Potassium channels in renal proximal tubule

Potassium channels are a diverse family of membrane proteins which are present within all cells of the body. They contain two subunits α, which determine the structure of the channel, and β, which can modify the properties of the channel. Those transmembrane proteins take part in K+ movement across cell membranes, via a highly selective pore. The kidneys have crucial role in maintaining total body potassium content, by matching its intake and excretion. K+ absorption in the proximal tubule is primarily passive and proportional to Na+ and water, so that changes in fluid and potassium transport are closely coupled. Proximal tubular K+ channels are crucial for the maintenance of a hyperpolarized membrane voltage. In leaky epithelia, such as the proximal tubule, the hyperpolarization of the basolateral membrane also results in the hyperpolarization of the apical membrane, due to increase in the K+ conductance of that barrier. They are also involved in regulating cell volume and in recycling potassium across the basolateral membrane. K+ channels of the KCNK and KCNJ gene families have been discovered in the basolateral membrane cell of various species. One of the primary functions of basolateral K+ channels is to recycle K+ across the basolateral membrane for proper function of the Na+-K+-ATPase. Activation by extracellular alkalinization has been associated with a role of TASK-2 in kidney proximal tubule bicarbonate reabsorption. In renal proximal tubules, luminal K+ channels play an important role for restoring the driving force of Na+-coupled transport systems (amino acids, glucose), which depolarize the luminal membrane. Some of these luminal K+ channels are activated directly by the transport-associated depolarization; others are regulated by mediators, second messenger pathways and cell volume

Efekat deprivacije REM faze spavanja na aktivnost acetilholinesteraze i NA+/K+-ATPaze sinaptozoma mozga pacova u hipotiroidizmu

Hipotireoidizam i deprivacija REM spavanja prepoznati su kao mogući faktori rizika za nastanak neurodegenerativnih oboljenja, pre svega Alchajmerove bolesti (AB). Enzimi acetilholinesteraza (AchE) i Na+ /K+ -ATPaza imaju ulogu u taloženju amiloidnih plakova, tako da je njihova ukupna aktivnost u mozgu obolelog od AB snižena. Cilj istraživanja bio je utvrđivanje mogućeg efekta hipotireoidizma i deprivacije REM faze spavanja na aktivnost AchE i Na+ /K+ -ATPaze. Koristili smo eksperimentalni model hipotireoidizma izazvan propiltiouracilom. Nakon uvođenja u hipotiroidizam, eksperimentalna grupa životinja (n = 12) podeljena je u dve podrupe. Životinje iz prve podgrupe (n = 6) boravile su 72 h na maloj platformi, koja ne dozvoljava REM spavanje, jer usled atonije životinja upada u vodu i budi se. Drugoj podgrupi životinja (n = 6) omogućeno je spavanje. Nakon 72 h, životinje su žrtvovane, i u sinaptozomalnoj frakciji mozga pacova određivana je aktivnost AchE i Na+ /K+ -ATPaze spektrofotometrijski. Rezultati pokazuju da hipotireoidizam dovodi do snižene aktivnosti AchE i Na+ /K+ -ATPaze u odnosu na kontrolu (p ≤ 0,01), dok zajednički efekat hipotireoidizma i deprivacije REM spavanja dovodi do snižene aktivnosti AchE i Na+ /K+ -ATPaze u odnosu na kontrolu (n = 6, p ≤ 0,01), ali ne u odnosu na hipotiroidizam (p > 0,05). Na osnovu dobijenih rezultata možemo zaključiti da hipotireoidizam smanjuje aktivnost enzima AchE i Na+ /K+ -ATPaze, a da deprivacija REM faze spavanja dodatno ne smanjuje aktivnost ovih enzima u hipotireoidizmu, tako je mogući način delovanja hipotireoidizma kao faktora rizika za nastanak AB upravo sniženje aktivnosti ovih enzima.Četvrti srpski kongres o štitastoj žlezdi : Zbornik sažetaka : Septembar, 2017, Zlatibor

