Understanding how a therapy will impact the injured kidney before being administered would be an asset to the clinical world. The work in this thesis advances the field of mathematical modeling of the kidneys to aid in this cause. The objectives of this work are threefold: 1) to develop and personalize a model to specific patients in different diseased states, via parameter estimation, in order to test therapeutic trajectories, 2) to use parameter estimation to understand the cause of different kidney diseases, differentiate between potential kidney diseases, and facilitate targeted therapies, and 3) to push forward the understanding of kidney physiology via physiology-based mathematical modeling techniques. To accomplish these objectives, we have developed two models of the kidneys: 1) a broad, steady-state, closed-loop model of the entire kidney with human physiologic parameters, and 2) a detailed, dynamic model of the proximal tubule, an important part of kidney, with rat physiologic parameters. To readily aid physicians, a human model would easily fit into the clinical workflow. Since there is a lack of invasive human renal data for validation and parameter estimation, we employ a minimal modeling approach. However, to aid in deeper understanding of renal function for future applications, targeted therapy testing, and potentially replace invasive measures, we develop a more detailed model. The development of such a model requires invasive data for validation and parameter estimation, and hence we model for rodents, where such invasive data are more readily available. The kidneys are composed of approximately one million functional units known as nephrons. The glomerular filtration rate (GFR) is the rate at which the kidney filters blood at the start of the nephron. This filtration rate is highly regulated via several control mechanisms and needs to be maintained within a small range in order to maintain a proper water and electrolyte balance. Hence, fluctuations of GFR are indicative of overall kidney health. In developing the human kidney model, we also sought to understand the relationship between blood pressure and GFR since many therapies affect blood pressure and subsequently GFR. This model describes steady-state conditions of the entire kidney, including renal autoregulation. Model validation is performed with experimental data from healthy subjects and severely hypertensive patients. The baseline model’s GFR simulation for normotensive and the manually tuned model’s GFR simulation for hypertensive intensive care unit patients had low root mean squared errors (RMSE) of 13.5 mL/min and 5 mL/min, respectively. These values are both lower than the error of 18 mL/min in GFR estimates, reported in previous studies. It has been shown that vascular resistance and renal autoregulation parameters are altered in severely hypertensive stages, and hence, a sensitivity analysis is conducted to investigate how changes in these parameters affect GFR. The results of the sensitivity analysis reinforce the fact that vascular resistance is inversely related to GFR and show that changes to either vascular resistance or renal autoregulation cause a significant change in sodium concentration in the descending limb of Henle. This is an important conclusion as it quantifies the mapping between hypertension parameters and two important kidney states, GFR and sodium urine levels. Glomerulonephritis is one of the two major intra-renal kidney diseases, characterized as a breakdown at the site of the glomerulus that affects GFR and subsequently other portions of the nephron. This disease accounts for 15% of all kidney injuries and one-fourth of end-stage renal disease patients. The human kidney model is used to estimate renal parameters of patients with glomerulonephritis. The model is an implicit system and in developing an optimization algorithm to use for parameter estimation, we modify in a novel way, the Levenberg-Marquardt optimization using the implicit function theorem in order to calculate the Jacobian and Hessian matrices needed. We further adapt the optimization algorithm to work for constrained optimization since our parameter values must be physiologically feasible within a certain range. The parameter estimation method we use is a three-step process: 1) manually adjusting parameters for the hypertension comorbidity, 2) iteratively estimating parameters that vary from person to person using no-kidney- injury (NKI) data, and 3) iteratively estimating parameters that are affected by glomerulonephritis using labeled diseased data. Such a process generates a model that is personalized to each given patient. This patient-specific model can then be used to simulate and evaluate outcomes of potential therapies (e.g., vasodilators) on the model in lieu of the patient, and observe how alterations in blood pressure or sodium level affect renal function. Parameter estimation in the presence of glomerulonephritis is a challenging task due to the complexity of the kidney physiology and the number of parameters to estimate. This is further complicated by comorbidities such as hypertension, cardiac arrythmia, and valvular disease, because they alter kidney physiology and hence, increase the number of parameters to estimate. We chose to focus on hypertension since it is very prevalent in hospitals and intensive care units. It was found that over all patients, average model estimates of GFR and urine output rate (UO) were within 9.2 mL/min and 0.71 mL/min for NKI data. These results are expectedly better than those achieved from the non-personalized model since the parameters are now specific to each patient. The results also demonstrate our ability to non-invasively estimate GFR with less error than the 18 mL/min currently possible. The estimations were validated by ensuring that the estimated parameter values were