Molecular Mechanisms of Proteinuria in Minimal Change Disease

Minimal change disease (MCD) is the most common type of idiopathic nephrotic syndrome in childhood and represents about 15% cases in adults. It is characterized by massive proteinuria, edema, hypoalbuminemia, and podocyte foot process effacement on electron microscopy. Clinical and experimental studies have shown an association between MCD and immune dysregulation. Given the lack of inflammatory changes or immunocomplex deposits in the kidney tissue, MCD has been traditionally thought to be mediated by an unknown circulating factor(s), probably released by T cells that directly target podocytes leading to podocyte ultrastructural changes and proteinuria. Not surprisingly, research efforts have focused on the role of T cells and podocytes in the disease process. Nevertheless, the pathogenesis of the disease remains a mystery. More recently, B cells have been postulated as an important player in the disease either by activating T cells or by releasing circulating autoantibodies against podocyte targets. There are also few reports of endothelial injury in MCD, but whether glomerular endothelial cells play a role in the disease remains unexplored. Genome-wide association studies are providing insights into the genetic susceptibility to develop the disease and found a link between MCD and certain human haplotype antigen variants. Altogether, these findings emphasize the complex interplay between the immune system, glomerular cells, and the genome, raising the possibility of distinct underlying triggers and/or mechanisms of proteinuria among patients with MCD. The heterogeneity of the disease and the lack of good animal models of MCD remain major obstacles in the understanding of MCD. In this study, we will review the most relevant candidate mediators and mechanisms of proteinuria involved in MCD and the current models of MCD-like injury

Murine models of renal disease: Possibilities and problems in studies using mutant mice

The elucidation of the pathogenesis of human renal disease at the molecular level has been facilitated by the growing field of gene targeting and the development of mouse strains with single-gene deletions - the `knock-out' mice. Experimental nephrology, therefore, requires well-characterized and reliable models of human renal disease that can be induced reproducibly in mice. Today surgical procedures for the induction of renal ischemia, chronic renal failure, and ureter obstruction are feasible in mice. Models of mesangioproliferative or crescentic glomerulonephritis, glomerulosclerosis, and tubulointerstitial disease are readily available; however, these depend heavily on the mouse genetic background. Attention to the genetic background and appropriate backcrossing are, therefore, of great importance in the design and interpretation of experimental studies, especially in transgenic mice. Simple murine models displaying the clinical features of other human renal diseases such as IgA nephropathy, membranous glomerulonephritis, and renal vasculitis are still lacking. Mouse strains that spontaneously develop distinct renal pathologies similar to lupus nephritis and focal-segmental glomerulosclerosis can be intercrossed with transgenic mice to study the impact of single-gene deletions on the renal phenotype. The present review provides a survey about currently available spontaneous and inducible murine models of renal disease with special attention to problems and future perspectives for their use in transgenic animals. Copyright (C) 2000 S. Karger AG, Basel

Role of interleukin-13 on the development of minimal change nephrotic syndrome - A novel animal model

Ph.DDOCTOR OF PHILOSOPH

An Update on Glomerulopathies

The book has fourteen chapters which are grouped under different sections: Immune System and Glomerulonephritis, Animal Models of Glomerulonephritis, Cytokines and Signalling Pathways, Role of Cells and Organelles in Glomerulonephritis and Miscellaneous. While the purpose of this volume is to serve as an update on recent advances in the etio-pathogenesis of glomerulopathies, the book offers the current and broad based knowledge in the field to readers of all levels in the nephrology community

Aging pathology in sprague dawley rats : background lesions and comparative study between wild type and transgenic rats with neuronal overexpression of human adenosine A2A receptors

