Podocytes are terminally differentiated epithelial cells which regulate glomerular permselectivity by their cell-cell junctions, known as slit diaphragms (SD). Notch signaling regulates podocyte cell fate specification and downregulation of Notch targets occurs with terminal podocyte differentiation. The effects of constitutive Notch activation in developing podocytes on podocyte differentiation and function were determined using a podocyte-specific Cre-lox-p(Neph/Cre) approach. Proteinuria was noted shortly after birth denoting loss of glomerular permselectivity in transgenic mice (CRE;NIC mice). Histologic and molecular analyses of CRE;NIC-expressing mice at onset of proteinuria, show morphologic and cellular changes in podocytes including de-differentiation, proliferation and de novo expression of Pax2. Prior to onset of proteinuria, lower protein levels of key SD proteins are observed while SD mRNA expression is preserved in CRE;NIC mice. Consequently, constitutive Notch signaling in developing podocytes opposes terminal differentiation with deleterious consequences on SD assembly and thereafter, glomerular permselectivity.

Podocytes are terminally differentiated epithelial cells which regulate glomerular permselectivity by their cell-cell junctions, known as slit diaphragms (SD). Notch signaling regulates podocyte cell fate specification and downregulation of Notch targets occurs with terminal podocyte differentiation. The effects of constitutive Notch activation in developing podocytes on podocyte differentiation and function were determined using a podocyte-specific Cre-lox-p(Neph/Cre) approach. Proteinuria was noted shortly after birth denoting loss of glomerular permselectivity in transgenic mice (CRE;NIC mice). Histologic and molecular analyses of CRE;NIC-expressing mice at onset of proteinuria, show morphologic and cellular changes in podocytes including de-differentiation, proliferation and de novo expression of Pax2. Prior to onset of proteinuria, lower protein levels of key SD proteins are observed while SD mRNA expression is preserved in CRE;NIC mice. Consequently, constitutive Notch signaling in developing podocytes opposes terminal differentiation with deleterious consequences on SD assembly and thereafter, glomerular permselectivity.

Podocytes play an important role in maintaining the structural integrity of glomerular capillaries. There is a limited capacity of podocytes to reproduce; therefore, they are required to support a greater capillary surface area in states of glomerular hypertrophy (i.e., reduced podocyte density). Such reductions in podocyte density can lead to podocyte dysfunction and is thought to substantially increase the susceptibility to hypertension-induced renal injury and progression of chronic kidney disease. However, the extent to which reduced podocyte density increases the susceptibility to hypertension-induced renal injury remains unknown. The goal of this study was to determine the susceptibility to hypertension-induced renal injury in a rodent model of chronic kidney disease with podocyte dysfunction. Male Sprague-Dawley rats were subjected to normotensive renal mass reduction and instrumented with a radiotelemeter for the continuous measurement of blood pressure. After a two week recovery from surgery to allow for completion of renal compensatory increases in size and function, groups of rats were administered a single dose of puromycin aminonucleoside (PAN, 75 mg/kg i.p.), which acutely injures podocytes, or saline (sham). Rats were followed for 4 weeks following PAN or saline injection. At one week post PAN injection, some groups of rats were administered antihypertensive regimens consisting of hydralazine (50-300 mg/L) + hydrochlorothiazide (HCTZ, 25-75 mg/L) or enalapril (50-300 mg/L) + HCTZ (25-75 mg/L) via the drinking water for the remainder of the study. At the end of the study, glomerulosclerosis (GS) was assessed in a blinded fashion and podocyte density was determined using immunofluorescence detection of the podocyte marker Wilms Tumor 1 (WT1) on paraffin-embedded sections from perfused-fixed kidneys. No differences were observed between antihypertensive groups, thus these data are presented as a singled group. The average systolic BP (mmHg) was higher (P<0.05) in rats administered PAN (144±3, n=12) vs. PAN + antihypertensives (127±2, n=20) and saline (130±4, n=17). The magnitude of glomerulosclerosis (GS, % glomeruli exhibiting GS in 100 evaluated) was greater (P<0.05) in rats administered PAN (64±7%) vs. PAN + antihypertensives (30±6%) vs. saline (6±2%). The slope of the relationship between BP and GS (Δ%GS vs. ΔmmHg) was significantly greater (P<0.05) in rats administered PAN (1.1) and PAN + antihypertensives (1.2) vs. saline (0.4). Podocyte density (podocytes/µm3x10-6) was assessed in non-injured glomeruli from a subset of rats from each group and was significantly greater (P<0.05) in those administered PAN + antihypertensives (63±3, n=8) and tended to be greater (P=0.09) in those administered saline (56±4, n=7) as compared to those administered PAN (47±4, n=7). The number of podocytes per glomerular tuft tended to be lower and the average glomerular tuft area tended to be higher in rats administered PAN vs. the other groups; however, these differences were not statistically significant. These data demonstrate that acute podocyte dysfunction increases the susceptibility to BP-induced GS by ~ 3-fold and that lowering BP in such states mitigates further reductions in podocyte density and progression of kidney disease.