physiologically sound and matched the literature in terms of expected values for different demographic groups. It is vital for a properly functioning kidney to maintain solute transport throughout the nephron. Kidney diseases in the nephron can manifest themselves via the solute transport mechanisms. To understand how these diseases affect the kidney and to simulate transporter- targeting therapies, we have developed a detailed model, starting from the human model previously developed, of one portion of the kidneys, the proximal tubule. The proximal tubule is the site of the most active transport within the nephron and the target for several therapies. Our goal is to study and understand the dynamic behavior of the proximal tubule when solute transporters breakdown and to investigate treatment therapies targeting certain solute transporters. The proposed model is dynamic and includes several solutes’ transport mechanisms, with parameters for rats. We chose to investigate diabetic nephropathy and the associated sodium-transporter alteration (knockout) therapy. Diabetic nephropathy is characterized as kidney damage due to diabetes and affects 30% of diabetics. In terms of reducing hyperfiltration, a potential cause of diabetic nephropathy where an overabundance of solutes and fluid are filtered at the glomerulus, the model demonstrates that knockout of this transporter results in a reduction in sodium and chloride reabsorptions in the proximal tubule, thereby preventing hyperfiltration. Further, we conclude that vital flows for maintaining kidney homeostasis, fluid and ammonium reabsorptions, are corrected to healthy values by a 50% knockout (impairment) of the sodium-hydrogen transporter. Next, we use the dynamic model to detect different diseased states of the proximal tubule transporters. We have accomplished this task by using Bayesian estimation to estimate transporter density parameters (a metric for kidney health) using measured signals from the proximal tubule. This approach is validated with experimental rat data, while further investigations are conducted into the performance of the estimation in the presence of varied input signals, signal resolutions, and noise levels. Estimation accuracy within 20% of true transporter density and within 4% of true fluid and solute reabsorption was achieved for all combinations of diseased transporters. We concluded that including chloride and bicarbonate concentrations improved estimation accuracy, whereas including formic acid did not. This is an important conclusion as it can help physicians determine which blood tests to order for diagnosing kidney disease; to our knowledge, this is a first. It was also found that sodium and glucose proximal tubule concentrations are most affected by changes in the sodium-hydrogen and sodium-bicarbonate transporters. This conclusion provides insight into the interplay between solute transporter density and sodium and glucose concentrations in the proximal tubule. Such knowledge paves the way for new transporter targeted therapies.

Fibrillary glomerulonephritis (FGN) is a rare disease identified in less than one percent of native kidney biopsy. FGN is characterized by hematuria, edema, renal insufficiency, and high-grade proteinuria. Renal biopsy results typically demonstrate deposition of randomly-arranged fibrils within the capillary wall or mesangium. Positive staining with DnaJ Heat Shock Protein Family Member B9 (DNAJB9) is considered diagnostic. Associations include malignancy, hepatitis C, dysproteinemia, autoimmune disorders, and diabetes mellitus. To our knowledge, this is the first case demonstrating association between fibrillary glomerulonephritis and vulvar squamous cell carcinoma. A 66-year-old year old Caucasian female presented to the emergency department for worsening renal injury. She was diagnosed with vulvar squamous cell carcinoma 5 years prior and underwent radical excision with inguinal lymphadenectomy and CO2 laser treatment. Over the years, she had multiple relapses and received wide local excision and adjuvant radiation, with the last treatment involving radiation being 2.5 years before admission. The patient had a recently identified non-malignant vulvar lesion at the time of admission, which was found to be lichen sclerosus et atrophicus. She was found to have renal dysfunction and nephrotic range proteinuria; her creatinine was 2.84 mg/dL (normal range 0.60–1.10 mg/dL), BUN 33 mg/dL (normal range 6–20 mg/dL), urine protein:creatinine ratio 3.9 mg/g (normal range < 0.15 mg/g). She was started on pulse dose methylprednisolone of 500 mg daily, but her creatinine worsened, necessitating renal biopsy. Renal biopsy findings indicated mesangial expansion and randomly-arranged non-branching fibril deposition. Glomerular immunofluorescence indicated positive staining for IgA, IgG, and DNAJB9. These findings confirmed the diagnosis of fibrillary glomerulonephritis. Screening for associations – coexistent malignancies, hepatitis C, multiple myeloma, and autoimmune disorders – was negative. The patient was started on rituximab and prednisone therapy after confirming the absence of underlying infection. One month after initial hospitalization, she was re-hospitalized for worsening kidney function and required initiation of dialysis, on which she remains dependent. FGN is rapidly progressing and results in end-stage-renal-disease within two years in 50% of individuals. It should be considered as a differential diagnosis in patients with a history of malignancy, especially vulvar squamous cell carcinoma. There is no definitive treatment for FGN. Rituximab can be used in conjunction with steroid therapy, but further research is needed to determine the proper treatment for FGN at various stages of disease manifestation. The original case is published in Cureus, and permission has been received to present this case.