Dissertação de Mestrado Integrado em Medicina VeterináriaAging is a complex phenomenon defined as a time-dependent functional decline, progressive loss of physiological integrity and progressive increase in disease susceptibly. Adenosine A2A receptors (A2AR) are G protein-couple receptors that, upon binding of adenosine, lead to different transducing signals. Although having a protective effect, A2AR also play an important role in neurodegenerative disorders and are upregulated in the brain of Alzheimer and Parkinson patients. Previous studies from our collaborators showed that transgenic rats with neuronal overexpression of human A2AR (Tg (CaMKIIhA2AR)) have depressive-like behavior, impaired hypothalamic-pituitary-adrenal (HPA) axis and, as a result of this, increased levels of circulating corticosteroids. The aim of this work was to evaluate, by histopathology, the impact of the neuronal overexpression of human A2AR in the onset of specific or age-associated lesions in transgenic Sprague Dawley rats. Comprehensive necropsy and histopathology were performed in 37 Wild-type (Wt) and 39 transgenic (Tg) rats, at specific time-points, ranging from 12 to 126 weeks of age. Univariate and multivariate statistical analysis were performed to investigate the association between the phenotype and genotype. Briefly we found that Tg rats are 2.7 times more likely to develop systemic pathology than Wt rats [Odds ratio (OR) 2.745, IC 95% 1.0.07-6.997; (p0.05)]. In blood vessels, mineralization was the most frequent lesion and Tg rats were 5.5 times more likely to develop this lesion than Wt [OR 5.486, IC 95% 1.776- 17.074; (p0.05)]. Regarding adrenal gland pathology, vacuolation of the cortical cells was the most frequent lesion and Tg rats were 4.3 times more likely to develop this pathology than Wt [OR 4.3, IC 95% 1.156-16.248; (p0.05)]. Nos vasos sanguíneos, a lesão mais frequente foi a mineralização da parede, sendo que os ratos Tg foram 5.5 vezes mais suscetíveis a desenvolver esta lesão que os Wt [OR 5.486, IC 95% 1.776- 17.074; (p0.05)]. Relativamente às adrenais, a lesão mais frequente foi a vacuolização das células da cortical e os ratos Tg foram 4.3 vezes mais suscetíveis para o desenvolvimento desta lesão que os Wt [OR 4.3, IC 95% 1.156-16.248; (p<0.05)]. Fibroadenoma mamário foi o tumor mais frequente, tendo sido observado em um rato Wt e cinco ratos Tg. Mesmo em casos onde não foi observada diferença entre ratos Wt e Tg, todas as lesões encontradas neste estudo são lesões muitas vezes associadas ao envelhecimento, típicas desta espécie e a sua incidência correlacionou-se com a idade. Os nossos resultados mostram uma relação clara entre a sobre expressão neuronal de A2AR e envelhecimento acelerado na nossa amostra, e apesar de não termos explorado os mecanismos específicos para tal acontecimento, poderá estar ligado ao facto dos ratos Tg terem disfunção do eixo hipotálamo-hipófise-adrenal e níveis elevados de corticosterona, o que se traduz em stress crónico. A conhecimento dos autores, este é o primeiro estudo a caracterizar as repercussões sistémicas da sobre expressão neuronal de A2AR, que é observada em várias doenças degenerativas durante o envelhecimento.N/

PROFILING MOLECULAR SIGNALS ASSOCIATED WITH PODOCYTE EFFACEMENT IN MINIMAL CHANGE NEPHROTIC SYNDROME FOLLOWING THE TH2 CYTOKINE STIMULATION

Ph.DDOCTOR OF PHILOSOPH

Inflammatory Response as a Mechanism of Perinatal Programming: Long-term Impact on Pulmonary and Renal Function?