It is well documented that the incidence of renal disease, and therefore renal dysfunction, increases with age in many species of mammals. Such alterations in renal structure and function may significantly affect long-term toxicology studies. The purpose of this study was to assess the temporal evolution of glomerulosclerosis, an important renal lesion, in laboratory housed dogs, an important model system in chronic toxicological studies. We histopathologically examined representative sections of dog kidneys, quantified glomerular lesions (using the 0-5 scale of the World Health Organization classification system) and performed of statistical analysis of the extent and distribution of such changes. The kidney samples were obtained by necropsy, and occasionally biopsy, procedures from a collection of 159 purebred Beagle dogs maintained for their entire lifespan in well-controlled conditions. The lesions were correlated with sex, age, and intra-renal location of affected glomeruli to determine the relationship of each in the development of glomerulosclerosis. All dogs examined had some degree of glomerulosclerosis. In the youngest (up to 2 years of age), this was minimal, but was more advanced by middle age (3-7 years). The condition progressed with further aging and was associated with progressive fibrosis and tubular loss. Location and advancing age were significantly related to the development of glomerulosclerosis such that as age increases, the incidence of glomerulosclerosis increases, with the inner medullary ray and inner cortex demonstrating the highest occurrence. Using immunohistochemical analysis, the percentage of type IV collagen within glomeruli was determined. No significant increase in type IV collagen in glomeruli due to age or location was seen. An increase in type III or type V collagen within glomeruli was not apparent either, upon visual examination. This study indicates that renal lesions, including glomerulosclerosis, occur commonly and progress over the lifetime in a genetically similar population of laboratory Beagle dogs maintained under optimal standard environmental conditions. Such typical, age-related change needs to be taken into consideration when conducting chronic toxicological experiments using such animals. / Master of Science

Introduction Podocytes are terminally differentiated, highly specialized glomerular cells that form the final barrier to protein loss. Podocyte injury is characterised by proteinuria. Proteinuria is an important prognostic marker in kidney diseases, and lowering proteinuria has become a principal clinical goal. Compelling evidence supports the notion that continuing loss of podocytes plays a major role in the initiation and progression of glomerular diseases. It is my hypothesis that interventions that reduce the disruption by rescuing susceptible podocytes next to injured ones are potential therapies to restore podocyte phenotype and filtration behaviour, thereby protecting the kidney from progressive deterioration. Prevention of this damage, or ways to aid its recovery, could therefore be important to improving the management of human kidney diseases. Methods Transgenic mice expressing the human diphtheria toxin receptor on podocytes had been previously generated in our laboratory. Characterization of two lines showed that graded specific podocyte injury could be induced by single intraperitoneal injection of diphtheria toxin. Eight-week intervention studies involved administration of oral drug in water or food from 24h after toxin injection. Two control groups received no drug or were non-transgenic (wild-type) littermates. Primary endpoints were glomerulosclerosis and kidney function (serum creatinine). Other readouts included blood pressure, albuminuria, serum albumin, podocyte quantification and collagen staining of kidney. The angiotensin converting enzyme inhibitor (ACEi) captopril was tested because of its proven protective effect on renal function in patients with proteinuria. Subsequently another proteinuria-reducing drug, the endothelin receptor A antagonist sitaxsentan was tested alone and in combination with captopril. Results Captopril reduced