BACKGROUND: Megalin (LRP2 receptor) mediates the endocytosis of radiolabeled peptides into proximal tubular kidney cells, which may cause nephrotoxicity due to the accumulation of a radioactive tracer. The study aimed to develop a cellular model of human kidney HK2 cells with LRP2 knockout (KO) using CRISPR/Cas9 technique. This model was employed for the determination of the megalin-mediated accumulation of 68Ga- and 99mTc-labeled 15-mer peptide developed to target the vascular endothelial growth factor (VEGF) receptor in oncology radiodiagnostics. RESULTS: The gene editing in the LRP2 KO model was verified by testing two well-known megalin ligands when higher viability of KO cells was observed after gentamicin treatment at cytotoxic concentrations and lower FITC-albumin internalization by the KO cells was detected in accumulation studies. Fluorescent-activated cell sorting was used to separate genetically modified LRP2 KO cell subpopulations. Moreover, flow cytometry with a specific antibody against megalin confirmed LRP2 knockout. The verified KO model identified both 68Ga- and 99mTc-radiolabeled 15-mer peptides as megalin ligands in accumulation studies. We found that both radiolabeled 15-mers enter LRP2 KO HK2 cells to a lesser extent compared to parent cells. Differences in megalin-mediated cellular uptake depending on the radiolabeling were not observed. Using biomolecular docking, the interaction site of the 15-mer with megalin was also described. CONCLUSION: The CRISPR/Cas9 knockout of LRP2 in human kidney HK2 cells is an effective approach for the determination of radiopeptide internalization mediated by megalin. This in vitro method provided direct molecular evidence for the cellular uptake of radiolabeled anti-VEGFR 15-mer peptides via megalin.

• Publikační typ
• časopisecké články MeSH

The synthetic analogs of regulatory peptides radiolabeled with adequate radionuclides are perspective tools in nuclear medicine. However, undesirable uptake and retention in the kidney limit their application. Specific in vitro methods are used to evaluate undesirable renal accumulation. Therefore, we investigated the usefulness of freshly isolated rat renal cells for evaluating renal cellular uptake of receptor-specific peptide analogs. Special attention was given to megalin as this transport system is an important contributor to the active renal uptake of the peptides. Freshly isolated renal cells were obtained from native rat kidneys by the collagenase method. Compounds with known accumulation in renal cells were used to verify the viability of cellular transport systems. Megalin expressions in isolated rat renal cells were compared to two other potential renal cell models by Western blotting. Specific tubular cell markers were used to confirm the presence of proximal tubular cells expressing megalin in isolated rat renal cell preparations by immunohistochemistry. Colocalization experiments on isolated rat kidney cells confirmed the presence of proximal tubular cells bearing megalin in preparations. The applicability of the method was tested by an accumulation study with several analogs of somatostatin and gastrin labeled with indium-111 or lutetium-177. Therefore, isolated rat renal cells may be an effective screening tool for in vitro analyses of renal uptake and comparative renal accumulation studies of radiolabeled peptides or other radiolabeled compounds with potential nephrotoxicity.

• Publikační typ
• časopisecké články MeSH

Vitamin D, either in its D2 or D3 form, is essential for normal human development during intrauterine life, kidney function and bone health. Vitamin D deficiency has also been linked to cancer development and some autoimmune diseases. Given this huge impact of vitamin D on human health, it is important for daily clinical practice and clinical research to have reliable tools to judge on the vitamin D status. The major circulating form of vitamin D is 25-hydroxyvitamin D (25(OH)D), although it is not the most active metabolite, the concentrations of total 25-hydroxyvitamin D in the serum are currently routinely used in clinical practice to assess vitamin D status. In the circulation, vitamin D - like other steroid hormones - is bound tightly to a special carrier - vitamin D-binding protein (DBP). Smaller amounts are bound to blood proteins - albumin and lipoproteins. Only very tiny amounts of the total vitamin D are free and potentially biologically active. Currently used vitamin D assays do not distinguish between the three forms of vitamin D - DBP-bound vitamin D, albumin-bound vitamin D and free, biologically active vitamin D. Diseases or conditions that affect the synthesis of DBP or albumin thus have a huge impact on the amount of circulating total vitamin D. DBP and albumin are synthesized in the liver, hence all patients with an impairment of liver function have alterations in their total vitamin D blood concentrations, while free vitamin D levels remain mostly constant. Sex steroids, in particular estrogens, stimulate the synthesis of DBP. This explains why total vitamin D concentrations are higher during pregnancy as compared to non-pregnant women, while the concentrations of free vitamin D remain similar in both groups of women. The vitamin D-DBP as well as vitamin D-albumin complexes are filtered through the glomeruli and re-uptaken by megalin in the proximal tubule. Therefore, all acute and chronic kidney diseases that are characterized by a tubular damage, are associated with a loss of vitamin D-DBP complexes in the urine. Finally, the gene encoding DBP protein is highly polymorphic in different human racial groups. In the current review, we will discuss how liver function, estrogens, kidney function and the genetic background might influence total circulating vitamin D levels and will discuss what vitamin D metabolite is more appropriate to measure under these conditions: free vitamin D or total vitamin D.