In the setting of an increasing incidence of endocarditis in the United States, we report a patient with necrotizing crescentic glomerulonephritis (GN) associated with native valve bacterial endocarditis due to Streptococcus parasanguinis. He was started on appropriate antibiotic treatment and subsequent blood cultures showed no growth. However, due to continuing decline in kidney function, immunosuppressive therapy was started. Despite immunosuppressive therapy and antibiotics, renal function did not improve and chronic hemodialysis was required. Due to rarity of condition, there are no definite treatment guidelines available. Antibiotics, steroids, immunosuppressive agents can be of help in most cases. Further research in this regard may help with early diagnosis and better treatment modalities.

No description available.

Κάθε χρόνο τα άτομα που υποφέρουν από κάποιας μορφής νεφρική νόσο αυξάνονται παρά τις εξελίξεις στον τομέα της ιατρικής. Έχει παρατηρηθεί ότι η νεφρική νόσος, στις περισσότερες περιπτώσεις μπορεί να εξελιχθεί σε νεφρική ανεπάρκεια, είτε με πιο αργούς είτε με πιο γρήγορους ρυθμούς. Η νεφρική ανεπάρκεια αντιμετωπίζεται είτε με αιμοκάθαρση είτε με μεταμόσχευση. Το ανοσοποιητικό σύστημα διαδραματίζει σημαντικό ρόλο τόσο στην αντιμετώπιση όσο και στη πρόοδο των διάφορων μορφών νεφρικής νόσου. Ωστόσο, οι διάφοροι λεμφοκυτταρικοί τύποι, και ιδιαίτερα αυτοί της φυσικής ανοσίας, δεν έχουν μελετηθεί ιδιαίτερα και πολλές φορές από τις λίγες μελέτες που υπάρχουν προκύπτουν αντικρουόμενα αποτελέσματα. Η μελέτη μας επικεντρώθηκε στα Τ, στα ΝΚ και στα ΝΚ-Τ λεμφοκύτταρα. Επιλέξαμε να ερευνήσουμε αρχικά και τελικά στάδια νεφρικής νόσου. Έτσι οι ασθενείς κατηγοριοποιούνται σε τρεις ομάδες: ασθενείς με σπειραμαρονεφρίτιδα (αρχικό στάδιο), ασθενείς που υποβάλλονται σε αιμοκάθαρση και μεταμοσχευμένοι ασθενείς (τελικά στάδια). Σε κάθε πείραμα γίνεται σύγκριση με μια ομάδα ελέγχου (control, που αποτελείται από υγιή άτομα). Στην περίπτωση της σπειραματονεφρίδας παρατηρήθηκαν φυσιολογικά ποσοστά των λεμφοκυτταρικών τύπων και της έκφρασης του NKG2D υποδοχέα. Παρατηρήθηκε, όμως, αυξημένη έκφραση του TNF-α. Στην περίπτωση της αιμοκάθαρσης αν και παρατηρήθηκε μειωμένο ποσοστό ΝΚ και Τ κυττάρων, βρέθηκε ότι παρουσιάζουν αυξημένη κυτταροτοξικότητα και λειτουργικότητα. Αυτό φαίνεται και από την αύξηση στην έκφραση του CD107α και από την αύξηση στη συγκέντρωση του TNF-α. Όσον αφορά τα ΝΚ-Τ κύτταρα, αν και το ποσοστό τους είναι φυσιολογικό, εμφανίζουν αυξημένη έκφραση του υποδοχέα NKG2D. Τέλος, όσον αφορά την ομάδα των μεταμοσχευμένων ασθενών, δεν παρατηρήθηκε μεταβολή στο ποσοστό των Τ και των ΝΚ-Τ κυττάρων. Όταν όμως οι ασθενείς κατηγοριοποιούνται με βάση την φαρμακευτική αγωγή, το ποσοστό των ΝΚ-Τ κυττάρων είναι αυξημένο σε ασθενείς που τους χορηγείται κυκλοσπορίνη. Επίσης, η έκφραση του υποδοχέα NKG2D είναι φυσιολογική και στις δύο κατηγορίες λεμφοκυττάρων. Από την άλλη, αν και το ποσοστό των ΝΚ κυττάρων μειώνεται, αυτά εμφανίζουν φυσιολογική λειτουργικότητα και κυτταροτοξικότητα, όπως φαίνεται από τη φυσιολογική έκφραση του NKG2D υποδοχέα, της πρωτεΐνης CD107α και του TNF-α. / Every year, people who suffer from some form of kidney disease are increasing despite advances in medicine. It has been observed that the kidney disease, in most cases might progress to renal failure, either slower or faster. The renal failure is treated