proteinuria and ameliorated scarring, with matrix accumulation and glomerulosclerosis falling almost to baseline. Podocyte counts were reduced after toxin administration and showed no significant recovery irrespective of captopril treatment. In the following sitaxsentan and captopril combined intervention study, glomerular scarring was significantly reduced in all drug-treated groups either alone or in combination, but only combination drug treatment reduced glomerular damage to levels comparable to wild-type controls, demonstrating a synergistic effect of the two agents. Similarly, serum creatinine was lowered further in combined but not single drug-treated groups. Blood pressure of all drug treated mice was lowered compared to the placebo group. Surprisingly in this second study there were no significant differences in proteinuria between treated and untreated groups. Conclusion These results support the hypothesis that continuing podocyte dysfunction is a key abnormality in proteinuric disease, and plays a major role in progressive glomerulosclerosis. Both captopril and sitaxsentan alone or in combination provided protection without substantial preservation or restoration of podocyte numbers at the degree of injury induced in these experiments. Combined therapy showed a synergistic effect in protecting the kidney from progressive damage. These results suggest that protection may be at least partly due to change in podocyte phenotype. The model is ideal for studying strategies to protect the kidney from progressive damage following specific podocyte injury. Further elucidations on the mechanism of action of the drugs may aid development of superior future therapeutic treatments in the field of renal diseases.

Cell surface receptors such as adhesion molecules and connexins are involved in interactions between cells and their surroundings. They play important roles in the normal function of healthy tissues and in the responses of cells to injury. In many cases aberrant repair mechanisms are thought to result in disease and this thesis will assess the expression of cell adhesion molecules (principally the pi integrins) and the connexin43 gap junction protein in normal and diseased kidney The expression of pi integrins on normal human mesangial cells was localised using the alkaline phosphatase anti-alkaline phosphatase immunochemical technique (APAAP) and Western blotting. Expression of mRNA coding for integrins was also assessed using reverse-transcription polymerase chain reaction (RT-PCR). Human mesangial cells in culture expressed the a2, a3, av, pi av and P3 integrin chains. Messenger RNA was detected for these integrin subunits plus the al, a4, a5 and a6 chains. Normal human kidney sections were stained using APAAP and monoclonal antibodies towards a wide range of integrin chains. Within the glomerulus, mesangial cells express al, a2 and pi, epithelial cells a3, av and pi and endothelial cells al, a5 and pi. Tubules express a2, a3, a6, av and pi and the interstitium al and pi. In renal biopsies from patients with IgA disease the main alterations in integrin expression were upregulation of a2, a3, av and pi on damaged tubules, with increased pi expression and de novo a5 and av staining within areas of interstitial damage. These changes were replicated in a wide range of other renal pathologies and correlate with the degree of tubulointerstitial histological damage. Connexin43 (Cx43) is distributed extensively on normal human kidney, particularly on glomerular epithelial cells and intra- and extra-glomerular endothelium. Human mesangial cells in vitro express Cx43 protein and its coding mRNA. There is, however, no expression of Cx43 by the mesangium in vivo. In biopsies from patients with inflammatory renal disease there is strong expression of Cx43 on infiltrating inflammatory cells, in areas of interstitial damage and on damaged tubules. The pattern of Cx43 expression in inflammatory renal disease was very similar to that of intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1. The work in this thesis has demonstrated the large repertoire of cell surface receptors expressed on normal kidney. The principal alterations in diseased kidney are found within the tubulointerstitum. The potential relevance of these changes in the pathogenesis of renal disease are discussed and possible future avenues of research are suggested.