), but against the idea of receiving (33.9%and 37.5%, respectively). There was no significant differences between groups. The age of the patients (< 35 years old or > 35 years old) did not influence the will do donate (58.2% and56.4% respectively) or receive eggs (36.9% and 33.0%, respectively).Conclusions: Our results help understand the factors that may influence the decision to participate in an egg-sharing scheme. We could speculate that patients who have previously undergone unsuccessful treatments are more open to egg-sharing, despite their age or educational background. It would also be relevant to investigate the psychosocial reasons that make couples more willing to donate eggs than receiving.

Objetivo: Avaliar a influência da idade, grau de escolaridade e tratamento anterior na decisão de doar ou receber óvulos. Pacientes e métodos: Mulheres atendidas em nosso serviço responderam à pergunta: “Vocêdoaria ou receberia óvulos?”. Avaliou-se a concordância de aceitação de ovodoação ou ovorecepção com a idade, o grau de escolaridade e tratamento anterior para infertilidade. Resultados: Foram incluídas 313 pacientes e a maioria (56,9%) respondeu que doaria óvulos enquanto apenas 34,5% receberiam. Houve correlação positiva entre doação e recepção (r = 0,537, p < 0,01). Pacientes submetidas a tratamento anterior de infertilidade se mostraram significativamente mais propensas à doação (63,4% sim vs 36,6% não, p < 0.05 vs sem tratamento anterior), mas não a receber (41,8% sim vs 58,2% não). Em níveis altos e baixos de escolaridade a maioria dos pacientes se mostrou a favor da doação (55,4% e 61,3%, respectivamente), mas contra a ideia de receber (37,5% e 33,9%, respectivamente), não houvediferenças significativas entre os grupos. A maioria das pacientes com menos ou mais de 35 anos de idade doaria (58,2% e 56,4%, respectivamente), mas não receberia (36,9% e 33,0%, respectivamente).Conclusões: Nossos resultados são relevantes para entender os fatores que podem influenciar na decisão de participar em um esquema de partilha de óvulos. Poderíamos especular que pacientes previamente submetidas a tratamentos mal sucedidos são mais aberta à ovodoação, apesar de sua idade ou formação educacional.

Tissue-specific expression of transgenic secreted ACE in vasculature can restore normal kidney functions, but not blood pressure, of Ace-/- mice.

Angiotensin-converting enzyme (ACE) regulates normal blood pressure and fluid homeostasis through its action in the renin-angiotensin-system (RAS). Ace-/- mice are smaller in size, have low blood pressure and defective kidney structure and functions. All of these defects are cured by transgenic expression of somatic ACE (sACE) in vascular endothelial cells of Ace-/- mice. sACE is expressed on the surface of vascular endothelial cells and undergoes a natural cleavage secretion process to generate a soluble form in the body fluids. Both the tissue-bound and the soluble forms of ACE are enzymatically active, and generate the vasoactive octapeptide Angiotensin II (Ang II) with equal efficiency. To assess the relative physiological roles of the secreted and the cell-bound forms of ACE, we expressed, in the vascular endothelial cells of Ace-/- mice, the ectodomain of sACE, which corresponded to only the secreted form of ACE. Our results demonstrated that the secreted form of ACE could normalize kidney functions and RAS integrity, growth and development of Ace-/- mice, but not their blood pressure. This study clearly demonstrates that the secreted form of ACE cannot replace the tissue-bound ACE for maintaining normal blood pressure; a suitable balance between the tissue-bound and the soluble forms of ACE is essential for maintaining all physiological functions of ACE.

Cardiovascular autonomic dysfunction in non-obese diabetic mice.

It is known that diabetes is associated with autonomic dysfunction; however, data about autonomic function in non-obese diabetic mice (NOD) remain scarce. We evaluated the autonomic profile of NOD mice. Female mice, 24-28 week old, were divided in two groups: NOD (n = 6) and control (n = 6, Swiss mice). NOD mice with glycemia ≥ 300 mg/dl were used. Heart rate variability (HRV) and arterial pressure variability (APV) in time and frequency domains, symbolic analysis of heart rate (HR) and baroreflex sensitivity were evaluated. HR and arterial pressure (AP) were similar between the groups; however, HRV (total variance of RR interval: NOD=21.07 ± 3.75 vs. C = 42.02 ± 6.54 ms(2)) and the vagal modulation index RMSSD were lower in NOD group (4.01 ± 0.32 vs. 8.28 ± 0.97 ms). Moreover, the absolute and normalized low-frequency (LF) components were also enhanced in NOD (normalized = 61.0 ± 4.0%) as compared to control mice (normalized = 20.0 ± 4.0%). Both the absolute and normalized high-frequency (HF) components were lower in NOD (normalized = 39.0 ± 4.0%) when compared to the control group (normalized = 80.0 ± 4.0). In the symbolic analysis the 0V pattern, an indication of sympathetic activity, was higher in NOD and 2 LV pattern, an indication of parasympathetic activity, was lower in the NOD than in the control group. Both bradycardic and tachycardic responses were decreased in NOD (3.01 ± 0.72 vs. 4.54 ± 0.36 bpm/mmHg and 2.49 ± 0.31 vs. C = 3.43 ± 0.33 bpm/mmHg) when compared to the control group. Correlation analysis showed negative correlations between vagal indexes (RMSSD, %HF and 2LV) and glycemic levels. In conclusion, NOD mice develop severe diabetes correlated with autonomic dysfunction.