either by hemodialysis or renal transplantation. The immune system plays an important role in the treatment and in the progression of various forms of renal disease. However, the various types of lymphocytes, and especially those of innate immunity have not been widely studied and often the few studies that exist result in conflicting results. Our study focused on T, NK and NK-T lymphocytes. We chose to investigate initial and final stages of renal disease. Patients were categorized into three groups: patients with glomerulonephritis (initial stage), patients on dialysis and transplanted patients (final stages). In each experiment patients were compared with a control group consisting of healthy individuals. In the case of glomerulonephritis, we observed normal percentages of the lymphocyte population examined and normal expression of the NKG2D receptor, as well as increased expression of TNF-α. In the case of hemodialysis, NK and T cell percentages were reduced, while NK cells exhibited enhanced cytotoxicity and functionality. This was shown by the increase in the expression of CD107α and by the increase in the concentration of TNF-α. Regarding the NK-T cells, although their percentage was normal, they showed increased expression of NKG2D receptor. Finally, concerning the group of transplanted patients, no change was observed in the percentage of T and NK-T cells. However, when patients were categorized based on the medication, the percentage of NK-T cells was increased in patients receiving cyclosporine. In addition, the expression of NKG2D receptor was normal on T and NK-T cells. On the other hand, although the percentage of NK cells was reduced, those cells exhibited normal functionality and cytotoxicity, as shown by the normal expression of NKG2D receptor, CD107α protein and TNF-α.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Os venenos das serpentes são compostos protéicos complexos com extensas atividades biológicas. Estas moléculas abrangem até 95% do peso seco do veneno, e compreendem enzimas, toxinas não enzimáticas e proteínas atóxicas. Dentre as complicações fisiológicas, a Insuficiência Renal Aguda - IRA é bem comum nos acidentes crotálicos, onde as concentrações renais do veneno se apresentam até 50% maiores do que na concentração plasmática. A fração tóxica responsável por essa complicação é a crotoxina. O veneno das serpentes, quando irradiado com raios gama de 60Co, tem a sua toxicidade diminuída, entretanto, são mantidas as suas propriedades imunológicas. Nossa hipótese é que a utilização de veneno irradiado em substituição ao nativo pode reduzir a taxa de lesões renais em animais de grande porte durante a produção de soros antiofídicos, propiciando melhor qualidade de vida e bem estar desses animais. No presente estudo realizou-se o isolamento da crotoxina por técnicas cromatográficas, a partir do veneno da serpente Crotalus durissus terrificus. Parte da toxina isolada foi reservada e denominada crotoxina nativa (CTXN) enquanto que outra parte foi irradiada com 2 kGy de raios gama oriundos de uma fonte de 60Co e denominada crotoxina irradiada (CTXI). Diferentes grupos de camundongos Balb/c receberam a toxina nativa ou irradiada e tiveram seus rins removidos em diferentes tempos pós-injeção da crotoxina. Realizou-se a nefrectomia dos animais e foi feito o preparo dos cortes histológicos em