8 ABSTRACT Author: Bc. Lucie Moravcová Supervisor: MUDr. Vít Řeháček Charles University, Faculty of Pharmacy in Hradec Králové Title of master's thesis: Kidney transplantation: donor-recipient pairing in University Hospital Hradec Králové Background: The aim of this study was to determine HLA, blood group, age and sex match in donor-recipient pairing in kidney transplantation. HLA alleles of deceased donors were typed in Transfusion Department of the University Hospital Hradec Králové. Methods: Donor's HLA-A*, HLA-B* and HLA-DRB1* alleles were typed by PCR - SSP method. Complex data evaluating and processing was then performed. Results: 97 deceased donors were tested between 2013 and 2018. A total of 98 kidneys received from them were subsequently transplanted to 98 recipients in University Hospital Hradec Králové. 60,2 % of the donors were men, 63,3 % of the recipients were men. Most of the donors, as well as the recipients, were 51-70 years old (50,0 % and 59,2 %, respectively). The most common diagnoses in the group of deceased donors were associated with brain damage (66,3 %), the most common cause of renal failure in the group of recipients was chronic inflammatory kidney disease (41,8 %). All 98 transplantations (100,0 %) were AB0 compatible. 74 transplantations (75,5 %) were RhD compatible. 5...

Renal cell carcinoma is a high risk and high mortality cancer. While the VHL pathway is frequently altered in renal cell carcinoma, emerging evidences points towards involvement of multiple complex pathways in the progression of renal cell carcinoma. In this present work, we aimed to investigate the role of newly identified protein, Major Intrinsically disordered NOTCH2-Associated Receptor 1 (MINAR1), in renal cell carcinoma. We used immunohistochemistry and demonstrated that MINAR1 is highly expressed in normal kidney epithelium. Furthermore, MINAR1 was expressed at variable levels in human renal cell carcinoma cell lines. More importantly, we found that MINAR1 was significantly downregulated in human samples of renal cell carcinoma. Although further studies are needed, our data suggests a potential role for MINAR1 in renal biology. Given that MINAR1 expression appears to be downregulated in renal cancer patients, it is suggestive that MINAR1 could function as a tumor suppressor. / 2020-06-17T00:00:00Z

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MMed (Paediatrics), Faculty of Health Sciences, University of the Witwatersrand / This prospective observational study analysed iron studies and vitamin C levels in patients with chronic kidney disease attending Johannesburg Hospital Paediatric Nephrology Clinic. The rationale behind this study was to determine the extent of iron deficiency among patients in chronic renal failure. Vitamin C deficiency is common among dialysis patients, it is easy to test for and easy to prevent. This study may assist in guiding future management with regards to vitamin C supplementation in patients with chronic renal insufficiency on dialysis. The study contained 45 patients of which 27 (60 %) were male and 18 (40 %) were female. The ages of the children varied from 2 years 1 month to 19 years and 7 months. The study included patients from all ethnic groups; 9 were Caucasian, 33 African, 2 Indian and 1 Coloured. Two male patients did not have Vitamin C levels analyzed. The patients were divided into 3 distinct groups; firstly those patients on haemodialysis (12 patients), those on peritoneal dialysis (22 patients) and those not yet dialysed (11 patients). In all patients who were not yet on dialysis the GFR ranged between 18.1 and 45 ml/min/1.73m2. There were no statistically significant differences between the three groups when the results of the iron studies were analysed. However, despite iron treatment 26.6 % of patients were iron deficient as indicated by their transferrin saturation which was less than 20 %. Vitamin C levels were also analysed in this study. Forty one percent of children in chronic renal failure were vitamin C deficient. There was no statistically significant variability among the three groups. Two patients (4.6%) were noted to be Vitamin C toxic. One of these patients was haemodialysed; the other was not yet on dialysis. Vitamin C deficiency in chronic renal insufficient patients on dialysis is easily correctable when identified. Vitamin C in specific well documented doses is safe to administer to this group of patients. It will also enhance the absorption of iron and thereby have an indirect effect on anaemia.