Overexpression of urinary N-domain ACE in chronic kidney dysfunction in Wistar rats.

Local activation of the renin-angiotensin system (RAS) has been implicated in the pathogenesis of several renal disorders. In this study we investigated how chronic kidney disease (CKD) modulates RAS components in an experimental model. Male Wistar rats were divided into three groups: sham, nephrectomized, and nephrectomized receiving losartan. Chronic kidney disease animals presented decreased renal N-domain angiotensin-converting enzyme (ACE) activity but overexpression of N-domain ACE in urine. Remnant kidneys presented high angiotensin II levels. Losartan treatment increased urine and tissue ACE activity and tissue levels of angiotensins, mainly angiotensin (1-7), and improved renal and histopathologic parameters. Taken together, the authors' results indicate that pathophysiological changes due to CKD could lead to an increased expression of somatic and N-domain ACE, mainly the 65 kDa isoform, suggesting that this enzyme could be used as a biological urinary marker in CKD.

Proteomic approaches in understanding a detected relationship between chemotherapy-induced nephrotoxicity and cell respiration in HK-2 cells.

BACKGROUND/AIMS: Nephrotoxicity is a prominent component of the profile of chemotherapeutic agents and to date proteomics has represented the main technique to identify protein profiles in response to xenobiotic exposure. METHODS: We made use of two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight analysis to evaluate chemotoxicity effects of cisplatin (CPT) and carboplatin (CB) on proteins from human renal proximal tubule epithelial cells (HK-2). RESULTS: Tandem mass spectrometry analysis showed that ATP synthase subunit α and serine hydroxymethyltransferase were only expressed in HK-2 cells exposed to CPT. Since CPT causes damage in cellular respiration, we suggest that this might be a protective adaptation to CPT-induced nephrotoxicity. Thioredoxin-dependent peroxide reductase disappeared in the CPT group and was upregulated in the CB group, suggesting that CB exposure stimulates preventive apoptotic mechanisms. We suggest a relationship between chemotherapeutic agent-induced nephrotoxicity and cell respiration. The identification of proteins differentially expressed in HK-2 cells, when exposed to CPT and CB, not only supplies important information to understand the molecular action mechanisms, which are triggered by metal-based drugs in cell nephrotoxicity, but also can lead to the design of more effective anticancer drugs. CONCLUSION: These results provide important insights into the investigation of possible biomarker(s) of toxicity that could eventually reduce the side effects of chemotherapeutic agents.

Renin-angiotensin system may trigger kidney damage in NOD mice.

Diabetic nephropathy is a complication of diabetes and one of the main causes of end-stage renal disease. A possible causal link between renin-angiotensin aldosterone system (RAAS) and diabetes is widely recognized but the mechanisms by which the RAAS may lead to this complication remains unclear. The aim of this study was to evaluate angiotensin-I converting enzyme (ACE) activity and expression in numerous tissues, especially kidney, of non-obese diabetic mouse. Kidney, lung, pancreas, heart, liver and adrenal tissues from diabetic and control female NOD mice were homogenized for measurement of ACE activity, SDS-PAGE and Western blotting for ACE and ACE2, immunohistochemistry for ACE and angiotensins I, II and 1-7 and bradykinin quantification. ACE activity was higher in kidney, lung and adrenal tissue of diabetic mice compared with control mice. In pancreas, activity was decreased in the diabetic group. Western blotting analysis indicated that both groups presented ACE isoforms with molecular weights of 142 and 69 kDa and a decrease in ACE2 protein expression. Angiotensin concentrations were not altered within groups, although bradykinin levels were higher in diabetic mice. The immunohistochemical study in kidney showed an increase in tubular ACE expression. Our results show that the RAAS is affected by diabetes and the elevated ACE/ACE2 ratio may contribute to renal damage.

Genetically determined angiotensin converting enzyme level and myocardial tolerance to ischemia.