Hematoxilina-Eosina (HE) e cortes de imunohistoquímica com anticorpo policlonal de coelho anticrotoxina. As alterações histopatológicas encontradas nas amostras foram glomerulonefrite (GN), congestão capilar (CC) e necrose tubular aguda (NTA). Verificou-se que a GN teve maior ocorrência no grupo CTXN que no grupo CTXI. A NTA foi dominante no grupo CTXN em relação ao grupo CTXI. A CC foi predominante no grupo CTXI que no grupo CTXN. Entretanto esta diferença não é estatisticamente significante segundo o teste t Student de amostras independentes. O tratamento imunohistoquímico revelou que a concentração de antígeno marcado com o anticorpo é decrescente segundo os intervalos de tempo de eutanásia dos animais do grupo inoculado com CTXI, pois a crotoxina quando irradiada tem a sua excreção renal facilitada. Conclui-se que as alterações histopatológicas GN e a NTA foram significativamente menos frequentes em rins de animais inoculados com CTXI. Diferentemente da crotoxina nativa, a crotoxina irradiada é detectada pelo anticorpo anticrotoxina apenas nos tempos intermediários pós-inoculação e em baixa concentração, indicando que a radiação gama promoveu mudanças na forma de ligação da toxina ao tecido renal, facilitando sua eliminação e diminuindo, portanto, as chances de lesão. Esses resultados reforçam as evidências de que o uso de veneno irradiado no processo de imunização garante uma melhor qualidade de vida e bem estar aos animais soroprodutores. / Snake venoms are complex mixtures of proteins and peptides with a wide spectrum of physiological targets such as the blood coagulation and cardiovascular systems and the motor end plate among others. Acute renal failure is a common complication in accidents with the South American rattlesnake. The toxin involved in this pathology is crotoxin, a major component of the venom in terms of concentration and toxicity. Snake venoms, when irradiated with 60Co gamma rays present a significant decrease in toxicity while the immunogenic properties of its components are preserved. The use of irradiated rattlesnake venom is an attractive alternative for antisera production since it might reduce the appearance of renal lesions improving the welfare and lifespan of animals employed for antivenom production. In the present work, we have isolated crotoxin from crude venom of C. d. terrificus and irradiates part of it with 2 kGy of gamma rays from 60Co and compared the effects of native (CTXN) and irradiated (CTXI) crotoxin on the mice renal function. We conducted nephrectomy of animals and preparation of histological sections with hematoxylin-eosin (HE) and immunohistochemistry with polyclonal rabbit anticrotoxin for analysis in optical microscopy. Histopatological changes found in the samples were glomerulonephritis (GN), capillary congestion (CC) acute kidney tubular necrosis (NTA). Immunohistochemistry indicates that the concentration of bound antibody labelled antigen decreases in a time dependent manner. The GN ante NTA were significantly less frequent in kidneys of animals inoculated with irradiated toxin. The capillary congestion was evidenced in all animals treated with irradiated or native crotoxin. Concluding, ionizing radiation appears to mitigate the nephrotoxic effects of crotoxin and may represent an alternative for antivenin production, improving welfare for seroproductor animal.

