Renal tubules represent an intercellular unit and function as a syncytium. When acute tubular necrosis was first visualized to occur through a process of synchronized regulated necrosis (SRN) in handpicked primary renal tubules, it became obvious that SRN actually promotes nephron loss. This realization adds to our current understanding of acute kidney injury (AKI)-chronic kidney disease (CKD) transition and argues for the prevention of AKI episodes to prevent CKD progression. Because SRN is triggered by necroptosis and executed by ferroptosis, 2 recently identified signaling pathways of regulated necrosis, a combination therapy employing necrostatins and ferrostatins may be beneficial for protection against nephron loss. Clinical trials in AKI and during the process of kidney transplantation are now required to prevent SRN. Additionally, necrotic cell death drives autoimmunity and necroinflammation and therefore represents a therapeutic target even for the prevention of antibody-mediated rejection of allografts years after the transplantation process.

Organoide aus adulten Mäusen sind vielversprechende Modelle für die Nierenforschung. Ihre Charakterisierung wurde jedoch nicht auf ein zufriedenstellendes Niveau gebracht. Hier habe ich ein langfristiges 3D-Maus-Organoid (Tubuloid)-Modell etabliert und charakterisiert, das die Erneuerung und die Reparatur sowie die Architektur und die Funktionalität der adulten tubulären Epithelien rekapituliert. In der Zukunft wird das Modell detaillierte Untersuchungen der Trajektorien selbsterneuernder Zellen sowohl zur teilweisen Wiederherstellung der Niere als auch zur malignen Transformation der Niere ermöglichen. Das klarzellige Nierenzellkarzinom (ccRCC) ist der häufigste und aggressivste Nierenkrebs. Die Inaktivierung des Tumorsuppressorgens Von Hippel-Lindau (VHL) ist der Haupttreiber des ccRCCs. Zuvor hatten wir die Hochregulation der Wnt- und Notch-Signalübertragung in den CXCR4+MET+CD44+-Krebsstammzellen (CSC) aus primären humanen ccRCC-Tumoren identifiziert. Das Blockieren von Wnt und Notch in von Patienten stammenden Xenotransplantaten, Organoiden und nicht-anhaftenden Sphären unter Verwendung von niedermolekularen Inhibitoren beeinträchtigte die Selbsterneuerung der CSC und das Tumorwachstum. Um CSC-gesteuertes humanes ccRCC in genetischen Mausmodellen nachzuahmen, begann ich mit der Erzeugung von zwei Doppelmausmutanten; β-Catenin-GOF; Notch-GOF und Vhl-LOF; β-Catenin-GOF. Sowohl die β-Catenin-GOF; Notch-GOF Mausmutante als auch die Vhl-LOF; β-Catenin-GOF Mausmutante entwickelten innerhalb einiger Monate schwere Krankheitssymptome. Überraschenderweise beobachtete ich weder Tumore oder Tumorvorläuferläsionen noch höhere Zellproliferationsraten in den mutierten Nieren. Weitere Analysen ergaben, dass die Mausmutanten Merkmale chronischer Nierenerkrankung (CKD) aufwiesen. / Adult mouse organoids are promising models for kidney research. However, their characterization has not been pushed forward to a satisfying level. Here, I have generated and characterized a long-term 3D mouse organoid (tubuloid) model, which recapitulates renewal and repair, and the architecture and functionality of the adult tubular epithelia. In the future, the model will allow detailed investigations of trajectories of self-renewing cells towards both the partial recreation and malignant transformation of the kidney. Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive kidney cancer. Inactivation of the Von Hippel-Lindau (VHL) tumor suppressor gene is the major driver of ccRCC. Earlier, we identified the upregulation of Wnt and Notch signaling in CXCR4+MET+CD44+ cancer stem cells (CSCs) from primary human ccRCCs. Blocking Wnt and Notch in patient-derived xenografts, organoids and non-adherent spheres using small-molecule inhibitors impaired self-renewal of CSCs and tumor growth. To mimic CSC-governed human ccRCC in genetic mouse models, I started from the generation of two double mouse mutants; β-catenin-GOF; Notch-GOF and Vhl-LOF; β-catenin-GOF. Surprizingly, I observed neither tumors or tumor precursor lesions nor higher cell proliferation rates in the mutant kidneys. Further analyses revealed that the mutant mice displayed features of chronic kidney disease (CKD). Thus, β-catenin-GOF; Notch-GOF and Vhl-LOF; β-catenin-GOF mouse mutants did not develop kidney tumors under the given experimental conditions.