<p> Intestinal phosphorus absorption is a key contributor to the body phosphorus pool, but much is unknown regarding physiological adaptations in intestinal phosphorus absorption that occur in vivo. We sought to measure changes in intestinal phosphorus absorption efficiency and phosphorus balance in adolescent females and in rats in response to several factors, using physiologically relevant assessment approaches including whole-body phosphorus balance techniques and <i>in situ</i> ligated intestinal loop absorption methods.</p> <p> We first assessed phosphorus balance and net phosphorus absorption in female adolescents from a controlled crossover study with two levels of calcium intake. Despite an increased calcium intake of 600 mg/day, there was no change in phosphorus balance, nor a significant change in net phosphorus absorption.</p> <p> Next, we measured intestinal phosphorus absorption efficiency with the <i>in situ</i> ligated loop method in healthy Sprague Dawley rats as well as the Cy/+ rat model of progressive kidney disease. We found 10-week-old healthy rats had a small but higher absorption efficiency of phosphorus compared to 20- and 30-week-old rats, while 20-week Cy/+ rats had higher absorption efficiency than 30-week-old. Each of these results corresponded to net phosphorus absorption from balance as well as the concentration of 1,25-dihydroxyvitamin D3. In healthy rats, there was no effect of altering the level of phosphorus in the diet on absorption efficiency. In Cy/+ rats, kidney disease produced a small <i>increase</i> in absorption efficiency, contrary to the predicted decrease that would occur with lower 1,25-dihydroxyvitamin D3 observed in CKD. Gene expression of the major intestinal phosphate transporter, NaPi-2b, largely followed absorption patterns.</p> <p> The utility of the Cy/+ model is limited to males as females do not begin to show signs of progressive kidney decline until a much older age. Therefore, we sought to test whether ovariectomy would accelerate kidney disease in Cy/+ females, with the aim of establishing a postmenopausal model of progressive kidney disease. Our results show that kidney disease is not accelerated by ovariectomy in this rat strain, as measured by kidney weight and biochemistries including blood urea nitrogen, creatinine, creatinine clearance, and plasma phosphorus and calcium.</p> <p> Our results utilizing <i>in situ</i> absorption measures as well as net absorption of phosphorus suggest that some of the factors that are understood to influence the intestinal absorption of phosphorus do not have a significant influence in a physiological context.</p>

BACKGROUND: Patients receiving haemodialysis (HD) treatment experience a significant symptom burden and their needs are multifaceted. In HD unit, patients receive treatment in a diverse cultural and open ward setting. However, patients‟ experiences of receiving HD treatment in an open ward setting are not known. OBJECTIVES: The central aim of this study was to explore and describe the experiences of patients receiving HD treatment in an open setting environment at an academic hospital in Johannesburg. DESIGN: A qualitative, exploratory and descriptive study was conducted at a public, tertiary level academic hospital in Johannesburg after obtaining ethical approval from Wits University and relevant authorities. SETTING: The research setting for this study was an adult Chronic Renal Dialysis Unit. POPULATION: Sixteen adult (age 18 and above) patients receiving chronic HD treatment were recruited purposively in this study. INTERVENTIONS: An in-depth semi-structured interview was conducted either before or after receiving HD treatment, which was audio-recorded, transcribed then analysed by using Giorgi Phenomenological method. FINDINGS: A mood enhancer appeared a major theme as participants positively valued the open setting environment for their overall and psychological well-being. Participants expressed by being with others and sharing experiences, a sense of community likened to a family developed. Common to all participants‟ language was the use of the “we” in relation to being in the open setting environment. This expression of the “we” by participants was interpreted as a community concept. The use of “we” associated with the concept of community described as a space to which every patient receiving HD belonged. They described their experiences in a collective manner. This was evident in repetitive reference to their common space, being together, sharing experiences, and finding identity from one another, being understood and a sense of being protected with personal relations that extend beyond 10 years for some. This open space contributed to shaping their perception of body image and illness. The nurse‟s role in timeously providing HD care was appreciated by many. However, being exposed to multiple situations of chronic illness and treatment a sense of fear developed. Any negative event experienced, watched, observed, or heard in the HD unit triggered fear in the patients. Two common fears were of HD complications and the constant threat of death. Complications such as clotting, muscle cramps and collapsing because of hypotension as well as watching somebody dying on the machine were all reported in this study and so psychological counselling was felt to be very important. CONCLUSION The recommendations proposed in this study hopefully will assist HD staff to intervene and make adjustments to support patients‟ holistic needs. Further studies into patients receiving HD in open settings and mixed-gender space are required for diversity of experiences and knowledge from different settings. Keywords: Haemodialysis, hospital environment, open setting, patient experiences