Os venenos das serpentes são compostos protéicos complexos com extensas atividades biológicas. Estas moléculas abrangem até 95% do peso seco do veneno, e compreendem enzimas, toxinas não enzimáticas e proteínas atóxicas. Dentre as complicações fisiológicas, a Insuficiência Renal Aguda - IRA é bem comum nos acidentes crotálicos, onde as concentrações renais do veneno se apresentam até 50% maiores do que na concentração plasmática. A fração tóxica responsável por essa complicação é a crotoxina. O veneno das serpentes, quando irradiado com raios gama de 60Co, tem a sua toxicidade diminuída, entretanto, são mantidas as suas propriedades imunológicas. Nossa hipótese é que a utilização de veneno irradiado em substituição ao nativo pode reduzir a taxa de lesões renais em animais de grande porte durante a produção de soros antiofídicos, propiciando melhor qualidade de vida e bem estar desses animais. No presente estudo realizou-se o isolamento da crotoxina por técnicas cromatográficas, a partir do veneno da serpente Crotalus durissus terrificus. Parte da toxina isolada foi reservada e denominada crotoxina nativa (CTXN) enquanto que outra parte foi irradiada com 2 kGy de raios gama oriundos de uma fonte de 60Co e denominada crotoxina irradiada (CTXI). Diferentes grupos de camundongos Balb/c receberam a toxina nativa ou irradiada e tiveram seus rins removidos em diferentes tempos pós-injeção da crotoxina. Realizou-se a nefrectomia dos animais e foi feito o preparo dos cortes histológicos em Hematoxilina-Eosina (HE) e cortes de imunohistoquímica com anticorpo policlonal de coelho anticrotoxina. As alterações histopatológicas encontradas nas amostras foram glomerulonefrite (GN), congestão capilar (CC) e necrose tubular aguda (NTA). Verificou-se que a GN teve maior ocorrência no grupo CTXN que no grupo CTXI. A NTA foi dominante no grupo CTXN em relação ao grupo CTXI. A CC foi predominante no grupo CTXI que no grupo CTXN. Entretanto esta diferença não é estatisticamente significante segundo o teste t Student de amostras independentes. O tratamento imunohistoquímico revelou que a concentração de antígeno marcado com o anticorpo é decrescente segundo os intervalos de tempo de eutanásia dos animais do grupo inoculado com CTXI, pois a crotoxina quando irradiada tem a sua excreção renal facilitada. Conclui-se que as alterações histopatológicas GN e a NTA foram significativamente menos frequentes em rins de animais inoculados com CTXI. Diferentemente da crotoxina nativa, a crotoxina irradiada é detectada pelo anticorpo anticrotoxina apenas nos tempos intermediários pós-inoculação e em baixa concentração, indicando que a radiação gama promoveu mudanças na forma de ligação da toxina ao tecido renal, facilitando sua eliminação e diminuindo, portanto, as chances de lesão. Esses resultados reforçam as evidências de que o uso de veneno irradiado no processo de imunização garante uma melhor qualidade de vida e bem estar aos animais soroprodutores. / Snake venoms are complex mixtures of proteins and peptides with a wide spectrum of physiological targets such as the blood coagulation and cardiovascular systems and the motor end plate among others. Acute renal failure is a common complication in accidents with the South American rattlesnake. The toxin involved in this pathology is crotoxin, a major component of the venom in terms of concentration and toxicity. Snake venoms, when irradiated with 60Co gamma rays present a significant decrease in toxicity while the immunogenic properties of its components are preserved. The use of irradiated rattlesnake venom is an attractive alternative for antisera production since it might reduce the appearance of renal lesions improving the welfare and lifespan of animals employed for antivenom production. In the present work, we have isolated crotoxin from crude venom of C. d. terrificus and irradiates part of it with 2 kGy of gamma rays from 60Co and compared the effects of native (CTXN) and irradiated (CTXI) crotoxin on the mice renal function. We conducted nephrectomy of animals and preparation of histological sections with hematoxylin-eosin (HE) and immunohistochemistry with polyclonal rabbit anticrotoxin for analysis in optical microscopy. Histopatological changes found in the samples were glomerulonephritis (GN), capillary congestion (CC) acute kidney tubular necrosis (NTA). Immunohistochemistry indicates that the concentration of bound antibody labelled antigen decreases in a time dependent manner. The GN ante NTA were significantly less frequent in kidneys of animals inoculated with irradiated toxin. The capillary congestion was evidenced in all animals treated with irradiated or native crotoxin. Concluding, ionizing radiation appears to mitigate the nephrotoxic effects of crotoxin and may represent an alternative for antivenin production, improving welfare for seroproductor animal.