Clinicopathological Relevance of PAX8 Expression Patterns in Acute Kidney Injury and Chronic Kidney Diseases

Transcription factor PAX8, expressed during embryonic kidney development, has been previously detected in various kidney tumors. In order to investigate expression of PAX8 transcription factor in acute kidney injury (AKI) and chronic kidney diseases (CKD), immunohistochemical analysis was performed. Presence, location and extent of PAX8 expression were analyzed among 31 human kidney samples of AKI (25 autopsy cases, 5 kidney biopsies with unknown etiology and 1 AKI with confirmed myoglobin cast nephropathy), as well as in animals with induced postischemic AKI. Additionally, expression pattern was analyzed in 20 kidney biopsy samples of CKD. Our study demonstrates that various kidney diseases with chronic disease course that results in the formation of tubular atrophy and interstitial fibrosis, lead to PAX8 expression in the nuclei of proximal tubules. Furthermore, patients with PAX8 detected within the damaged proximal tubuli would be carefully monitored, since deterioration in kidney function was observed during follow-up. We also showed that myoglobin provoked acute kidney injury followed with large extent of renal damage, was associated with strong nuclear expression of PAX8 in proximal tubular cells. These results were supported and followed by data obtained in experimental model of induced postischemic acute kidney injury. Considering these findings, we can assume that PAX8 protein might be involved in regeneration process and recovery after acute kidney injury. Thus, accordingly, all investigation concerning PAX8 immunolabeling should be performed on biopsy samples of the living individuals

Hyperbaric oxygen preconditioning and the role of NADPH oxidase inhibition in postischemic acute kidney injury induced in spontaneously hypertensive rats

Renal ischemia/reperfusion injury is a common cause of acute kidney injury (AKI) and hypertension might contribute to the increased incidence of AKI. The purpose of this study was to investigate the effects of single and combined hyperbaric oxygen (HBO) preconditioning and NADPH oxidase inhibition on oxidative stress, kidney function and structure in spontaneously hypertensive rats (SHR) after renal ischemia reperfusion injury. HBO preconditioning was performed by exposing to pure oxygen (2.026 bar) twice a day for two consecutive days for 60 minutes, and 24h before AKI induction. For AKI induction, the right kidney was removed and ischemia was performed by clamping the left renal artery for 45 minutes. NADPH oxidase inhibition was induced by apocynin (40 mg/kg b.m., intravenously) 5 minutes before reperfusion. AKI significantly increased renal vascular resistance and reduced renal blood flow, which were significantly improved after apocynin treatment. Also, HBO preconditioning, with or without apocynin treatment showed improvement on renal hemodynamics. AKI significantly increased plasma creatinine, urea, phosphate levels and lipid peroxidation in plasma. Remarkable improvement, with decrease in creatinine, urea and phosphate levels was observed in all treated groups. HBO preconditioning, solitary or with apocynin treatment decreased lipid peroxidation in plasma caused by AKI induction. Also, combined with apocynin, it increased catalase activity and solitary, glutathione reductase enzyme activity in erythrocytes. While AKI induction significantly increased plasma KIM- 1 levels, HBO preconditioning, solitary or with apocynin decreased its levels. Considering renal morphology, significant morphological alterations present after AKI induction were significantly improved in all treated groups with reduced tubular dilatation, tubular necrosis in the cortico-medullary zone and PAS positive cast formation. Our results reveal that NADPH oxidase inhibition and hyperbaric oxygen preconditioning, with or without NADPH oxidase inhibition may have beneficial effects, but their protective role should be evaluated in further studies

Immunohistochemical Analysis of 4-HNE, NGAL, and HO-1 Tissue Expression after Apocynin Treatment and HBO Preconditioning in Postischemic Acute Kidney Injury Induced in Spontaneously Hypertensive Rats