• MeSH
• biologické markery krev   MeSH
• lidé MeSH
• nedostatek vitaminu D krev   diagnóza   MeSH
• protein vázající vitamin D metabolismus   MeSH
• vitamin D analogy a deriváty   krev   MeSH
• vitaminy krev   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

• Publikační typ
• souhrny MeSH

Autosomal-dominant polycystic kidney disease (ADPKD) is an inherited disease that results in multiple kidney cysts, and it is a common cause of end-stage renal disease. Recent studies have shown that disease progression can be slowed by simultaneous disruption of the primary cilium and polycystins. The exact genetic mechanism of this process is still unknown. The aim of the present study was to characterize the mutation profile of ciliary signalling pathways in the renal epithelial cells of ADPKD patients. In our study, we performed an analysis of 110 genes encoding the components of Sonic Hedgehog, Hippo, Notch, Wnt and planar cell polarity signalling (PCP) by targeted next-generation sequencing. We analysed 10 formalin-fixed, paraffinembedded (FFPE) tissue samples of patients with ADPKD. We identified a unique mutation profile in each of the analysed ADPKD samples, which was characterized by the presence of pathogenic variants in eight to 11 genes involved in different signalling pathways. Despite the significant genetic heterogeneity of ADPKD, we detected five genes whose genetic variants affected most ADPKD samples. The pathogenic variants in NCOR2 and LRP2 genes were present in all analysed samples of ADPKD. In addition, eight out of 10 samples showed a pathogenic variant in the MAML2 and FAT4 genes, and six out of 10 samples in the CELSR1 gene. In our study, we identified the signalling molecules that may contribute to the cystogenesis and may represent potential targets for the development of new ADPKD treatments.

• MeSH
• DNA vazebné proteiny genetika   MeSH
• dospělí MeSH
• jaderné proteiny genetika   MeSH
• kadheriny genetika   MeSH
• kationtové kanály TRPP genetika   MeSH
• korepresor 2 jaderného receptoru genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mutace genetika   MeSH
• nádorové supresorové proteiny genetika   MeSH
• pilotní projekty MeSH
• polarita buněk genetika   fyziologie   MeSH
• polycystická choroba ledvin genetika   metabolismus   patologie   MeSH
• polycystické ledviny autozomálně dominantní genetika   metabolismus   patologie   MeSH
• progrese nemoci MeSH
• protein 2 související s LDL-receptory genetika   MeSH
• signální transdukce genetika   fyziologie   MeSH
• transkripční faktory genetika   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

INTRODUCTION: Radiolabeled receptor-targeting peptides are a useful tool for the diagnostic imaging and radiotherapy of some malignancies. However, the retention of radioactivity in the kidney may result in renal radiotoxic injury. This study seeks to evaluate the role of endocytic receptor megalin, renal SLC influx transporters and fluid phase endocytosis (FPE) in the cellular accumulation of radiolabeled peptides. METHODS: In vitro transport cellular studies using megalin ligands (RAP, albumin), fluid phase endocytosis (FPE) inhibitor rottlerin and low temperature were employed to evaluate the transport mechanisms of the peptides. Cells transfected with hOAT1 or hOCT2 were used to analyze the role of these SLC transporters. Somatostatin ((177)Lu-DOTA-[Tyr(3)]octreotate, (177)Lu-DOTA-[1-Nal(3)]octreotide), gastrin ((177)Lu-DOTA-sargastrin) and bombesin ((177)Lu-DOTA-[Pro(1),Tyr(4)]bombesin, (177)Lu-DOTA-[Lys(3)]bombesin, (177)Lu-PCTA-[Lys(3)]bombesin) analogues were involved in the study. RESULTS: RAP, albumin and low temperature decreased the accumulation of all the studied peptides significantly. With one exception, rottlerin caused the concentration dependent inhibition of the cellular accumulation of the radiopeptides. No significant differences in the uptake of the peptides between the control cells and those transfected with hOAT1 or hOCT2 were observed. CONCLUSION: The study showed that active transport mechanisms are decisive for the cellular accumulation in all tested (177)Lu-labeled somatostatin, gastrin and bombesin analogues. Besides receptor-mediated endocytosis by megalin, FPE participates significantly in the uptake. The tested types of renal SLC transporters are not involved in this process.