Background: Acute kidney injury (AKI) is a highly prevalent clinical disorder with significant mortality and no current treatment. The Burns Lab has previously shown that endothelial colony forming cells (ECFCs) release exosomes highly enriched in pro-survival micro-RNA-486-5p. In our mouse model of AKI, intravenous (i.v.) injection of ECFCs or their exosomes protects against kidney ischemic injury, associated with reduction in PTEN, a target of miR-486-5p. Mechanisms mediating recruitment and retention of exosomes are unclear. The interaction of CXC chemokine receptor type 4 (CXCR4) with stromal cell-derived factor (SDF)-1α promotes ECFC adhesion and migration in hypoxic endothelial cells. Whether exosomal miR-486-5p is critical to the prevention of ischemic injury is unclear. The current study aimed to investigate biodistribution and targeting mechanisms of ECFC-derived exosomes, to investigate the delivery and therapeutic potential of miR-486-5p alone, and to determine whether sex differences alter the treatment efficacy. Methods: ECFC-derived exosomes were isolated from cultured media by differential centrifugation and characterized using nanoparticle tracking analysis and immunoblot. Kidney ischemic injury was induced in male and female FVB mice by bilateral renal vascular clamping (30 min). Exosomes (20 µg) or Invivofectamine-mimic complex containing miR-486-5p (1mg/kg) were injected at the start of kidney reperfusion via tail vein. Organs were removed and assays were performed to identify structure and function. In vitro cell studies were also used when necessary. Results: ECFC-derived exosomes preferentially target the ischemic kidney, its endothelium and tubular epithelium, which correlates with increases in miR-486-5p. The transfer of exosomes may be mediated by macropinocytosis by target cells. The SDF-1α/CXCR4 axis plays a role in targeting exosomes to the site of injury. miR-486-5p alone has a similar therapeutic efficacy in preventing ischemia/reperfusion injury as ECFC-exosomes in the mouse model of AKI. Both male and female mice respond to both therapies, however female mice are protected against ischemia reperfusion injury. Conclusions: These results suggest that the protective effects of ECFCs or their exosomes in ischemic AKI may be largely mediated by pro-survival miR-486-5p. These data provide further support for the promising therapeutic potential of ECFC-derived exosomes and miR-486-5p in human AKI.

A lesão renal induzida pela I/R é a principal causa de IRA nos rins nativos e nos rins transplantados e estudos enfatizam a participação de células inflamatórias na sua patogênese, através da caracterização de lesão endotelial, infiltração leucocitária e a geração de mediadores inflamatórios pelas células epiteliais tubulares. Evidências recentes mostram que as células T CD4+ exercem um papel fundamental como mediadoras da agressão renal na I/R, ressaltando-se o envolvimento do paradigma Th1/Th2 como um possível mecanismo efetor. O presente estudo foi realizado com o objetivo de estudar a participação de algumas citocinas Th1 e Th2 no desenvolvimento da lesão de I/R renal. Para tanto, nós nos propusemos a desenvolver um modelo experimental de I/R renal em camundongos deficientes em IL-12, IFN-, e duplo deficientes em IFN- e IL-12 (representando defeito da via de ativação Th1), camundongos deficientes em IL-4 e IL-10 (representando defeito da via de ativação Th2) e duplo deficientes em IL-10 e IL-12, tendo como controles camundongos normais (selvagens). Todos os animais foram submetidos a uma lesão de I/R por ligadura reversível do pedículo renal por 45 minutos seguidos de 24 horas de reperfusão. Após a indução da isquemia, nós analisamos as alterações funcionais (creatinina e uréia por método bioquímico colorimétrico) e morfológicas renais (morfometria renal), além de investigar a expressão molecular de HO-1 (um gene de proteção tecidual), de t-bet (um transcrito envolvido na diferenciação Th1), de GATA-3 (um transcrito envolvido na diferenciação Th2), citocina pró-inflamatória IL-6 e de uma quimiocina pró-inflamatória MCP-1, visando caracterizar a influência da polarização Th1/Th2 da resposta imune na lesão renal induzida pela I/R. Nós mostramos que os camundongos deficientes em IL-4, IFN-, IL-10/IL-12 e IL-10 apresentaram uma disfunção renal importante, caracterizada por altos níveis séricos de creatinina e uréia e por um alto grau de agressão morfológica renal, avaliada pela percentagem de área de necrose tubular. Todos