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13193号 / 論医博第2157号 / 新制||医||1030(附属図書館) / (主査)教授 浅野 雅秀, 教授 岩井 一宏, 教授 小川 修 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Diabetic nephropathy (DN) affects around 40% of diabetic patients worldwide and has become a major health concern due to its high morbidity and mortality. The progression of DN is characterized by the thickening of glomerular basement membrane, albuminuria and the development of glomerulosclerosis. Renal function is eventually compromised. Due to various hemodynamic and metabolic changes, especially the elevated blood glucose level in diabetic patients, glomerular mesangial cells have been shown to upregulate transforming growth factor-β1 (TGF-β1) level and signaling, resulting in the excessive production of extracellular matrix (ECM) proteins. The atypical expression of the 78-kDa glucose-regulated protein (GRP78) on the cell surface may be associated with this pro-fibrotic effect through its interaction with the TGF-β1 activation process. However, there is no current literature demonstrating the role of cell surface GRP78 (csGRP78) in the pathogenesis of diabetic renal diseases. The purpose of my MSc project was to determine the role of csGRP78 in TGF-β1 synthesis and activation and thereby in the progression of DN. We hypothesized that the increased expression of csGRP78 in response to high glucose exposure stimulates TGF-β1 upregulation through intracellular signaling, as well as its activation through interaction with the latent complex, which leads to the expansion of mesangial matrix. / Thesis / Master of Science (MSc) / Diabetic kidney disease affects around 40% of diabetic patients worldwide and is a major health concern. A major feature of the disease is glomerulosclerosis, which is the scarring of glomeruli. The glomeruli filter blood passing through blood vessels in the kidneys to remove waste, which will then be excreted into urine. In diabetic patients, high blood glucose causes the fibrosis of glomeruli and damages the filtration barrier. As a result, a large amount of proteins leak from the blood into the urine. It has been discovered that TGF-β1 is one of the key molecules mediating the generation of scar tissue in the glomerulus. It promotes the growth of mesangial cells, a major type of kidney glomerular cells, and stimulates their production of extracellular matrix proteins. Our results showed that GRP78, a protein that is primarily expressed in the endoplasmic reticulum and assists with protein folding, moves from the inside of cells to the surface in response to a high glucose environment. Here, we found that it facilitated TGF-β1 signaling. Based on our studies, we propose that when GRP78 is at the cell surface, it enables the release of latent TGF-β1, increasing TGF-β1 activity and thus promoting the development of disease.

INTRODUÇÃO: A Glomerulosclerose Segmentar e Focal (GESF) é a glomerulopatia primária mais prevalente no Brasil e sua incidência vem aumentando no mundo inteiro. Na sua forma primária, caracteriza-se clinicamente por acometer pessoas jovens e causar proteinúria acentuada, geralmente acompanhada de síndrome nefrótica. O mecanismo patogênico tem como evento principal a lesão ao podócito, desencadeado por fatores de natureza variada: vírus, drogas/medicamentos, imunológicos, etc. Em 2004, foi publicada a classificação de Columbia, propondo 5 variantes morfológicas distintas na GESF: colapsante (COL), usual (NOS), apical ou tip lesion (TIP), perihilar (PHI) e variante celular (CEL). Diversos estudos comprovam alterações moleculares em podócitos na GESF. Essas alterações são observadas em diversos sítios podocitários: em moléculas envolvidas na fenda de filtração (slit diaphragm), por exemplo, nefrina, podocina e CD2AP; em moléculas do citoesqueleto podocitário, como a -actinina-4 e sinaptopodina; em moléculas marcadoras de diferenciação dos podócitos, como CD10 e WT-1; e ainda em