Bibliography: leaves 119-136.

Η παρουσία λευκωματουρίας αποτελεί δυσμενή προγνωστικό παράγοντα για τους ασθενείς με σπειραματονεφρίτιδα (ΣΝ) χωρίς εντούτοις αυτό να επιβεβαιώνεται πάντα. Συνεπώς, είναι επιβεβλημένη η εύρεση περισσότερο ειδικών προγνωστικών δεικτών που να αντανακλούν τη διαδικασία επούλωσης της βλάβης ή εξέλιξής της σε ίνωση. Σκοπός, της παρούσας μελέτης ήταν η αναγνώριση πρώιμων δεικτών εξέλιξης της νεφρικής βλάβης σε ασθενείς με σημαντικού βαθμού λευκωματουρία. Ως τέτοιοι προγνωστικοί δείκτες μελετήθηκαν η έκφραση της τρανσγελίνης (SM22) και της τρανσγλουταμινάσης–2 (TG2). Η SM22 είναι μια πρωτεΐνη που εκφράζεται στα λεία μυϊκά κύτταρα και αποτελεί δείκτη της επιθήλιο-μεσεγχυματικής μετάπτωσης. Η TG2 αποτελεί ένα ένζυμο που επάγει τη νεφρική ίνωση μεταβάλλοντας την ομοιόσταση της εξωκυτταρίου θεμέλιας ουσίας. Η έκφραση της SM22 μελετήθηκε με τη μέθοδο της ανοσοϊστοχημείας και του ανοσοφθορισμού, σε νεφρικές βιοψίες 67 ασθενών με διάφορους τύπους ΣΝ, και συσχετίστηκε με την κλινική τους πορεία μετά πάροδο πενταετούς παρακολούθησης. Επιπλέον, εξετάστηκε με τη μέθοδο του συνεντοπισμού, κατά πόσον η έκφραση της SM22 συμπίπτει με αυτήν της α–ακτίνης των λείων μυϊκών ινών (α–SMA). Η έκφραση της TG–2 μελετήθηκε με ανοσοφθορισμό σε νεφρικές βιοψίες από 32 ασθενείς με μεμβρανώδη νεφροπάθεια (ΜΝ). Όλοι οι ασθενείς με ΜΝ έλαβαν αγωγή με κορτικοειδή και κυκλοσπορίνη για 24 μήνες και σε 14 από αυτούς διενεργήθηκε επαναληπτική βιοψία και έγινε περαιτέρω μελέτη της TG2. Ως ομάδα ελέγχου για την έκφραση των παραπάνω μορίων χρησιμοποιήθηκαν τομές από υγιείς περιοχές νεφρών. Από τους 67 ασθενείς που μελετήθηκε η έκφραση της SM22, οι 46 διατήρησαν σταθερή νεφρική λειτουργία. Στις βιοψίες ελέγχου η έκφραση της SM22 περιορίστηκε στο τοίχωμα των αγγείων ενώ στους ασθενείς με ΣΝ επεκτάθηκε εντός των σπειραμάτων και του διάμεσου χώρου. Η SM22 βρέθηκε να σχετίζεται με το βαθμό σπειραματικής σκλήρυνσης και ίνωσης του διάμεσου χώρου, με το βαθμό μεσαγγειακής υπερπλασίας και την έκβαση της νεφρικής λειτουργίας. Η μελέτη συνεντοπισμού της SM22 και της α-SMA ανέδειξε περιοχές όπου οι δύο πρωτεΐνες εκφράζονταν ταυτόχρονα ενώ σε άλλες κάθε πρωτεΐνη εκφραζόταν χωριστά. Κατά το διάστημα παρακολούθησης οι 22 από τους 32 ασθενείς με ΜΝ παρουσίασαν σταθερή νεφρική λειτουργία. Η έκφραση της TG2 βρέθηκε αυξημένη στις βιοψίες των ασθενών με ΜΝ σε σχέση με την ομάδα ελέγχου. Η έκφραση της TG2 στις βιοψίες διάγνωσης ήταν περισσότερο αυξημένη στους ασθενείς με πιο έντονη διάμεση ίνωση και σπειραματική σκλήρυνση. Τέλος, η TG2 βρέθηκε αυξημένη στους περισσότερους ασθενείς στην επαναληπτική βιοψία, ενώ οι ασθενείς που παρουσίαζαν έντονη αύξηση της διάμεσης ίνωσης στην επαναληπτική βιοψία είχαν και εντονότερη έκφραση της TG2 κατά τη διάγνωση της νόσου. Συμπερασματικά, η έκφραση της SM22 βρέθηκε αυξημένη στο νεφρικό ιστό ασθενών με ΣΝ, ενώ η μελέτη συνεντοπισμού της SM22 και της α–SMA ανέδειξε την παρουσία πιθανώς διαφορετικών υποπληθυσμών μυοϊνοβλαστών. Όσον αφορά την TG2, η έντονη έκφραση της στο νεφρικό ιστό ασθενών με ΜΝ, η οποία καθίσταται εντονότερη μετά τη χορήγηση ανοσοκατασταλτικής αγωγής, υποδηλώνει τη συμμετοχή της TG2 στους μηχανισμούς βλάβης και εξέλιξης της ΜΝ. Επιπρόσθετα, η πρώιμη ανίχνευση της TG2 πιθανόν να έχει ιδιαίτερη σημασία στη ΜΝ καθώς η αυξημένη έκφρασή της