A new method for large scale isolation of kidney glomeruli from mice.
01 Sep 2002-American Journal of Pathology (American Society for Investigative Pathology)-Vol. 161, Iss: 3, pp 799-805
TL;DR: The method was applicable also to newborn mice, which allows for the isolation of immature developmental stage glomeruli and makes feasible transcript profiling and proteomic analysis of the developing, healthy and diseased mouse glomerulus.
Abstract: Here we report a new isolation method for mouse glomeruli. The method is fast and simple and allows for the isolation of virtually all glomeruli present in the adult mouse kidney with minimal contamination of nonglomerular cells. Mice were perfused through the heart with magnetic 4.5- micro m diameter Dynabeads. Kidneys were minced into small pieces, digested by collagenase, filtered, and collected using a magnet. The number of glomeruli retrieved from one adult mouse was 20,131 +/- 4699 (mean +/- SD, n = 14) with a purity of 97.5 +/- 1.7%. The isolated glomeruli retained intact morphology, as confirmed by light and electron microscopy, as well as intact mRNA integrity, as confirmed by Northern blot analysis. The method was applicable also to newborn mice, which allows for the isolation of immature developmental stage glomeruli. This method makes feasible transcript profiling and proteomic analysis of the developing, healthy and diseased mouse glomerulus.
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TL;DR: The cellular components are the key players in restricting solute transport, while the GBM is responsible for most of the resistance to water flow across the glomerular barrier.
Abstract: This review focuses on the intricate properties of the glomerular barrier. Other reviews have focused on podocyte biology, mesangial cells, and the glomerular basement membrane (GBM). However, since all components of the glomerular membrane are important for its function, proteinuria will occur regardless of which layer is affected by disease. We review the properties of endothelial cells and their surface layer, the GBM, and podocytes, discuss various methods of studying glomerular permeability, and analyze data concerning the restriction of solutes by size, charge, and shape. We also review the physical principles of transport across biological or artificial membranes and various theoretical models used to predict the fluxes of solutes and water. The glomerular barrier is highly size and charge selective, in qualitative agreement with the classical studies performed 30 years ago. The small amounts of albumin filtered will be reabsorbed by the megalin-cubulin complex and degraded by the proximal tubular cells. At present, there is no unequivocal evidence for reuptake of intact albumin from urine. The cellular components are the key players in restricting solute transport, while the GBM is responsible for most of the resistance to water flow across the glomerular barrier.
771 citations
Cites methods from "A new method for large scale isolat..."
...For a more large-scale approach, glomeruli can be obtained by passing renal tissue through stainless steel sieves with specific mesh sizes (13) or by using magnetic beads (305)....
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TL;DR: It is postulate that constitutive and induced autophagy is a major protective mechanism against podocyte aging and glomerular injury, representing a putative target to ameliorate human glomersular disease and aging-related loss of renal function.
Abstract: Injury and loss of podocytes are leading factors of glomerular disease and renal failure. The postmitotic podocyte is the primary glomerular target for toxic, immune, metabolic, and oxidant stress, but little is known about how this cell type copes with stress. Recently, autophagy has been identified as a major pathway that delivers damaged proteins and organelles to lysosomes in order to maintain cellular homeostasis. Here we report that podocytes exhibit an unusually high level of constitutive autophagy. Podocyte-specific deletion of autophagy-related 5 (Atg5) led to a glomerulopathy in aging mice that was accompanied by an accumulation of oxidized and ubiquitinated proteins, ER stress, and proteinuria. These changes resulted ultimately in podocyte loss and late-onset glomerulosclerosis. Analysis of pathophysiological conditions indicated that autophagy was substantially increased in glomeruli from mice with induced proteinuria and in glomeruli from patients with acquired proteinuric diseases. Further, mice lacking Atg5 in podocytes exhibited strongly increased susceptibility to models of glomerular disease. These findings highlight the importance of induced autophagy as a key homeostatic mechanism to maintain podocyte integrity. We postulate that constitutive and induced autophagy is a major protective mechanism against podocyte aging and glomerular injury, representing a putative target to ameliorate human glomerular disease and aging-related loss of renal function.
597 citations
Cites methods from "A new method for large scale isolat..."
...Glomeruli were isolated using Dynabead perfusion and were glass-glass homogenized in lysis buffer (containing 20 mM CHAPS and 1% Triton X-100) (53, 54)....
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...buffer (containing 20 mM CHAPS and 1% Triton X-100) (53, 54)....
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TL;DR: It is suggested that mTOR inhibition can protect podocytes and prevent progressive diabetic nephropathy and demonstrate the requirement for tightly balanced mTOR activity in podocyte homeostasis.
Abstract: Chronic glomerular diseases, associated with renal failure and cardiovascular morbidity, represent a major health issue. However, they remain poorly understood. Here we have reported that tightly controlled mTOR activity was crucial to maintaining glomerular podocyte function, while dysregulation of mTOR facilitated glomerular diseases. Genetic deletion of mTOR complex 1 (mTORC1) in mouse podocytes induced proteinuria and progressive glomerulosclerosis. Furthermore, simultaneous deletion of both mTORC1 and mTORC2 from mouse podocytes aggravated the glomerular lesions, revealing the importance of both mTOR complexes for podocyte homeostasis. In contrast, increased mTOR activity accompanied human diabetic nephropathy, characterized by early glomerular hypertrophy and hyperfiltration. Curtailing mTORC1 signaling in mice by genetically reducing mTORC1 copy number in podocytes prevented glomerulosclerosis and significantly ameliorated the progression of glomerular disease in diabetic nephropathy. These results demonstrate the requirement for tightly balanced mTOR activity in podocyte homeostasis and suggest that mTOR inhibition can protect podocytes and prevent progressive diabetic nephropathy.
