Publications by authors named "Christopher E Pedigo"

13 Publications

  • Page 1 of 1

Inhibiting calpain 1 and 2 in cyclin G associated kinase-knockout mice mitigates podocyte injury.

JCI Insight 2020 11 19;5(22). Epub 2020 Nov 19.

Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USA.

Evidence for reduced expression of cyclin G associated kinase (GAK) in glomeruli of patients with chronic kidney disease was observed in the Nephroseq human database, and GAK was found to be associated with the decline in kidney function. To examine the role of GAK, a protein that functions to uncoat clathrin during endocytosis, we generated podocyte-specific Gak-knockout mice (Gak-KO), which developed progressive proteinuria and kidney failure with global glomerulosclerosis. We isolated glomeruli from the mice carrying the mutation to perform messenger RNA profiling and unearthed evidence for dysregulated podocyte calpain protease activity as an important contributor to progressive podocyte damage. Treatment with calpain inhibitor III specifically inhibited calpain-1/-2 activities, mitigated the degree of proteinuria and glomerulosclerosis, and led to a striking increase in survival in the Gak-KO mice. Podocyte-specific deletion of Capns1, essential for calpain-1 and calpain-2 activities, also improved proteinuria and glomerulosclerosis in Gak-KO mice. Increased podocyte calpain activity-mediated proteolysis of IκBα resulted in increased NF-κB p65-induced expression of growth arrest and DNA-damage-inducible 45 beta in the Gak-KO mice. Our results suggest that loss of podocyte-associated Gak induces glomerular injury secondary to calcium dysregulation and aberrant calpain activation, which when inhibited, can provide a protective role.
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http://dx.doi.org/10.1172/jci.insight.142740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710277PMC
November 2020

Murine Epsins Play an Integral Role in Podocyte Function.

J Am Soc Nephrol 2020 12 13;31(12):2870-2886. Epub 2020 Oct 13.

Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut

Background: Epsins, a family of evolutionarily conserved membrane proteins, play an essential role in endocytosis and signaling in podocytes.

Methods: Podocyte-specific , , triple-knockout mice were generated to examine downstream regulation of serum response factor (SRF) by cell division control protein 42 homolog (Cdc42).

Results: Podocyte-specific loss of epsins resulted in increased albuminuria and foot process effacement. Primary podocytes isolated from these knockout mice exhibited abnormalities in cell adhesion and spreading, which may be attributed to reduced activation of cell division control protein Cdc42 and SRF, resulting in diminished integrin expression. In addition, podocyte-specific loss of resulted in severe albuminuria and foot process effacement, and defects in cell adhesion and spreading, along with decreased integrin expression.

Conclusions: Epsins play an indispensable role in maintaining properly functioning podocytes through the regulation of Cdc42 and SRF-dependent integrin expression.
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http://dx.doi.org/10.1681/ASN.2020050691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790213PMC
December 2020

Inhibition of Endocytosis of Clathrin-Mediated Angiotensin II Receptor Type 1 in Podocytes Augments Glomerular Injury.

J Am Soc Nephrol 2019 12 11;30(12):2307-2320. Epub 2019 Sep 11.

Section of Nephrology, Department of Internal Medicine, Yale School of Medicine, New Haven, Connecticut;

Background: Inhibition of the renin-angiotensin system remains a cornerstone in reducing proteinuria and progression of kidney failure, effects believed to be the result of reduction in BP and glomerular hyperfiltration. However, studies have yielded conflicting results on whether podocyte-specific angiotensin II (AngII) signaling directly induces podocyte injury. Previous research has found that after AngII stimulation, -arrestin-bound angiotensin II receptor type 1 (AT1R) is internalized in a clathrin- and dynamin-dependent manner, and that and double-knockout mice exhibit impaired clathrin-mediated endocytosis.

Methods: We used podocyte-specific double-knockout mice to examine AngII-stimulated AT1R internalization and signaling in primary podocytes and controls. We also examined the effect of AngII in these double-knockout mice through renin-angiotensin system blockers and through deletion of (which encodes the predominant AT1R isoform expressed in kidney, AT1aR). We tested calcium influx, Rac1 activation, and lamellipodial extension in control and primary podocytes of double-knockout mice treated with AngII.

Results: We confirmed augmented AngII-stimulated AT1R signaling in primary double-knockout podocytes resulting from arrest of clathrin-coated pit turnover. Genetic ablation of podocyte in double-knockout mice demonstrated improved albuminuria and kidney function compared with the double-knockout mice. Isolation of podocytes from double-knockout mice revealed abnormal membrane dynamics, with increased Rac1 activation and lamellipodial extension, which was attenuated in double-knockout podocytes lacking AT1aR.

Conclusions: Our results indicate that inhibiting aberrant podocyte-associated AT1aR signaling pathways has a protective effect in maintaining the integrity of the glomerular filtration barrier.
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http://dx.doi.org/10.1681/ASN.2019010053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900791PMC
December 2019

ATP-binding cassette A1 deficiency causes cardiolipin-driven mitochondrial dysfunction in podocytes.

J Clin Invest 2019 07 22;129(8):3387-3400. Epub 2019 Jul 22.

Katz Family Division of Nephrology and Hypertension/ Drug Discovery Center, Department of Medicine, University of Miami, Miami, Florida, USA.

Fibroblasts from patients with Tangier disease carrying ATP-binding cassette A1 (ABCA1) loss-of-function mutations are characterized by cardiolipin accumulation, a mitochondrial-specific phospholipid. Suppression of ABCA1 expression occurs in glomeruli from patients with diabetic kidney disease (DKD) and in human podocytes exposed to DKD sera collected prior to the development of DKD. We demonstrated that siRNA ABCA1 knockdown in podocytes led to reduced oxygen consumption capabilities associated with alterations in the oxidative phosphorylation (OXPHOS) complexes and with cardiolipin accumulation. Podocyte-specific deletion of Abca1 (Abca1fl/fl) rendered mice susceptible to DKD, and pharmacological induction of ABCA1 improved established DKD. This was not mediated by free cholesterol, as genetic deletion of sterol-o-acyltransferase-1 (SOAT1) in Abca1fl/fl mice was sufficient to cause free cholesterol accumulation but did not cause glomerular injury. Instead, cardiolipin mediates ABCA1-dependent susceptibility to podocyte injury, as inhibition of cardiolipin peroxidation with elamipretide improved DKD in vivo and prevented ABCA1-dependent podocyte injury in vitro and in vivo. Collectively, we describe a pathway definitively linking ABCA1 deficiency to cardiolipin-driven mitochondrial dysfunction. We demonstrated that this pathway is relevant to DKD and that ABCA1 inducers or inhibitors of cardiolipin peroxidation may each represent therapeutic strategies for the treatment of established DKD.
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http://dx.doi.org/10.1172/JCI125316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6668702PMC
July 2019

Podocyte histone deacetylase activity regulates murine and human glomerular diseases.

J Clin Invest 2019 03 18;129(3):1295-1313. Epub 2019 Feb 18.

Department of Internal Medicine, and.

We identified 2 genes, histone deacetylase 1 (HDAC1) and HDAC2, contributing to the pathogenesis of proteinuric kidney diseases, the leading cause of end-stage kidney disease. mRNA expression profiling from proteinuric mouse glomeruli was linked to Connectivity Map databases, identifying HDAC1 and HDAC2 with the differentially expressed gene set reversible by HDAC inhibitors. In numerous progressive glomerular disease models, treatment with valproic acid (a class I HDAC inhibitor) or SAHA (a pan-HDAC inhibitor) mitigated the degree of proteinuria and glomerulosclerosis, leading to a striking increase in survival. Podocyte HDAC1 and HDAC2 activities were increased in mice podocytopathy models, and podocyte-associated Hdac1 and Hdac2 genetic ablation improved proteinuria and glomerulosclerosis. Podocyte early growth response 1 (EGR1) was increased in proteinuric patients and mice in an HDAC1- and HDAC2-dependent manner. Loss of EGR1 in mice reduced proteinuria and glomerulosclerosis. Longitudinal analysis of the multicenter Veterans Aging Cohort Study demonstrated a 30% reduction in mean annual loss of estimated glomerular filtration rate, and this effect was more pronounced in proteinuric patients receiving valproic acid. These results strongly suggest that inhibition of HDAC1 and HDAC2 activities may suppress the progression of human proteinuric kidney diseases through the regulation of EGR1.
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http://dx.doi.org/10.1172/JCI124030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391095PMC
March 2019

Vinculin is required to maintain glomerular barrier integrity.

Kidney Int 2018 03 12;93(3):643-655. Epub 2017 Dec 12.

Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USA. Electronic address:

Cell-matrix interactions and podocyte intercellular junctions are key for maintaining the glomerular filtration barrier. Vinculin, a cytoplasmic protein, couples actin filaments to integrin-mediated cell-matrix adhesions and to cadherin-based intercellular junctions. Here, we examined the role of vinculin in podocytes by the generation of a podocyte-specific knockout mouse. Mice lacking podocyte vinculin had increased albuminuria and foot process effacement following injury in vivo. Analysis of primary podocytes isolated from the mutant mice revealed defects in cell protrusions, altered focal adhesion size and signaling, as well as impaired cell migration. Furthermore, we found a marked mislocalization of the intercellular junction protein zonula occludens-1. In kidney sections from patients with focal segmental glomerulosclerosis, minimal change disease and membranous nephropathy, we observed dramatic differences in the expression levels and localization of vinculin. Thus, our results suggest that vinculin is necessary to maintain the integrity of the glomerular filtration barrier by modulating podocyte foot processes and stabilizing intercellular junctions.
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http://dx.doi.org/10.1016/j.kint.2017.09.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5846847PMC
March 2018

Direct Measurement of Free and Esterified Cholesterol Mass in Differentiated Human Podocytes: A TLC and Enzymatic Assay-Based Method.

Methods Mol Biol 2017 ;1609:51-56

Division of Nephrology and Hypertension and Peggy and Harold Katz Drug Discovery Center, University of Miami Miller School of Medicine, Batchelor Bldg., 6th Floor, 1580 NW, 10th Ave., Miami, FL, 33136, USA.

Esterified cholesterol content is often lower than free cholesterol content in biological systems and thus the determination of the esterified cholesterol content of cells is often challenging. Traditional methods use enzymatic assays in which an indirect measurement of the esterified cholesterol content is obtained by subtracting the measurements of the free from the total cholesterol content. However, this approach fails in the case where the total cholesterol content of cells is unchanged while the ratio of free to esterified cholesterol changes such that total and free cholesterol content are very similar and thus the difference may fall within the background noise of the enzymatic assay. To overcome this challenge, we here describe a method that utilizes a TLC-based technique to isolate esterified cholesterol. Isolated esterified cholesterol can then be measured using traditional enzymatic methods. Therefore, this method provides a practical and more sensitive assay to measure esterified cholesterol content in cellular extracts.
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http://dx.doi.org/10.1007/978-1-4939-6996-8_6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792302PMC
March 2018

Local TNF causes NFATc1-dependent cholesterol-mediated podocyte injury.

J Clin Invest 2016 09 2;126(9):3336-50. Epub 2016 Aug 2.

High levels of circulating TNF and its receptors, TNFR1 and TNFR2, predict the progression of diabetic kidney disease (DKD), but their contribution to organ damage in DKD remains largely unknown. Here, we investigated the function of local and systemic TNF in podocyte injury. We cultured human podocytes with sera collected from DKD patients, who displayed elevated TNF levels, and focal segmental glomerulosclerosis (FSGS) patients, whose TNF levels resembled those of healthy patients. Exogenous TNF administration or local TNF expression was equally sufficient to cause free cholesterol-dependent apoptosis in podocytes by acting through a dual mechanism that required a reduction in ATP-binding cassette transporter A1-mediated (ABCA1-mediated) cholesterol efflux and reduced cholesterol esterification by sterol-O-acyltransferase 1 (SOAT1). TNF-induced albuminuria was aggravated in mice with podocyte-specific ABCA1 deficiency and was partially prevented by cholesterol depletion with cyclodextrin. TNF-stimulated free cholesterol-dependent apoptosis in podocytes was mediated by nuclear factor of activated T cells 1 (NFATc1). ABCA1 overexpression or cholesterol depletion was sufficient to reduce albuminuria in mice with podocyte-specific NFATc1 activation. Our data implicate an NFATc1/ABCA1-dependent mechanism in which local TNF is sufficient to cause free cholesterol-dependent podocyte injury irrespective of TNF, TNFR1, or TNFR2 serum levels.
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http://dx.doi.org/10.1172/JCI85939DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5004940PMC
September 2016

Metabolism, energetics, and lipid biology in the podocyte - cellular cholesterol-mediated glomerular injury.

Front Endocrinol (Lausanne) 2014 14;5:169. Epub 2014 Oct 14.

Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Diabetes Research Institute, University of Miami , Miami, FL , USA.

Chronic kidney disease (CKD) is associated with a high risk of death. Dyslipidemia is commonly observed in patients with CKD and is accompanied by a decrease in plasma high-density lipoprotein, and an increase in plasma triglyceride-rich lipoproteins and oxidized lipids. The observation that statins may decrease albuminuria but do not stop the progression of CKD indicates that pathways other than the cholesterol synthesis contribute to cholesterol accumulation in the kidneys of patients with CKD. Recently, it has become clear that increased lipid influx and impaired reverse cholesterol transport can promote glomerulosclerosis, and tubulointerstitial damage. Lipid-rafts are cholesterol-rich membrane domains with important functions in regulating membrane fluidity, membrane protein trafficking, and in the assembly of signaling molecules. In podocytes, which are specialized cells of the glomerulus, they contribute to the spatial organization of the slit diaphragm (SD) under physiological and pathological conditions. The discovery that podocyte-specific proteins such as podocin can bind and recruit cholesterol contributing to the formation of the SD underlines the importance of cholesterol homeostasis in podocytes and suggests cholesterol as an important regulator in the development of proteinuric kidney disease. Cellular cholesterol accumulation due to increased synthesis, influx, or decreased efflux is an emerging concept in podocyte biology. This review will focus on the role of cellular cholesterol accumulation in the pathogenesis of kidney diseases with a focus on glomerular diseases.
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http://dx.doi.org/10.3389/fendo.2014.00169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4196552PMC
October 2014

Sphingomyelinase-like phosphodiesterase 3b expression levels determine podocyte injury phenotypes in glomerular disease.

J Am Soc Nephrol 2015 Jan 12;26(1):133-47. Epub 2014 Jun 12.

Department of Medicine, Division of Nephrology and Hypertension, Peggy and Harold Katz Family Drug Discovery Center and Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, Florida;

Diabetic kidney disease (DKD) is the most common cause of ESRD in the United States. Podocyte injury is an important feature of DKD that is likely to be caused by circulating factors other than glucose. Soluble urokinase plasminogen activator receptor (suPAR) is a circulating factor found to be elevated in the serum of patients with FSGS and causes podocyte αVβ3 integrin-dependent migration in vitro. Furthermore, αVβ3 integrin activation occurs in association with decreased podocyte-specific expression of acid sphingomyelinase-like phosphodiesterase 3b (SMPDL3b) in kidney biopsy specimens from patients with FSGS. However, whether suPAR-dependent αVβ3 integrin activation occurs in diseases other than FSGS and whether there is a direct link between circulating suPAR levels and SMPDL3b expression in podocytes remain to be established. Our data indicate that serum suPAR levels are also elevated in patients with DKD. However, unlike in FSGS, SMPDL3b expression was increased in glomeruli from patients with DKD and DKD sera-treated human podocytes, where it prevented αVβ3 integrin activation by its interaction with suPAR and led to increased RhoA activity, rendering podocytes more susceptible to apoptosis. In vivo, inhibition of acid sphingomyelinase reduced proteinuria in experimental DKD but not FSGS, indicating that SMPDL3b expression levels determined the podocyte injury phenotype. These observations suggest that SMPDL3b may be an important modulator of podocyte function by shifting suPAR-mediated podocyte injury from a migratory phenotype to an apoptotic phenotype and that it represents a novel therapeutic glomerular disease target.
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http://dx.doi.org/10.1681/ASN.2013111213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4279736PMC
January 2015

Cyclodextrin protects podocytes in diabetic kidney disease.

Diabetes 2013 Nov 8;62(11):3817-27. Epub 2013 Jul 8.

Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of Medicine, Miami, Florida.

Diabetic kidney disease (DKD) remains the most common cause of end-stage kidney disease despite multifactorial intervention. We demonstrated that increased cholesterol in association with downregulation of ATP-binding cassette transporter ABCA1 occurs in normal human podocytes exposed to the sera of patients with type 1 diabetes and albuminuria (DKD(+)) when compared with diabetic patients with normoalbuminuria (DKD(-)) and similar duration of diabetes and lipid profile. Glomerular downregulation of ABCA1 was confirmed in biopsies from patients with early DKD (n = 70) when compared with normal living donors (n = 32). Induction of cholesterol efflux with cyclodextrin (CD) but not inhibition of cholesterol synthesis with simvastatin prevented podocyte injury observed in vitro after exposure to patient sera. Subcutaneous administration of CD to diabetic BTBR (black and tan, brachiuric) ob/ob mice was safe and reduced albuminuria, mesangial expansion, kidney weight, and cortical cholesterol content. This was followed by an improvement of fasting insulin, blood glucose, body weight, and glucose tolerance in vivo and improved glucose-stimulated insulin release in human islets in vitro. Our data suggest that impaired reverse cholesterol transport characterizes clinical and experimental DKD and negatively influences podocyte function. Treatment with CD is safe and effective in preserving podocyte function in vitro and in vivo and may improve the metabolic control of diabetes.
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http://dx.doi.org/10.2337/db13-0399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3806621PMC
November 2013

Bioaccessibility of arsenic in various types of rice in an in vitro gastrointestinal fluid system.

J Environ Sci Health B 2012 ;47(2):74-80

Department of Sciences, John Jay College, The City University of New York, 445 W59th Street, New York, NY 10019, USA.

Rice can be a major contributor to dietary arsenic exposure because of the relatively high total arsenic concentration compared to other grains, especially for people whose main staple is rice. This study employed in vitro gastrointestinal fluid digestion to determine bioaccessible or gastrointestinal fluid extractable arsenic concentration in rice. Thirty-one rice samples, of which 60 % were grown in the United States, were purchased from food stores in New York City. Total arsenic concentrations in these samples ranged from 0.090 ± 0.004 to 0.85 ± 0.03 mg/kg with a mean value of 0.275 ± 0.161 mg/kg (n = 31). Rice samples with relatively high total arsenic (>0.20 mg/kg, n = 18) were treated by in vitro artificial gastrointestinal fluid digestion, and the extractable arsenic ranged from 53 % to 102 %. The bioaccessibility of arsenic in rice decreases in the general order of extra long grain, long grain, long grain parboiled, to brown rices.
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http://dx.doi.org/10.1080/03601234.2012.611431DOI Listing
May 2012

Evaluating the use of 3'-(p-Aminophenyl) fluorescein for determining the formation of highly reactive oxygen species in particle suspensions.

Geochem Trans 2009 Aug 11;10. Epub 2009 Aug 11.

Center for Environmental Molecular Science, Stony Brook University, Stony Brook, NY 11794-2100, USA.

Background: Given the importance of highly reactive oxygen species (hROS) as reactants in a wide range of biological, photochemical, and environmental systems there is an interest in detection and quantification of these species. The extreme reactivity of the hROS, which includes hydroxyl radicals, presents an analytical challenge. 3'-(p-Aminophenyl) fluorescein (APF) is a relatively new probe used for measuring hROS. Here, we further evaluate the use of APF as a method for the detection of hydroxyl radicals in particle suspensions.

Results: Particle-generated hROS can be quantified with an estimated detection limit of 50 nM. Measurements of hROS in two National Institute of Standards and Technology (NIST 2709 and 2710) soil suspensions and a pyrite suspension show non-linear particle dose-response curves for hROS generation. APF can also be used in solutions containing no dissolved molecular oxygen (O2) to determine the role of O2 in the formation of hROS. Results confirm that O2 is mechanistically important in the formation of hROS by dissolved ferrous iron and in pyrite suspensions.

Conclusion: Given the non-linear dose-response curves for particle generation of hROS, we recommend using several particle loadings in experiments aimed to compare particles for their hROS generation potential. The method presented here is specific to hROS and simple to perform. The analysis can be conducted in mobile labs as only basic laboratory equipment is required.
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http://dx.doi.org/10.1186/1467-4866-10-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2736975PMC
August 2009