Aims: 1. Determination of AGEs (Advanced Glycation End products) in patients with various types of nephropathy. 2. Association AGEs with nutritional parameters and anemia. 3. Influence of renal parameters on sRAGE (soluble form of Receptor for Advanced Glycation End products) levels. 4. Technics and proceeding methods of the podocytes cultivation. 5. Determination of urine podocytes. Methods: We determined fluorescent AGEs by spectrofluorometry, sRAGE by Enzyme-Linked ImmunoSorbent Assay (ELISA). Podocytes were passaged and identified immunocytochemically. Podocytes in urine were specified by flow cytometry method. Results: 1. We did not find significant differences in AGEs serum levels among various types of nephropathy, even though the pathogenesis differs. 2. The albumin and prealbumin levels positively and haemoglobin levels negatively correlate with AGEs in patients with CKD grade 1-5, without necessity of dialysis. 3. Serum sRAGE levels are increased in patients with decreased renal function independently on the course of renal disease. 4. We implemented the methods and technics of podocyte cultivation. 5. Urine podocytes observation and confirmation that podocyturia relates to disease activity. Conclusion: We confirmed that AGEs serum levels depend more on renal function than the type of...

<p>Urine-based biomarkers offer numerous advantages for clinical analysis, including non-invasive collection, a suitable sample source for longitudinal disease monitoring, a better screenshot of disease heterogeneity, higher sample volumes, faster processing times, and lower rejection rates and costs. They will be extremely useful in a clinical trial context, which can be applied alone or in combination with other methods as long as they demonstrate clear reproducibility across cohorts. While biofluids such as urine present enormous challenges with a wide dynamic range and extreme complex typically dominated by a few highly abundant proteins, we have demonstrated that the analytical issue can be efficiently addressed by focusing on extracellular vesicles (EVs), tiny packages released by all kinds of cells. These tiny packages contain different kinds of molecules from inside the cells. Here, we established a robust EV isolation and characterization platform to screen and validate Parkinson’s Disease (PD) and Renal Cell Carcinoma (RCC) biomarkers from urine. PD is a progressive neurological disorder affecting body movement because some brain cells stop producing dopamine. PD is often not diagnosed until it has advanced, making early detection crucial. We investigated urinary EVs from 138 individuals to enable early detection and found several proteins involved in PD development that could be biological indicators for early disease detection. Several biochemical techniques were applied to verify our findings. In the second project, we attempted to develop a novel diagnostic technique for early intervention of RCC. Here, we made our efforts to develop a quantitative method based on data-independent acquisition (DIA) mass spectrometry to analyze urinary EV phosphoproteomics for non-invasive RCC biomarker screening. Combined with our in-house EVtrap method for EV isolation and PolyMAC enrichment of phosphopeptides, we quantified 2,584 unique phosphosites. We observed unique upregulated phosphosites and pathways differentiating healthy control (HC), chronic kidney disease (CKD), low-grade, and high-grade clear cell RCC. These applications have a significant promise for early PD and RCC diagnosis and monitoring based on actual functional proteins with urine as the source. These studies might provide a viable path to developing urinary EV-based disease diagnosis.</p>

Chronic kidney disease (CKD) progresses faster in males than females; however, the underlying mechanisms remain poorly understood. Sex differences in glomerular capillary morphology has been hypothesized to contribute, in part, to the increased susceptibility to hypertension-induced renal injury and CKD progression in males, but this has not been investigated. The goal of the present study was to assess glomerular capillary morphology in male vs. female rats with intact kidneys and after uninephrectomy (UNX). We hypothesized that glomerular capillary radii (RCAP) and length (LCAP) would be greater in male rats. Male (n=4) and female (n=4) with intact kidneys and UNX (n=4 males, n=4 females) provided a 0.4% NaCl diet and water ad libitum. Kidneys were perfusion-fixed, the left kidney was excised, and a 3 mm transverse section through the midline of the kidney was selected for further processing. Multiple 1 mm3 cubes were randomly excised from the left, middle, and right regions of the outer cortex, embedded in EPONTM, sectioned (1 μm), and stained with toluidine blue. Four glomeruli from each region were randomly selected for stereological analysis. Glomerular tuft volume (VG), RCAP, and LCAP were assessed. In rats with intact kidneys, no significant sex differences were observed in VG, RCAP, or LCAP. VG, RCAP, and LCAP were significant greater in both male and female rats with UNX vs. respective rats with intact kidneys. In rats with UNX, males exhibited a significantly greater VG and LCAP, but not RCAP, as compared to females despite no significant differences in relative kidney weight. These data indicate that males exhibit greater compensatory increases in LCAP following UNX. The greater capillary length may lead to reduced podocyte density, a well-known mechanism that increases the susceptibility to CKD progression.