marcadores de divisão celular como Ki-67 e PCNA. Os objetivos desse estudo foram: 1-) classificar as lesões morfológicas de GESF em biópsia renais nas 5 variantes da GESF propostas na Classificação de Columbia; e 2-) analisar a ocorrência de alterações moleculares podocitárias nestes casos. MÉTODOS: Foram selecionados 131 casos de biópsias renais com diagnóstico de GESF primária no período de 1996 a 2006. Os casos foram classificados de acordo com os critérios de Columbia e posteriormente submetidos a reações imuno-histoquímicas para os marcadores CD10, WT-1, vimentina, sinaptopodina, -actinina-4, GLEPP-1, citoqueratina 8/18, citoqueratina 19 e Ki-67. Os resultados foram submetidos à análise estatística através do teste qui-quadrado. RESULTADOS: A classificação das variantes da GESF se distribuiu da seguinte forma: 38,2% de variante NOS, 36,6% de variante COL, 14,5% de variante TIP, 6,9% de variante PHI e 3,8% de variante CEL. Os casos da variante COL se destacaram das demais variantes pela perda de expressão de marcadores de diferenciação celular, como o CD10 e o WT-1 (p<0,01), perda da molécula do citoesqueleto -actinina-4 (p<0,01) e neo-expressão de citoqueratinas 8-18 (p<0,05) e 19 (p<0,01). Adicionalmente, os casos das variantes COL e CEL se destacam das outras variantes pela expressão do marcador de divisão celular Ki-67 (p<0,05). CONCLUSÃO: a variante COL destacou-se das demais em relação às alterações moleculares observadas na análise imuno-histoquímica. O diagnóstico diferencial desta forma de GESF tem importância clínica por ela estar associada a pior evolução e prognóstico em relação às demais variantes. A integração destes marcadores na rotina diagnóstica pode auxiliar no diagnóstico diferencial da GESF COL / INTRODUCTION: Focal segmental glomerulosclerosis (FSGS) is the most prevalent primary glomerulopathy in Brazil and its incidence is increasing worldwide. Primary FSGS is characterized clinically by affecting young people and causing severe proteinuria, often accompanied by nephrotic syndrome. The pathogenesis is related to podocyte injury, which may be due to several factors: viruses, drugs, immunological, etc. In 2004, the Columbia classification of FSGS identified five histological variants of the disease: collapsing (COL), usual (NOS), tip lesion (TIP), perihilar (PHI) and cellular variant (CEL). Several studies have demonstrated molecular changes in podocytes of FSGS patients, which were observed in molecules involved in the filtering function of these cells (nephrin, podocina and CD2AP), in podocyte cytoskeleton molecules (-actinin-4, and synaptopodin), as well as in molecular markers of podocyte differentiation (CD10 and WT-1) and of cell division (Ki-67 and PCNA). The aim of this study was to classify the FSGS biopsies according to the Columbia classification and to analyze the occurrence of molecular changes in the five morphological variants. METHODS: 131 cases of renal biopsies with a diagnosis of primary FSGS in the period 1996 to 2006 were classified according to the criteria of Columbia and then submitted to immunohistochemical reactions with the following antibodies: CD10, WT-1, Vimentin, Synaptopodin, -actinin-4, GLEPP-1, cytokeratin 8-18, cytokeratin 19, and Ki-67. RESULTS: FSGS cases were classified into five variants as follows: 38.2% of NOS variant, 36.6% COL, 14.5% TIP, 6.9% PHI and 3.8% CEL. The COL variant cases distinguished themselves among the other for having lost the expression of CD10 and WT-1 (p <0.01), and also of -actinin-4 (p <0, 01). Furthermore, they gained expression of the cytokeratin 8-18 (p <0.05) and 19 (p <0.01). The group of CEL and COL variants together differed from the other variants regarding the expression of cell division marker Ki-67 (p <0.05). CONCLUSION: COL variant of FSGS presents molecular changes that differs from others and can be demonstrated by immunohistochemistry. The differential diagnosis of this variant is important because of the worse clinical outcome and prognosis it presents in comparison with other variants. The identification of these markers by immunohistochemical on the routine practice may be useful in the diagnosis of COL FSGS