Oxidative stress has been considered as a central aggravating factor in the development of postischemic acute kidney injury (AKI). The aim of this study was to perform the immunohistochemical analysis of 4-hydroxynonenal (4-HNE), neutrophil gelatinase-associated lipocalin (NGAL), and heme oxygenase-1 (HO-1) tissue expression after apocynin (APO) treatment and hyperbaric oxygenation (HBO) preconditioning, applied as single or combined protocol, in postischemic acute kidney injury induced in spontaneously hypertensive rats (SHR). Twenty-four hours before AKI induction, HBO preconditioning was carried out by exposing to pure oxygen (2.026 bar) twice a day, for 60 min in two consecutive days. Acute kidney injury was induced by removal of the right kidney while the left renal artery was occluded for 45 min by atraumatic clamp. Apocynin was applied in a dose of 40 mg/kg body weight, intravenously, 5 min before reperfusion. We showed increased 4-HNE renal expression in postischemic AKI compared to Sham-operated (SHAM) group. Apocynin treatment, with or without HBO preconditioning, improved creatinine and phosphate clearances, in postischemic AKI. This improvement in renal function was accompanied with decreased 4-HNE, while HO-1 kidney expression restored close to the control group level. NGAL renal expression was also decreased after apocynin treatment, and HBO preconditioning, with or without APO treatment. Considering our results, we can say that 4-HNE tissue expression can be used as a marker of oxidative stress in postischemic AKI. On the other hand, apocynin treatment and HBO preconditioning reduced oxidative damage, and this protective effect might be expected even in experimental hypertensive condition

Hyperbaric Oxygen Preconditioning Upregulates Heme OxyGenase-1 and Anti-Apoptotic Bcl-2 Protein Expression in Spontaneously Hypertensive Rats with Induced Postischemic Acute Kidney Injury

Renal ischemia and reperfusion (I/R) injury is the most common cause of acute kidney injury (AKI). Pathogenesis of postischemic AKI involves hemodynamic changes, oxidative stress, inflammation process, calcium ion overloading, apoptosis and necrosis. Up to date, therapeutic approaches to treat AKI are extremely limited. Thus, the aim of this study was to evaluate the effects of hyperbaric oxygen (HBO) preconditioning on citoprotective enzyme, heme oxygenase-1 (HO-1), pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins expression, in postischemic AKI induced in normotensive Wistar and spontaneously hypertensive rats (SHR). The animals were randomly divided into six experimental groups: SHAM-operated Wistar rats (W-SHAM), Wistar rats with induced postischemic AKI (W-AKI) and Wistar group with HBO preconditioning before AKI induction (W-AKI + HBO). On the other hand, SHR rats were also divided into same three groups: SHR-SHAM, SHR-AKI and SHR-AKI + HBO. We demonstrated that HBO preconditioning upregulated HO-1 and anti-apoptotic Bcl-2 protein expression, in both Wistar and SH rats. In addition, HBO preconditioning improved glomerular filtration rate, supporting by significant increase in creatinine, urea and phosphate clearances in both rat strains. Considering our results, we can also say that even in hypertensive conditions, we can expect protective effects of HBO preconditioning in experimental model of AKI

Effects of NADPH oxidase blockade and hyperbaric oxygen preconditioning on 4-HNE, NGAL, and HO-1 tissue expression in postischemic acute kidney injury induced in spontaneously hypertensive rat