Short summary: Background: High cardiovascular risk in patients with chronic kidney disease is partly due to mineral dysbalance, microinflammation and oxidative stress. CKD patients accumulate traditional and non-traditional CV risk factors. FGF23, MMPs and PlGF belong among these non-traditional biomarkers of CV risk. FGF23 is a phosphaturic hormone and inhibitor of calcitriol synthesis. It is associated with vascular calcifications. Matrix-metalloproteinases (e.g. MMP-2, MMP-9) are proteolytic, proinflammatory enzymes, contributing to myocardial remodelation. Placental growth factor (PlGF) is a proangiogenic cytokine that is associated with LV hypertrophy in animal model. Plasmatic FGF23, MMPs and PlGF are elevated in CKD. Aim: We aimed to describe dynamic changes between several novel biomarkers of CV risk (FGF23, MMP-2, MMP-9 and PlGF) in CKD stages 1-5, to describe their mutual correlations and possible association with traditional CV risk markers. We studied possible association of laboratory and echocardiographic parameters in patients with CKD stages 2-4. Methods: In a cross-sectional study we evaluated 80 patiens with CKD 1-5 and 44 healthy controls. In a prospective study we evaluated echocardiographic and laboratory parameters in 62 patients with CKD 2-4 for an average study period of 36±10...

Aims: The thesis deals with the biomarkers of early renal injury, namely albuminuria and neutrophil gelatinase-associated lipocalin (NGAL). The aims in the case of albuminuria were the implementation of HPLC method, comparing HPLC with immunoturbidimetric (IT) method and monitoring the relationship to the diagnosis of diabetes mellitus. The aim of urinary NGAL (and eventually other markers) examination was to verify its reliability in the prediction of acute kidney injury (AKI). Methods: We investigated albuminuria in fresh urine samples in the groups of 636 diabetics and 456 nondiabetics using the HPLC method (Agilent 1200, Agilent Technologies, USA) and immunoturbidimetrically (Cobas Integra 400, Roche Diagnostics); we studied the correlations and relationships between albuminuria and glycated hemoglobin HbA1c. We investigated urinary NGAL by chemiluminescent microparticle immunoassay (Architect i4000, Abbott) in children's groups: 1) after renal transplantation (N = 15), 2) with acute or chronic kidney disease (N = 28); and in adult patient's groups: 1) after cardiac surgery (N = 10) and 2) post angiography (N = 41). Results: Albuminuria determined by HPLC was statistically significantly higher than albuminuria determined by IT. We excluded nonspecificity of the HPLC method. Results indicate...

Short summary: Background: High cardiovascular risk in patients with chronic kidney disease is partly due to mineral dysbalance, microinflammation and oxidative stress. CKD patients accumulate traditional and non-traditional CV risk factors. FGF23, MMPs and PlGF belong among these non-traditional biomarkers of CV risk. FGF23 is a phosphaturic hormone and inhibitor of calcitriol synthesis. It is associated with vascular calcifications. Matrix-metalloproteinases (e.g. MMP-2, MMP-9) are proteolytic, proinflammatory enzymes, contributing to myocardial remodelation. Placental growth factor (PlGF) is a proangiogenic cytokine that is associated with LV hypertrophy in animal model. Plasmatic FGF23, MMPs and PlGF are elevated in CKD. Aim: We aimed to describe dynamic changes between several novel biomarkers of CV risk (FGF23, MMP-2, MMP-9 and PlGF) in CKD stages 1-5, to describe their mutual correlations and possible association with traditional CV risk markers. We studied possible association of laboratory and echocardiographic parameters in patients with CKD stages 2-4. Methods: In a cross-sectional study we evaluated 80 patiens with CKD 1-5 and 44 healthy controls. In a prospective study we evaluated echocardiographic and laboratory parameters in 62 patients with CKD 2-4 for an average study period of 36±10...