estes resultados foram significativamente mais acentuados que obtidos nos camundongos deficientes em IL-12 e IFN-/IL-12, sendo comparáveis aos animais selvagens. Por outro lado, a capacidade de reparação renal, medida pela percentagem de área de regeneração tubular, foi mais precoce nos camundongos deficientes em IFN-, quando comparada com os camundongos deficientes em IL-12 e IFN-/IL-12. A análise molecular quantitativa, utilizando-se o PCR em tempo real mostrou uma expressão significantimente maior de RNAm de HO-1, IL-6 MCP-1 e do fator transcricional pata Th2 (GATA-3) nos camundongos deficientes em IL-4, IFN-, IL-10/IL-12 e IL-10, em comparação com os camundongos deficientes em IL-12, IFN-/IL-12 que apresentaram baixa expressão do fator transcricional para Th1 (T-bet), em 24 horas após a I/R renal. Quando fomos fazer a transferência adotiva de médula óssea de animais deficientes em IL-12 e IL-4 para animais selvagens previamente com depleção subletal celular, esses animais vieram apresentar resultados semelhantes aos animais deficientes em IL-12 e IL-4, onde os animais selvagens reconstituídos com medula óssea dos animais IL-12 apresentaram menor níveis de uréia sérica e menor expressão de RNAm IL-6, enquanto que os animais selvagens recontituídos com medula óssea dos animais IL-4 apresentaram maior níveis de uréia sérica e maior expressão de RNAm IL-6, esses dados comprovam que realmente a citocina IL-12 parece ser a principal citocina envolvida no processo inflamatório desencadeado pela lesão de isquemia e reperfusão renal. Tais achados favorecem a hipótese dos efeitos deletérios das células com per_l Th1 e o papel protetor das células Th2 na lesão isquêmica renal. Estes novos episódios podem fornecer a base para uma melhor compreensão fisiopatológicos, bem como para o desenvolvimento de métodos preventivos e terapêuticos para a insuficiência renal aguda isquêmica. / Renal ischemia/reperfusion injury (I/R) is the major cause of acute renal failure (ARF) in the native as well as in the transplanted kidneys, with a complex pathogenesis that involves many components of inflammatory response such as leukocyte infiltration and generation of inflammatory mediators by tubular cells. Recent evidences show a critical role of the CD4+ T cell, with the Th1/Th2 paradigm as a possible ejectors mechanism. The present study has the objective to investigate the participation of some main Th1 and Th2 cytokines in the development of the renal of I/R. For that, we developed an experimental model of renal I/R in deficient in IL-12, IFN-, double deficient IFN- and IL-12 (representing defect of the pathway Th1), IL-4 and IL-10 (representing defect of the pathway Th2) and in double deficient IL-10 and IL-12 knockout mice, having wild-type mice as controls. All animals were submitted to renal I/R by reversible ligation of the renal pedicles for 45 minutes followed by 24 hours of reperfusion. After induction of the ischemia, we analyzed renal function and morphometric histological analyzes. Furthermore, we quantified the expression of HO-1 (cytoprotection gene), t-bet (transcription factor involved in the differentiation Th1), GATA-3 (transcription factor involved in the differentiation Th2), and cytokine pro-inflammatory IL-6 and chemokine pro-inflammatory MCP-1, aiming to characterize the influence of Th1/Th2 polarization in the immune response driven by the renal I/R. We showed that the IL-4, IFN-, IL-10 and IL-10/IL-12-de_cient mice presented an important kidney dysfunction, characterized by higher levels serum creatinine and tubular necrosis, being comparable to the wild-type animals. The molecular analyses, utilizing the real-time PCR, showed a significantly higher expression of HO-1, IL-6, MCP-1 and transcription factor involved in the differentiation Th2 (GATA-3) in the in IL-4, IFN-, IL-10 and IL-10/IL-12-defficient mice, in comparison with the in IL-12, IFN-/IL-12-de_cient mice showed lower expression transcription factor involved in the differentiation Th1 (T-bet) . Moreover, the renal dysfunction seen in Th2-de_cient mice was followed by a higher expression of IL-6. When we make the transfer adoptive of bone marrow of animals deficient in IL-12 and IL-4 to wild animals previously with sublethal cell depletion, these animals produce similar results to animals deficient in IL-12 and IL-4, where the animals reconstituted wild animals with bone marrow IL-12 showed lower levels of serum urea and lower expression mRNA of IL-6, while the wild animals reconstituted with bone marrow of animals IL-4 showed higher levels of serum urea and greater expression of mRNA IL-6. These data show that really the cytokine IL-12 appears to be the main cytokine involved in the inflammatory process triggered by the injury of ischemia and reperfusion kidney. Such _ndings favor the hypothesis of the deleterious effects of Th1 cells with prole and the protective role of Th2 cells in ischemic renal injury. These novel insights may provide basis for the better pathophysiological understanding, as well as for development of preventive and therapeutic methods for the ischemic acute renal failure.