RATIONALE: Temporal changes in the fetal environment, such as malnutrition and placental insufficiency induce intrauterine growth restriction (IUGR) and lead to a permanent changes of physiological processes later in life. Interestingly, epidemiological studies demonstrated an impairment of lung and renal function in young infants subsequent to IUGR. Complementary, experimental studies showed that IUGR induces a perinatal programming of the developing lung with persisting impairment of pulmonary structure and function. Besides IUGR, early postnatal hyperalimentation (pHA) is discussed as a crucial factor of IUGR-associated diseases. Both extracellular matrix (ECM) and inflammatory processes have been shown to be dysregulated following IUGR and early pHA. However, the underlying molecular mechanisms of IUGR-associated diseases and the potential linkage of ECM and inflammation have not been addressed so far. Therefore, the ultimate goal of this project was to elucidate the role of regulation of ECM and inflammatory cytokines subsequent to IUGR and early pHA. AIMS: There are three specific aims: (1) to analyze the role of the TGF-β signaling in lung development subsequent to IUGR, (2) to determine the regulation of inflammatory cytokines and ECM-molecules in lungs subsequent to IUGR, and (3) to characterize the pathomechanistic role of early pHA using the example of the kidney. METHODS: Two simultaneous sets of animal experiments were used. One animal model addressed pre- and postnatal nutritional intervention, the other was restricted to postnatal interventions only: (A) IUGR was induced in Wister rats by isocaloric low protein diet (8% casein; IUGR) during gestation. The control group received normal protein diet (17% casein; Co). At birth the litter size was reduced to 6 male pups to induce early pHA. During lactation the mothers of both groups were fed standard chow. (B) Early pHA was induced by litter size reduction to 6 (LSR6) or 10 (LSR10) male neonates. Home-cage control (HCC; mean litter size of 16) animals without any postnatal manipulation during lactation were included. At postnatal day (P) 28 as well as P70 animals underwent whole body plethysmography and in addition metabolic cages at P70. Serum and samples of lungs and kidney were obtained at P1, P12, P21, P42, and P70 for mRNA extraction, protein extraction as well as histological analyses. RESULTS: Both respiratory system resistance and compliance were impaired subsequent to IUGR at P28; this impairment was even more significant at P70. (1) These changes were accompanied by persistent attenuated activity of the TGF-β signaling, assessed by phosphorylation of Smad2 and Smad3. Expression analysis of TGF-β-regulated ECM components in the lungs of IUGR animals at P1, such as collagen I, elastin, and tenascin N, revealed a significant deregulation. Consistently, in vitro inhibition of TGF-β signaling in NIH/3T3, MLE 12 and endothelial cells by adenovirus-delivered Smad7 demonstrated a direct effect on the expression of ECM components. Interestingly, however, not just a deregulation of ECM components was detected at P1, but also attenuated apoptotic processes, e.g. decreased cleavage of PARP. (2) Since the TGF-β signaling has potent anti-inflammatory effects, we next determined the dynamic expression of pro-inflammatory and pro-fibrotic markers as well of ECM components in the lung subsequent to IUGR at P1, P42 and P70. The expression of ECM components and metabolizing enzymes was markedly deregulated and the deposition of collagen I was strikingly increased at P70. Concomitantly to the pro-fibrotic processes in the lung subsequent to IUGR, the expression of inflammatory cytokines and both the activity and the expression of target genes of Stat3 signaling were dynamically regulated, with unaltered or decreased expression at P1 and significantly increased expression art P70. (3) Assessment of renal function at postnatal day 70 revealed decreased glomerular filtration rate, proteinuria, and increased fractional sodium and potassium secretion following early pHA (LSR6). Moreover, the deposition of ECM molecules, such as collagen I, was increased. Interestingly, despite the elevated expression of pro-inflammatory leptin and IL-6 expression the phosphorylation of Stat3 and ERK1/2 in the kidney, however, was decreased after LSR6. In accordance, neuropeptide Y (NPY) gene expression – sympathetic co-neurotransmitter regulated by Stat3 signaling – was down-regulated. In accordance, suppressor of cytokine signaling (SOCS)3 protein expression, an inhibitor of Stat3 and Erk1/2 signaling, was strongly elevated and colocalized with NPY. Interestingly, NPY is co- localized with SOCS3 in the distal tubules in the cortex and outer medulla, and in the proximal tubules, but no expression of SOCS3 was detectable in glomeruli. CONCLUSION: Taken together, IUGR has a direct and strong negative impact on respiratory resistance and compliance of the lung. There are two major underlying mechanisms linking IUGR and deregulation of ECM: first, the attenuated TGF-β signaling during late lung development; and second, the increased expression of inflammatory cytokines subsequent to IUGR. In addition, the missing anti-inflammatory effect of TGF-β signaling could contribute to the increased inflammatory response following IUGR. Furthermore we demonstrated that early pHA leads to organ-intrinsic increased expression of NPY via a postreceptor-leptin-receptor leptin resistance. This leptin resistance could contribute to the observed profibrotic processes, and ultimately to a long-term impairment of renal function. Thus, both IUGR and early postnatal hyperalimentation have a strong impact on perinatal programming of multi-organ inflammatory response

Pathophysiological lessons from rare associations of immunological disorders

Rare associations of immunological disorders can often tell more than mice and rats about the pathogenesis of immunologically mediated human kidney disease. Cases of glomerular disease with thyroiditis and Graves’ disease and of minimal change disease with lymphoepithelioma-like thymic carcinoma and lymphomatoid papulosis were recently reported in Pediatric Nephrology. These rare associations can contribute to the unraveling of the pathogenesis of membranous nephropathy (MN) and minimal change disease (MCD) and lead to the testing of novel research hypotheses. In MN, the target antigen may be thyroglobulin or another thyroid-released antigen that becomes planted in the glomerulus, but other scenarios can be envisaged, including epitope spreading, polyreactivity of pathogenic antibodies, and dysregulation of T regulatory cells, leading to the production of a variety of auto-antibodies with different specificities [immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX syndrome)]. The occurrence of MCD with hemopathies supports the role of T cells in the pathogenesis of proteinuria, although the characteristics of those T cells remain to be established and the glomerular permeability factor(s) identified