Chronic kidney disease (CKD) and acute kidney injury (AKI) are major public health problems. It is important to be able to identify those at high risk of adverse outcome, CKD progression and associated cardiovascular disease. The aim of the thesis was to study novel promising biomarkers, their relationship to kidney function, chronic inflammation and/or cardiovascular risk - placental growth factor (PlGF), pregnancy associated plasma protein A (PAPP-A), matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), soluble receptor for advanced glycation end products (sRAGE), calcium binding protein S100A12 or extracellular newly identified RAGE binding protein (EN-RAGE), and high mobility group box protein-1 (HMGB-1) in patients with renal diseases including CKD, haemodialysis (HD), AKI patients, and healthy controls for comparison. First study revealed that PlGF is elevated in patients with decreased renal function. Second study demonstrated the association of MMP-2 and PAPP-A with proteinuria in patients with CKD. Moreover, serum MMP-2, MMP-9 and PAPP-A levels significantly differed in patients with various nephropathies. EN-RAGE levels are not elevated in patients with CKD, but are related to inflammatory status. PAPP-A, EN-RAGE and HMGB-1 levels are significantly elevated, but sRAGE and PlGF...

Text in English with abstracts in English and isiZulu / Poor adherence to their prescribed diet, medications and treatment contributes to increased mortality and morbidity in patients with end-stage renal disease. These patients must change their diet when receiving dialysis treatment, but cultural beliefs and practices can affect their adherence to the prescribed diet. The purpose of this qualitative, descriptive, exploratory study was to improve health education on prescribed diet adherence to patients living on haemodialysis at a haemodialysis unit in eThekwini Municipality. Data from a sample of 20 patients was collected using semi-structured interviews and analysed using qualitative content analysis. The findings revealed that haemodialysis patients’ prescribed diet adherence was influenced by cultural and religious views, and by family support. Food availability, patients’ geographical location and patients’ financial means hindered their adherence to their prescribed diet. A multi-disciplinary health care team including nurses, should be sensitive to patients’ different cultural beliefs and practices when providing health education. / Ukungabambeleli endleleni emisiwe yokudla, amakhambi nasekwelashweni kunomthelela ekwandiseni izimpawu zesifo sezinso esingapheli (ESRD) futhi kwandisa isibalo sabantu ababulawa yilesisifo. Iziguli ezinalesisifo zidinga ukushitsha indlela yokuphila, iziphuzo kanye nokulandela indlela emisiwe yokudla kakhulukazi mabe ngaphansi kokwelashwa ngokuhlanzwa kwegazi ngomshini (dialysis). Izinkolelo zamasiko nendlela zokuphila ezihambisana namasiko kwenze imfundiso nge ezempilo maqondana nendlela emisiwe yokudla yaba lukhuni. Inhloso yalolucwaningo bekuwukwandisa ulwazi nemfundiso ngezempilo mayelana nokulandela indlela emisiwe yokudla kwiziguli izithola ukulashwa ngokuhlanzwa kwegazi ngomshini (haemodialysis) esikhungweni esikuMasipala weTheku. Kusetshenziwe indlela yokwenza ucwaningo esezingeni elifanele, Imininingwano eqoqiwe eqembini (sample) leziguli ezingamashumi amabili (20) ezithola ukwelashwange haemodialyisis. Imininigwane iqoqwe kusetshenziswa izingxoxo ezihleliwe. Imigomo elawula ukuhlaziya ilandeliwe yonke ngenkathi kwenziwe lolucwaningo. Lolucwaningo luveze ukuthi indlela yokudla emisiwe yeziguli ezikwi dialysis iphazanyiswa imobono yamasiko, inkolo kanye nokusekelwa nokuzimbandakanya kwamalungu omndeni. Izinselelo ezinjengokutholakala, indawo isiguli esihlala kuyo nezinkinga zemali zivimbela ukubambelela endleleni emisiwe yokudla. Abasebenzi bezempilo kumele banakekele indima edlalwa izinkolelo namasiko uma befundisa ngezempilo ezigulini eziphethwe izinso. / Nursing Science / M.A. (Nursing Science)