φαίνεται να προηγείται της διάμεσης ίνωσης. / It is well established that albuminuria is an unfavorable prognostic factor for patients with various types of glomerulonephritis (GN), however, this is not observed in all patients. It is therefore important to identify more specific prognostic markers that reflect the healing process of injury or the progression to fibrosis. The aim of this study was to identify early markers of progression to renal injury in patients with significant degree of albuminuria. As such markers the expression of transgelin (SM22) and transglutaminase–2 (TG2) was studied. SM22 is expressed specifically in smooth muscle cells and is an early marker of epithelial to mesenchymal transition. TG–2 is an enzyme that contributes to renal scarring through altering extracellular matrix homeostasis. SM22 was studied in 67 patients with various kinds of GN and a 5 year follow up. SM22 was identified in kidney sections at the time of diagnosis using immunohistochemistry and immunofluorescence. SM22 expression was examined concerning its correlation with the clinical course of GNs. The expression of alpha smooth muscle actin (a–SMA) and co-localization with SM22 was also investigated. TG2 expression was studied by immunofluorescence in kidney sections from 32 patients with MN. These MN patients were subsequently treated by combination of cyclosporine and prednisolone for 24 months with a repeat biopsy taken in 14. Kidney sections from the normal part of kidneys were used as normal controls for the expression of the aforementioned molecules. Forty six out of 67 patients, that transgelin was studied, showed stable renal function. In control biopsies SM22 and a-SMA were restricted to vascular wall whereas in patients with GN expression was extended within glomeruli and the interstitium. SM22 expression correlated to the degree of glomerular sclerosis and interstitial fibrosis, to the degree of mesangial proliferation and renal function outcome. Double staining for co-localization of both SMM22 and a-SMA showed that in some areas of kidney tissue both proteins were identified whereas in other areas the expression of either SM22 or a-SMA was predominant. Twenty two out of 32 patients with MN showed stable renal function. TG2 immunostaining was increased in sections from patients with MN compared to healthy controls. TG2 at diagnosis was more intense in patients with more severe interstitial fibrosis and advanced glomerular sclerosis. TG2 significantly increased in most patients in the repeat biopsies whereas patients that showed a marked increase in interstitial fibrosis in the repeat biopsy had significantly more TG2 expression in the first biopsy. In conclusion, intense SM22 expression was observed in the renal tissue of patients with different types of GN. The co-localization study of SM22 and a–SMA suggests that different subpopulations of myofibroblasts might be involved in the development of kidney injury. As far it concerns TG2, its’ expression is increased in MN patients and continues to increase despite treatment which is suggestive of its’ involvement in the development and progression of renal scarring. Moreover, early detection of TG2 might be of value in MN since increased TG2 production seems to precede extensive interstitial fibrosis.