• MeSH
• biologický transport MeSH
• bombesin chemie   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• endocytóza * MeSH
• gastriny chemie   metabolismus   MeSH
• HeLa buňky MeSH
• izotopové značení MeSH
• lidé MeSH
• lutecium * MeSH
• peptidové hormony chemie   metabolismus   MeSH
• prasata MeSH
• přenašeče organických aniontů nezávislé na sodíku metabolismus   MeSH
• protein 1 přenášející organické anionty metabolismus   MeSH
• somatostatin chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Lipokaliny tvoří velkou skupinu malých extracelulárních proteinů. Vykazují velkou rozmanitost na úrovni sekvenčního uspořádání, terciární struktura je oproti tomu vysoce konzervovaná a představuje ji β-válec tvořený osmi antiparalelními strukturami β-skládaného listu. Vazebné místo ligandu se nachází uvnitř β-válce. Zastávají rozličné funkce, např. transportují retinoidy, steroidy, biliny, feromony, účastní se syntézy prostaglandinů, tvorby ochranného zbarvení bezobratlých, vytváření čichových vjemů, jsou zapojené do regulace buněčné homeostázy a modulace imunitní odpovědi. Lipokalin-2 (neutrofilní s gelatinázou asociovaný lipokalin), časný marker různých typů renálního poškození, vykazuje pleiotropní biologické aktivity v různých typech buněk a tkání. Hraje významnou roli v angiogenezi, apoptóze, organogenezi, zánětu, hematopoeze, v procesu hojení ran, v renální fyziologii, v nádorové a reprodukční biologii.

The lipocalin protein family is a large group of small extracellular proteins. The family demonstrates great diversity at sequence level; however, lipocalin crystal structures are highly conserved and comprise a single eight-stranded antiparallel β-barrel, which encloses an internal ligand-binding site. They have been associated with a variety of different functions, among them transport of retinoids, steroids, bilins, pheromones, enzymic synthesis of prostaglandins, cryptic coloration, olfaction, immune response, cell regulation. Lipocalin-2 (also known as neutrophil gelatinase-associated lipocalin), which is a useful biomarker for early detection of various renal injuries, shows pleiotropic bioactivities in a variety of different cell types and tissues. It plays important roles in angiogenesis, apoptosis, organogenesis, inflammation, hematopoiesis, wound healing, renal physiology, tumor and reproductive biology.

• MeSH
• akutní poškození ledvin diagnóza   MeSH
• dospělí MeSH
• extracelulární matrix - proteiny MeSH
• fyziologická neovaskularizace MeSH
• genetická pleiotropie MeSH
• glykoproteiny biosyntéza   MeSH
• hematopoéza fyziologie   MeSH
• hojení ran fyziologie   MeSH
• kreatinin analýza   krev   MeSH
• lidé MeSH
• lipokaliny * fyziologie   chemie   klasifikace   ultrastruktura   MeSH
• matrixová metaloproteinasa 9 fyziologie   MeSH
• molekulární medicína MeSH
• nádorová transformace buněk sekrece   MeSH
• neutrofily fyziologie   MeSH
• protein 2 související s LDL-receptory MeSH
• proteiny akutní fáze * MeSH
• protoonkogenní proteiny MeSH
• terciární struktura proteinů * MeSH
• transportní proteiny MeSH
• vazebná místa MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

