Methods Knockout animals. The inactivation of the murineThe LIM-homeodomain transcription factor Lmx1b plays a crucial role in podocytes Patients with nail-patella syndrome often suffer from a nephropathy, which ultimately results in chronic renal failure. The finding that this disease is caused by mutations in the transcription factor LMX1B, which in the kidney is expressed exclusively in podocytes, offers the opportunity for a better understanding of the renal pathogenesis. In our analysis of the nephropathy in nail-patella syndrome, we have made use of the Lmx1b knockout mouse. Transmission electron micrographs showed that glomerular development in general and the differentiation of podocytes in particular were severely impaired. The glomerular capillary network was poorly elaborated, fenestrae in the endothelial cells were largely missing, and the glomerular basement membrane was split. In addition podocytes retained a cuboidal shape and did not form foot processes and slit diaphragms. Expression of the α4 chain of collagen IV and of podocin was also severely reduced. Using gel shift assays, we demonstrated that LMX1B bound to two AT-rich sequences in the promoter region of NPHS2, the gene encoding podocin. Our results demonstrate that Lmx1b regulates important steps in glomerular development and establish a link between three hereditary kidney diseases: nail-patella syndrome (Lmx1b), steroid-resistant nephrotic syndrome (podocin), and Alport syndrome (collagen IV α4). COOH-terminal trans-activation domain (9). Heterozygous mice were maintained on a C57BL/6 background. Homozygous mice are born at the expected Mendelian ratio but do not survive beyond postnatal day 1; therefore, on the day of birth offspring were killed by decapitation, and the kidneys were removed and processed as described below. Electron microscopy. For transmission electron microscopy, kidneys were immersion-fixed overnight in 1× PBS/2% glutaraldehyde and then embedded in Epon. Ninety-nanometer-thick sections were used for transmission electron microscopy. Counterstaining was done with osmium and uranyl acetate.Morphometry. To determine the glomerular tuft area, kidneys were fixed 4 hours at room temperature in 1× PBS/4% paraformaldehyde and then paraffin-embedded. Four-micrometer-thick sections were prepared and stained with hematoxylin and eosin. Approximately ten glomerular tufts (defined as those profiles that had progressed beyond the S-shaped body stage) were counted randomly on every 24th kidney section, thus keeping the same glomerular from being counted twice. For every kidney, 100 glomerular tufts were analyzed and the mean tuft area calculated.Immunohistochemistry. For cryosections, kidneys were fixed overnight in 1× PBS/4% paraformaldehyde except when anti-collagen IV antibodies were used (see below). After equilibration in 1× PBS/18% sucrose, the kidneys were frozen in liquid nitrogen and stored at -80°C until further use. Seven-to 10-µm-thick cryosections were blocked in 1× PBS/2% BSA before being stained with a primary anti...
Methods Knockout animals. The inactivation of the murineThe LIM-homeodomain transcription factor Lmx1b plays a crucial role in podocytes Patients with nail-patella syndrome often suffer from a nephropathy, which ultimately results in chronic renal failure. The finding that this disease is caused by mutations in the transcription factor LMX1B, which in the kidney is expressed exclusively in podocytes, offers the opportunity for a better understanding of the renal pathogenesis. In our analysis of the nephropathy in nail-patella syndrome, we have made use of the Lmx1b knockout mouse. Transmission electron micrographs showed that glomerular development in general and the differentiation of podocytes in particular were severely impaired. The glomerular capillary network was poorly elaborated, fenestrae in the endothelial cells were largely missing, and the glomerular basement membrane was split. In addition podocytes retained a cuboidal shape and did not form foot processes and slit diaphragms. Expression of the α4 chain of collagen IV and of podocin was also severely reduced. Using gel shift assays, we demonstrated that LMX1B bound to two AT-rich sequences in the promoter region of NPHS2, the gene encoding podocin. Our results demonstrate that Lmx1b regulates important steps in glomerular development and establish a link between three hereditary kidney diseases: nail-patella syndrome (Lmx1b), steroid-resistant nephrotic syndrome (podocin), and Alport syndrome (collagen IV α4). COOH-terminal trans-activation domain (9). Heterozygous mice were maintained on a C57BL/6 background. Homozygous mice are born at the expected Mendelian ratio but do not survive beyond postnatal day 1; therefore, on the day of birth offspring were killed by decapitation, and the kidneys were removed and processed as described below. Electron microscopy. For transmission electron microscopy, kidneys were immersion-fixed overnight in 1× PBS/2% glutaraldehyde and then embedded in Epon. Ninety-nanometer-thick sections were used for transmission electron microscopy. Counterstaining was done with osmium and uranyl acetate.Morphometry. To determine the glomerular tuft area, kidneys were fixed 4 hours at room temperature in 1× PBS/4% paraformaldehyde and then paraffin-embedded. Four-micrometer-thick sections were prepared and stained with hematoxylin and eosin. Approximately ten glomerular tufts (defined as those profiles that had progressed beyond the S-shaped body stage) were counted randomly on every 24th kidney section, thus keeping the same glomerular from being counted twice. For every kidney, 100 glomerular tufts were analyzed and the mean tuft area calculated.Immunohistochemistry. For cryosections, kidneys were fixed overnight in 1× PBS/4% paraformaldehyde except when anti-collagen IV antibodies were used (see below). After equilibration in 1× PBS/18% sucrose, the kidneys were frozen in liquid nitrogen and stored at -80°C until further use. Seven-to 10-µm-thick cryosections were blocked in 1× PBS/2% BSA before being stained with a primary anti...
Transcanial sonography (TCS) is increasingly applied in the diagnosis of Parkinson's disease (PD), but investigator bias may influence the results of examination. Blinding the sonographer to the clinical diagnosis of 42 PD patients and 35 controls, we obtained a positive predictive value of 85.7% and a negative predictive value of 82.9% in the diagnosis of PD solely by interpreting the results of TCS, indicating that TCS is a valuable additional tool in the diagnosis of PD.
Methods Knockout animals. The inactivation of the murineThe LIM-homeodomain transcription factor Lmx1b plays a crucial role in podocytes Patients with nail-patella syndrome often suffer from a nephropathy, which ultimately results in chronic renal failure. The finding that this disease is caused by mutations in the transcription factor LMX1B, which in the kidney is expressed exclusively in podocytes, offers the opportunity for a better understanding of the renal pathogenesis. In our analysis of the nephropathy in nail-patella syndrome, we have made use of the Lmx1b knockout mouse. Transmission electron micrographs showed that glomerular development in general and the differentiation of podocytes in particular were severely impaired. The glomerular capillary network was poorly elaborated, fenestrae in the endothelial cells were largely missing, and the glomerular basement membrane was split. In addition podocytes retained a cuboidal shape and did not form foot processes and slit diaphragms. Expression of the α4 chain of collagen IV and of podocin was also severely reduced. Using gel shift assays, we demonstrated that LMX1B bound to two AT-rich sequences in the promoter region of NPHS2, the gene encoding podocin. Our results demonstrate that Lmx1b regulates important steps in glomerular development and establish a link between three hereditary kidney diseases: nail-patella syndrome (Lmx1b), steroid-resistant nephrotic syndrome (podocin), and Alport syndrome (collagen IV α4). COOH-terminal trans-activation domain (9). Heterozygous mice were maintained on a C57BL/6 background. Homozygous mice are born at the expected Mendelian ratio but do not survive beyond postnatal day 1; therefore, on the day of birth offspring were killed by decapitation, and the kidneys were removed and processed as described below. Electron microscopy. For transmission electron microscopy, kidneys were immersion-fixed overnight in 1× PBS/2% glutaraldehyde and then embedded in Epon. Ninety-nanometer-thick sections were used for transmission electron microscopy. Counterstaining was done with osmium and uranyl acetate.Morphometry. To determine the glomerular tuft area, kidneys were fixed 4 hours at room temperature in 1× PBS/4% paraformaldehyde and then paraffin-embedded. Four-micrometer-thick sections were prepared and stained with hematoxylin and eosin. Approximately ten glomerular tufts (defined as those profiles that had progressed beyond the S-shaped body stage) were counted randomly on every 24th kidney section, thus keeping the same glomerular from being counted twice. For every kidney, 100 glomerular tufts were analyzed and the mean tuft area calculated.Immunohistochemistry. For cryosections, kidneys were fixed overnight in 1× PBS/4% paraformaldehyde except when anti-collagen IV antibodies were used (see below). After equilibration in 1× PBS/18% sucrose, the kidneys were frozen in liquid nitrogen and stored at -80°C until further use. Seven-to 10-µm-thick cryosections were blocked in 1× PBS/2% BSA before being stained with a primary anti...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.