454 citations
TL;DR: In cultured human and mouse mesangial cells to high glucose and transforming growth factor‐β to simulate the diabetic milieu, microRNA‐377 was consistently up‐regulated relative to controls and can lead to increased fibronectin production in diabetic nephropathy.
Abstract: Intrinsic glomerular cells in a diabetic milieu have transcriptional activation of genes that influence the development of diabetic nephropathy. The cellular repertoire of microRNAs can regulate translation of these expressed genes into proteins. Fibronectin is a key matrix protein accumulated in excess in diabetic nephropathy. Here, we exposed cultured human and mouse mesangial cells to high glucose and transforming growth factor-β to simulate the diabetic milieu. In these conditions in vitro, as well as in mouse diabetic nephropathy models in vivo, microRNA-377 was consistently up-regulated relative to controls. Through a combination of computational and biological approaches, we identified relevant miR-377 target genes. Although fibronectin was induced by miR-377, it was not a direct target of miR-377. However, miR-377 led to reduced expressions of p21-activated kinase and superoxide dismutase, which enhanced fibronectin protein production. Thus, overexpression of miR-377 in diabetic nephropathy indirectly leads to increased fibronectin protein production; as such, miR-377 can have a critical role in the pathophysiology of this prevalent human disease.—Wang, Q., Wang, Y., Minto, A. W., Wang, J., Shi, Q., Li, X., Quigg, R. J. MicroRNA-377 is up-regulated and can lead to increased fibronectin production in diabetic nephropathy.
381 citations
Cites methods from "A new method for large scale isolat..."
...In some instances, glomeruli were isolated magnetically after intraarterial injection of Dynabeads (13), and then either studied directly for protein expression or used to culture mesangial cells using standard techniques (14)....
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TL;DR: The results suggest that targeting hyperactive Wnt/beta-catenin signaling may represent a novel therapeutic strategy for proteinuric kidney diseases, and upregulation of Wnt1 and active beta-Catenin in podocytes is observed.
Abstract: Podocyte dysfunction, one of the major causes of proteinuria, leads to glomerulosclerosis and end stage renal disease, but its underlying mechanism remains poorly understood. Here we show that Wnt/β-catenin signaling plays a critical role in podocyte injury and proteinuria. Treatment with adriamycin induced Wnt and activated β-catenin in mouse podocytes. Overexpression of Wnt1 in vivo activated glomerular β-catenin and aggravated albuminuria and adriamycin-induced suppression of nephrin expression, whereas blockade of Wnt signaling with Dickkopf-1 ameliorated podocyte lesions. Podocyte-specific knockout of β-catenin protected against development of albuminuria after injury. Moreover, pharmacologic activation of β-catenin induced albuminuria in wild-type mice but not in β-catenin-knockout littermates. In human proteinuric kidney diseases such as diabetic nephropathy and focal segmental glomerulosclerosis, we observed upregulation of Wnt1 and active β-catenin in podocytes. Ectopic expression of either Wnt1 or stabilized β-catenin in vitro induced the transcription factor Snail and suppressed nephrin expression, leading to podocyte dysfunction. These results suggest that targeting hyperactive Wnt/β-catenin signaling may represent a novel therapeutic strategy for proteinuric kidney diseases.
349 citations
References
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TL;DR: The results of an international collaboration to produce and make freely available a draft sequence of the human genome are reported and an initial analysis is presented, describing some of the insights that can be gleaned from the sequence.
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TL;DR: Serial analysis of gene expression (SAGE) should provide a broadly applicable means for the quantitative cataloging and comparison of expressed genes in a variety of normal, developmental, and disease states.
Abstract: PROBLEM TO BE SOLVED: To provide a method for preparing a short nucleotide sequence (tag) which is useful to identify a cDNA oligonucleotide and is derived from a restricted position in a mRNA or a cDNA. SOLUTION: This is the method of preparing a tag for identifying the cDNA oligonucleotide. The above method comprises preparing the cDNA oligonucleotide bearing 5' and 3' terminals, collecting cDNA fragments by cutting the cDNA oligonucleotide with a restriction enzyme at the first restriction endonuclease site, separating a cDNA oligonucleotide bearing 5' or 3' terminal and connecting an oligonucleotide linker to the isolated cDNA fragment bearing the cDNA oligonucleotide 5' or 3' terminal. Here, the oligonucleotide linker contains the recognition site of the second restriction endonuclease enzyme and the isolated cDNA fragment is cut with the second restriction endonuclease enzyme which cuts the cDNA fragment in a section separated from the recognition site to obtain the tag for identifying the cDNA oligonucleotide.
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TL;DR: A crucial role for this protein in the development or function of the kidney filtration barrier is demonstrated and by Northern and in situ hybridization was shown to be specifically expressed in renal glomeruli.
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TL;DR: Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems are indicated.
Abstract: A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies—a whole-genome assembly and a regional chromosome assembly—were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional ∼12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.
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