• MeSH
• chronická renální insuficience * patofyziologie   MeSH
• intestinální absorpce * fyziologie   MeSH
• lidé MeSH
• membránové glykoproteiny fyziologie   MeSH
• modely nemocí na zvířatech MeSH
• nedostatek vitaminu B12 patofyziologie   MeSH
• protein 2 související s LDL-receptory fyziologie   MeSH
• proximální tubuly ledvin fyziologie   MeSH
• receptory buněčného povrchu * fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• komentáře MeSH

1) Zjištění změn chemorezistence/chemosensitivity ovariálních nádorových buněk v průběhu léčby pomocí MTT-WST-1 testu a stanovení "proteinů rezistence" (MRP, LRP, p170). 2) Vzorky solidního nádoru nebo ascitu budou odebírány při primární operaci před zahájením chemoterapie první linie a při každé další operaci či punkci ascitu následovaném zahájením nové linie chemoterapie. 3)Zhodnocení vztahu chemorezistence/chemosensitivity ovariálních nádorových buněk s délkou bezpříznakového období, dobou přežití, histologickým typem nádoru a výskytem komplikací v průběhu chemoterapie; 1)The changes in chemoresistance/chemosensitivity of ovarian cancer cells by means of MTT -WST-1 test and assessment of resistance proteins (MRP, LRP, p170) in a course of treatment. 2) Samples of ascitic fluid or solid tissue will be taken during each surgical intervation and before the initiation of a new round of chemotherapy. 3) The evaluation of the relation between chemoresistance/chemosensitivity of ovarian carcinoma cells and the length of disease free interval, overall survival, histologic typeand the occurrence of complications in the course of chemotherapy.

• MeSH
• chemorezistence MeSH
• cytotoxické testy imunologické MeSH
• geny MDR MeSH
• mnohočetná léková rezistence MeSH
• nádory vaječníků farmakoterapie   MeSH
• P-glykoprotein MeSH
• prognóza MeSH
• protein 2 související s LDL-receptory MeSH
• screeningové testy protinádorových léčiv MeSH

• Konspekt
• Farmacie. Farmakologie
• NLK Obory
• gynekologie a porodnictví
• onkologie
• farmacie a farmakologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

Cílená radionuklidová terapie uživající radioaktivně značené pro somatostatinové receptory specifické peptidy je již užívanou a dále se rozvíjející metodou léčby některých typů tumorů. Avšak nežádoucí retence radioaktivity v ledvinné tkáni vedoucí k radiotoxickému poškození ledvin limituje úspěšnost léčby. Projekt by měl poskytnout data o možnostech ovlivnění ledvinné akumulace získáním znalostí o mechanismech transportu těchto látek v ledvinách. Tyto informace by mohly být využitelné v klinické praxi pro snížení nežádoucích nefrotoxických účinků radioterapeutik z této skupiny. Bude použita řada experimentálních metod na úrovni orgánové, buněčné a molekulární. Na základě poznání mechanismů chování v ledvinách budou testovány možné inhibitory ledvinné retence radiopeptidů. Výsledky projektu budou sloužit k optimalizaci dalšího klinického použití radioaktivně značených pro somatostatinové receptory specifických peptidů v nukleární medicíně.; The targeted radionuclide therapy using radiolabelled somatostatin receptor-specific peptides is a used and developing method to treat some type of tumours. However, undesirable retention of radioactivity in the kidney with subsequent radiotoxicologicalrenal impairment limits the benefit of the therapy. The project should provide data regarding possibility how to affect renal retention by means of knowledge in transport mechanisms of the agents in the kidney. This information may be useful in clinicalpractice to limit the undesirable nephrotoxic effect of the radiotherapeutics from this group. Several experimental methods at organ, cellular and molecula levels will be used in the project. On the basis of knowledge in renal transport mechanisms of radiopeptides, potential inhibitors of the radiopeptide renal retention will be studied. The results of the project may serve for optimalization of further clinical use of radiolabelled somatostatin receptor-specific peptides in nuclear medicine.

• MeSH
• adjuvantní radioterapie MeSH
• akutní poškození ledvin MeSH
• nádory terapie   MeSH
• nežádoucí účinky léčiv MeSH
• protein 2 související s LDL-receptory MeSH
• radiofarmaka toxicita   MeSH
• somatostatin MeSH

• Konspekt
• Farmacie. Farmakologie
• NLK Obory
• farmacie a farmakologie
• onkologie
• nefrologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR