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Cold Spring Harbor, NY, United States

Li X.,Mount Sinai School of Medicine | Li X.,Tongji University | Chuang P.Y.,Mount Sinai School of Medicine | D'Agati V.D.,Columbia University | And 10 more authors.
Journal of the American Society of Nephrology | Year: 2015

Nephrin is required during kidney development for the maturation of podocytes and formation of the slit diaphragm junctional complex. Because nephrin expression is downregulated in acquired glomerular diseases, nephrin deficiency is considered a pathologic feature of glomerular injury. However, whether nephrin deficiency exacerbates glomerular injury in glomerular diseases has not been experimentally confirmed. Here, we generatedmicewith inducible RNA interference-mediated nephrin knockdown. Short-term nephrin knockdown (6 weeks), starting after the completion of kidney development at 5weeks of age, did not affect glomerular structure or function. In contrast, mice with long-term nephrin knockdown (20 weeks) developed mild proteinuria, foot process effacement, filtration slit narrowing, mesangial hypercellularity and sclerosis, glomerular basement membrane thickening, subendothelial zone widening, and podocyte apoptosis. When subjected to an acquired glomerular insult induced by unilateral nephrectomy or doxorubicin,mice with short-term nephrin knockdown developed more severe glomerular injury compared with mice without nephrin knockdown. Additionally, nephrin-knockdown mice developed more exaggerated glomerular enlargement when subjected to unilateral nephrectomy and more podocyte apoptosis and depletion after doxorubicin challenge. AKT phosphorylation, which is a slit diaphragm-mediated and nephrin-dependent pathway in the podocyte, was markedly reduced in mice with long-term or short-term nephrin knockdown challenged with uninephrectomy or doxorubicin. Taken together, our data establish that under the basal condition and in acquired glomerular diseases, nephrin is required tomaintain slit diaphragmintegrity and slit diaphragm-mediated signaling to preserve glomerular function and podocyte viability in adult mice. Copyright © 2015 by the American Society of Nephrology.

Chuang P.Y.,Mount Sinai School of Medicine | Xu J.,Mount Sinai School of Medicine | Dai Y.,Fudan University | Jia F.,Nanjing University | And 6 more authors.
American Journal of Pathology | Year: 2014

The silent mating type information regulation 2 homolog 1 gene (Sirt1) encodes an NAD-dependent deacetylase that modifies the activity of well-known transcriptional regulators affected in kidney diseases. Sirt1 is expressed in the kidney podocyte, but its function in the podocyte is not clear. Genetically engineered mice with inducible and reversible Sirt1 knockdown in widespread, podocyte-specific, or tubular-specific patterns were generated. We found that mice with 80% knockdown of renal Sirt1 expression have normal glomerular function under the basal condition. When challenged with doxorubicin (Adriamycin), these mice develop marked albuminuria, glomerulosclerosis, mitochondrial injury, and impaired autophagy of damaged mitochondria. Reversal of Sirt1 knockdown during the early phase of Adriamycin-induced nephropathy prevented the progression of glomerular injury and reduced the accumulation of dysmorphic mitochondria in podocytes but did not reverse the progression of albuminuria and glomerulosclerosis. Sirt1 knockdown mice with diabetes mellitus, which is known to cause mitochondrial dysfunction in the kidney, developed more albuminuria and mitochondrial dysfunction compared with diabetic mice without Sirt1 knockdown. In conclusion, these results demonstrate that our RNA interference-mediated Sirt1 knockdown models are valid and versatile tools for characterizing the function of Sirt1 in the kidney; Sirt1 plays a role in homeostatic maintenance of podocytes under the condition of mitochondrial stress/injury. © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Fellmann C.,Mirimus Inc. | Lowe S.W.,Sloan Kettering Cancer Center
Nature Cell Biology | Year: 2014

RNA interference has become an indispensable tool for loss-of-function studies across eukaryotes. By enabling stable and reversible gene silencing, shRNAs provide a means to study long-term phenotypes, perform pool-based forward genetic screens and examine the consequences of temporary target inhibition in vivo. However, efficient implementation in vertebrate systems has been hindered by technical difficulties affecting potency and specificity. Focusing on these issues, we analyse current strategies to obtain maximal knockdown with minimal off-target effects. © 2014 Macmillan Publishers Limited.

Knott S.R.V.,Howard Hughes Medical Institute | Maceli A.R.,Howard Hughes Medical Institute | Erard N.,Howard Hughes Medical Institute | Chang K.,Howard Hughes Medical Institute | And 10 more authors.
Molecular Cell | Year: 2014

The strength of conclusions drawn from RNAi-based studies is heavily influenced by the quality of tools used to elicit knockdown. Prior studies have developed algorithms to design siRNAs. However, to date, no established method has emerged to identifyeffective shRNAs, which have lower intracellular abundance than transfected siRNAs and undergo additional processing steps. We recently developed a multiplexed assay for identifying potent shRNAs and used this method to generate ~250,000 shRNA efficacy data points. Using these data, we developed shERWOOD, an algorithm capable of predicting, for any shRNA, the likelihood that it will elicit potent target knockdown. Combined with additional shRNA design strategies, shERWOOD allows the ab initio identification of potent shRNAs that specifically target the majority of each gene's multiple transcripts. We validated the performance of our shRNA designs using several orthogonal strategies and constructed genome-wide collections of shRNAs for humans and mice based on our approach. © 2014 Elsevier Inc.

Mirimus Inc. | Date: 2012-10-30

Live animals, namely, genetically engineered mice for laboratory and clinical trial purposes. Medical and scientific research, namely, the design for others of genetically engineered mice for laboratory and clinical trial purposes.

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