Angiotensin I-converting enzyme (ACE; kininase II) levels in humans are genetically determined. ACE levels have been linked to risk of myocardial infarction, but the association has been inconsistent, and the causality underlying it remains undocumented. We tested the hypothesis that genetic variation in ACE levels influences myocardial tolerance to ischemia. We studied ischemia-reperfusion injury in mice bearing 1 (ACE1c), 2 (ACE2c, wild type), or 3 (ACE3c) functional copies of the ACE gene and displaying an ACE level range similar to humans. Infarct size in ACE1c was 29% lower than in ACE2c (P<0.05). Pretreatment with a kinin B2 receptor antagonist suppressed this reduction. In ACE3c, infarct size was the same as in ACE2c. But ischemic preconditioning, which reduced infarct size in ACE2c (-63%, P<0.001) and ACE1c (-52%, P<0.05), was not efficient in ACE3c (-2%, NS, P<0.01 vs. ACE2c). In ACE3c, ischemic preconditioning did not decrease myocardial inflammation or cardiomyocyte apoptosis. Pretreatment with a renin inhibitor had no cardioprotective effect in ACE2c, but in ACE3c partially restored (38%) the cardioprotection of ischemic preconditioning. Thus, a modest genetic increase in ACE impairs myocardial tolerance to ischemia. ACE level plays a critical role in cardiac ischemia, through both kinin and angiotensin mediated mechanisms.

Tissue kallikrein deficiency and renovascular hypertension in the mouse.

The kallikrein kinin system (KKS) is involved in arterial and renal functions. It may have an antihypertensive effect in both essential and secondary forms of hypertension. The role of the KKS in the development of two-kidneys, one-clip (2K1C) hypertension, a high-renin model, was investigated in mice rendered deficient in tissue kallikrein (TK) and kinins by TK gene inactivation (TK-/-) and in their wild-type littermates (TK+/+). Four weeks after clipping the renal artery, blood flow was reduced in the clipped kidney (2K1C-TK+/+: -90%, 2K1C-TK-/-: -93% vs. sham-operated mice), and the kidney mass had also decreased (2K1C-TK+/+: -65%, 2K1C-TK-/-: -66%), whereas in the unclipped kidney, blood flow (2K1C-TK+/+: +19%, 2K1C-TK-/-: +17%) and kidney mass (2K1C-TK+/+: +32%, 2K1C-TK-/-: +30%) had both increased. The plasma renin concentration (2K1C-TK+/+: +78%, 2K1C-TK-/-: +65%) and renal renin content of the clipped kidney (2K1C-TK+/+: +58%, 2K1C-TK-/-: +65%) had increased significantly. There was no difference for these parameters between 2K1C-TK+/+ and 2K1C-TK-/- mice. Blood pressure monitored by telemetry and by plethysmography, rose immediately after clipping in both genotypes, and reached similar levels (2K1C-TK+/+: +24%, 2K1C-TK-/-: +21%). 2K1C-TK+/+ and 2K1C-TK-/- mice developed similar concentric left ventricular hypertrophy (+24% and +17%, respectively) with normal cardiac function. These findings suggest that in the context of chronic unilateral reduction in renal blood flow, TK and kinins do not influence the trophicity of kidneys, the synthesis and secretion of renin, blood pressure increase, and cardiac remodeling due to renin angiotensin system activation.

Diabetes induces changes of catecholamines in primary mesangial cells.

Diabetes mellitus is a frequent cause of kidney function damage with diabetic nephropathy being predominantly related to glomerular dysfunction. Diabetes is capable of interfering with distinct hormonal systems, as well as catecholamine metabolism. Since mesangial cells, the major constituent of renal glomerulus, constitute a potential site for catecholamine production, the present study was carried out to investigate alterations in catecholamine metabolism in cultured mesangial cells from the nonobese diabetic mouse, a well-established model for type I diabetes. We evaluated mesangial cells from normoglycemic and hyperglycemic nonobese diabetic mice, as well as cells from normoglycemic Swiss mice as control. Mesangial cells from normoglycemic mice presented similar profiles concerning all determinations. However, cells isolated from hyperglycemic animals presented increased dopamine and norepinephrine production/secretion. Among the studied mechanisms, we observed an upregulation of tyrosine hydroxylase expression accompanied by increased tetrahydrobiopterin consumption, the tyrosine hydroxylase enzymatic cofactor. However, this increase in synthetic pathways was followed by decreased monoamine oxidase activity, which corresponds to the major metabolic pathway of catecholamines in mesangial cells. In addition, whole kidney homogenates from diabetic animals also presented increased dopamine and norepinephrine levels when compared to normoglycemic animals. Thus, our results suggest that diabetes alters catecholamine production by interfering with both synthesizing and degrading enzymes, suggesting a possible role of catecholamine in the pathogenesis of acute and chronic renal complications of diabetes mellitus.