O transplante de órgãos é hoje uma opção de tratamento de várias doenças terminais. Apesar de todos os progressos no campo do transplante, o principal problema enfrentado ainda é a rejeição. As principais moléculas responsáveis pela resposta alogeneica e subsequente rejeição ao enxerto, são os antígenos leucocitários humanos (HLA, do inglês Human Leucocyte Antigens). Porém, existem evidências que anticorpos dirigidos a antígenos não-HLA estão associados com rejeição de transplantes. Neste estudo, foi investigada a presença de anticorpos anti-célula endotelial (AACE) em 11 pacientes que perderam seus rins transplantados devido à rejeição humoral irreversível e em 2 com perda por trombose de veia renal. A ausência de anticorpos anti-HLA contra o doador foi verificada antes do transplante, da rejeição e antes e depois da transplantectomia, através da realização de provas cruzadas usando as técnicas mais sensíveis. Anticorpos não-HLA presentes em nove eluatos reagiram com EAHy.926. Eluatos positivos e negativos contra linhagem EAHy.926 foram testados contra cortes histológicos de 6 rins sadios para detecção de quais estruturas renais são reconhecidas por esses anticorpos. A reação foi avaliada pelo método de imunofluorescência indireta. Dos 13 eluatos testados, 4 (isotipo IgG) e 5 (isotipo IgM) reagiram com forte fluorescência nos glomérulos e endotélio arterial, mas não foi verificada reação na cápsula de Bowman e no epitélio tubular. Não foi observado polimorfismo na reatividade dos eluatos. Em onclusão, verificamos que os anticorpos não-HLA têm um importante papel na rejeição humoral. Estes estão reconhecendo antígenos de um sistema provavelmente não-polimórfico nas células de endoteliais presentes, principalmente, nos capilares glomerulares. / The transplant of organs is today an option of treatment to several terminal diseases. In spite of all the progress in the field of the transplants, the rejection remains a problem to be solved. The main target molecules for the allogenic response and subsequent allograft rejection are the human leukocyte antigens (HLA). However, there are growing evidences that non-HLA antibodies are associated with transplant rejection. In this study it was investigated the presence of anti-endothelial cell antibodies (AECA) in 11 patients who had early lost their transplanted kidney by irreversible humoral rejection and in 2 ones from renal venal thrombosis. The absence of anti-HLA antibodies against the donor was verified by the negativity of crossmatches performed using the most sensitive assays, at the transplant, at the rejection, and before and after the transplantectomy Antibodies from 9 eluates bound to EAHy.926. Positive and negatives eluates were tested against frozen sections from 6 normal kidneys in order to define the structures to which they were reactive. The reactivity was identified by indirect immunofluorescence method. From 13 eluates evaluated, 4 (isotipe IgG) and 5 (isotipe IgM) reacted to the glomerulus and renal arterial endothelium with intense fluorescence but they did not react to the Bowmans capsule and tubular epithelium. No polymorphism was observed in eluates reactivity. In conclusion, we have shown that non-HLA antibodies may represent a cause of the humoral rejection. These antibodies are probably recognizing antigens of a nonpolymorphic system in endothelial cells present, mainly, in the glomerular capillaries.

PURPOSE: Doppler renal resistive index (RRI) has emerged in the last decade as a useful prognostic indicator for transient (fluid responsive) and persistent acute kidney injury (AKI). The determinants of RRI are largely systemic and recent studies confirm that RRI measurement could also be a useful early marker for sub-clinical AKI and post procedural AKI risk. This study aimed to determine the feasibility of RRI measurement in an Emergency Department (ED) resuscitation room setting using a point­of­care ultrasound system. METHODS: In this prospective single centre study, RRI measurement was attempted in 20 non-consecutive patients (meeting the inclusion criteria) by a single expert sonographer. RRI measurements were evaluated against context specific feasibility criteria and target outcomes. RESULTS: 20 patients (11 male, 9 female) were recruited to the study. Age of patients ranged from 33 years to 91 years (mean 62.3 years). Adequate visualisation of both kidneys was achieved in 60% of patients (n=12). In patients where it was not possible to achieve adequate views of both kidneys (n=8), limiting technical factors were shortness of breath (SOB) (n=6), high BMI (n=2). At least one measurement of RRI was achieved in 70% of patients (n=14). However, in 9 of these patients (64.3%) the Doppler spectral traces achieved were substandard and did not meet the measurement criteria for RRI as specified in the study protocol. In 30% of patients (n=6) no usable spectral trace was achieved and it was not possible to measure RRI. SOB was noted as a technical difficulty in 60% of patients (n=12) including three for whom RRI measurements were achieved. In 9 patients (45%) SOB was recorded as the primary reason for failure to acquire a usable Doppler trace. All criteria for RRI measurements were met in only 3 patients (15%). CONCLUSION: Measurement of RRI was not feasible in patients requiring resuscitation room care using a current point of care ultrasound system. If RRI is to play a useful role in this high priority patient group, adaptation of the available technology is required to mitigate the problem of image blur due to patient breathing movement.