Kidney disease represents a serious global health problem. Free radicals and prooxidants produced during ischemic acute kidney injury (AKI) may further aggravate the course of the disease and play a role in the pathogenesis of subsequent complications. The aims of our study were to examine the importance of NADPH oxidase blockade and to determine the effect of hyperbaric oxygen preconditioning on the immunohistochemical analysis of 4-hydroxynonenal (4-HNE), neutrophil gelatinase-associated lipocalin (NGAL), and heme oxygenase-1 (HO-1) tissue expression in postischemic acute kidney injury induced in spontaneously hypertensive rats (SHR). Twenty-four hours before AKI induction, HBO preconditioning was carried out by exposing to pure oxygen (2.026bar) twice a day, for 60 min in two consecutive days. Acute kidney injury was induced by removal of the right kidney while the left renal artery was occluded for 45 min by atraumatic clamp. NADPH oxidase blockade was performed by Apocynin (40 mg/kg body weigh)t, intravenously, 5 min before reperfusion. We showed increased 4-HNE renal expression in postischemic AKI compared to Sham operated (SHAM) group. Apocynin treatment, with or without HBO preconditioning, improved creatinine and phosphate clearances, in postischemic AKI. This improvement in renal function was accompanied with decreased 4-HNE, while HO-1 kidney expression restored close to the control group level. NGAL renal expression was also decreased after apocynin treatment, and HBO preconditioning,with or without APO treatment. Considering our results, we can say that 4-HNE tissue expression can be used as a marker of oxidative stress in postischemic AKI. On the other hand, NADPH oxidase blockade and HBO preconditioning reduced oxidative damage, and this protective effect might be expected even in experimental hypertensive condition

Serum beta(2)-microglobulin as a marker of congenital toxoplasmosis and cytomegalovirus infection in preterm neonates

Background: Fetal serum beta(2)-microglobulin (beta M-2) has been reported as a reliable indicator of fetal infectious diseases. Objectives: To evaluate serum beta M-2 as a marker of congenital toxoplasmosis or cytomegalovirus (CMV) infection in neonates. Methods: beta M-2 was retrospectively measured in 72 neonatal serum samples from preterm neonates. Of these, 32 originated from neonates with serological evidence of congenital toxoplasmosis (n = 12) and CMV infection (n = 20), while 40 samples from neonates in which both infections were serologically excluded served as controls. beta M-2 levels were compared between the infection and control groups. Results: Mean (+/-SEM) beta M-2 levels were significantly higher in the groups of neonates infected with Toxoplosma (5.64 +/- 0.61 mg/l) (p = 0.014) and CMV (6.06 +/- 0.66 mg/l) (p lt 0.0001) than in the control group (3.80 +/- 0.2). Against the cut-off level of 5 Mg/l, beta M-2 was normal in 36 of the 40 uninfected neonates examined, indicating a specificity of 90%. In contrast, it was elevated in 66.7% (8/12) and 65% (13/20) of the Toxoplasma and CMV-infected neonates, respectively, indicating an overall sensitivity of 66%. Conclusions: In the absence of urogenital disorders, an increase in beta M-2 in neonates is likely to be infection-induced. We showed that serum beta M-2 is increased in congenital toxoplasmosis and CMV infection in the first weeks of life. Copyright (C) 2008 S. Karger AG, Basel

Effects of Automation on Sustainability of Immunohistochemistry Laboratory

The COVID-19 pandemic that hit the world recently caused numerous changes affecting the health system in every department. Reduced staff numbers, mostly due to illness, led to an increase in automation at every stage of laboratory work. The immunohistochemistry (IHC) laboratory conducts a high volume of slide staining every day. Therefore, we analyzed time and total costs required to obtain IHC slides in both the manual and automated way, comparing their efficiency by processing the same sample volume (48 microscope slides—the maximum capacity that an automated immunostainer—DAKO, Autostainer Link 48, Part No AS48030—can process over a single cycle). The total IHC procedure time to run 48 slides manually by one technician was 460 min, while the automated process finished a cycle within 390 min (15.22% less time). The final cost of a single manual IHC slide was 12.26 EUR and 7.69 EUR for slides labeled in the automated immunostainer, which reduced final costs by 37.27%. Thus, automation of the IHC procedure reduces the time and costs of the IHC process, contributing significantly to the sustainability of the healthcare system during the COVID-19 pandemic, overcoming insufficient human resources