Dataset Information

Brain death associated activation of Calpain-Talin axis in donor kidneys underlies podocyte matrix degradation and graft dysfunction in kidney transplantation

ABSTRACT: Deceased kidney donation after brain death (DBD) is the main source of transplants, yet these grafts yield inferior transplant outcomes when compared to living donation. In brain death, cerebral injury contributes to systemic biological dysregulation, causing significant cellular stress in donor kidneys that adversely impacts the quality of grafts. Here, we hypothesized that proteolytic processes in DBD kidneys might lead to podocyte damage with subsequent development of post-transplant dysfunction. Using protein topography and migration analysis platform (PROTOMAP), we mapped degradation profiles of cytoskeletal proteins in DBD kidneys. Cytoskeletal proteolytic degradation was further studied by Immunoblotting on a separate cohort of deceased and living donor kidney biopsies. To investigate potential mechanism of kidney cytoskeletal protein degradation, in-vitro human podocytes and ex-vivo precision-cut human kidney slices were employed. We found novel proteolytic profiles of key podocyte cytoskeletal proteins in donor kidneys associated with suboptimal posttransplant function. These were unique to brain-death and were not observed in circulatory-death or living-donor kidneys. Talin-specific protein degradation in DBD kidneys indicated Calpain-1 activation may have a key role in proteolytic processes observed in the dysfunctional kidneys. Investigation of the underlying mechanism suggests that Transforming-Growth Factor-Î² (TGFÎ²) induces Calpain-1 activation, leading to brain-death specific podocyte degradation patterns and dysregulation of actin cytoskeleton; events that were prevented, in-vitro, by Calpain inhibition. Conclusion Our data demonstrate that podocyte protein degradation impacts the quality of DBD kidneys, propose a role of TGFÎ² mediated Calpain-1 proteolytic processing of cytoskeletal Talin-1, suggesting therapeutic opportunities to prevent kidney dysfunction.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Glomerular Visceral Epithelial Cell, Kidney

SUBMITTER: Philip Charles

LAB HEAD: Maria Kaisar

PROVIDER: PXD022074 | Pride | 2022-08-12

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	HF0018_MariaK_D01_1.mgf	Mgf
	HF0018_MariaK_D01_1.mzid.gz	Mzid
	HF0018_MariaK_D01_1.raw	Raw
	HF0018_MariaK_D01_2.mgf	Mgf
	HF0018_MariaK_D01_2.mzid.gz	Mzid

Items per page:

1 - 5 of 289

Publications

Cytoskeletal protein degradation in brain death donor kidneys associates with adverse posttransplant outcomes.

Vaughan Rebecca H RH Kresse Jean-Claude JC Farmer Louise K LK Thézénas Marie L ML Kessler Benedikt M BM Lindeman Jan H N JHN Sharples Edward J EJ Welsh Gavin I GI Nørregaard Rikke R Ploeg Rutger J RJ Kaisar Maria M

American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons 20220204 4

In brain death, cerebral injury contributes to systemic biological dysregulation, causing significant cellular stress in donor kidneys adversely impacting the quality of grafts. Here, we hypothesized that donation after brain death (DBD) kidneys undergo proteolytic processes that may deem grafts susceptible to posttransplant dysfunction. Using mass spectrometry and immunoblotting, we mapped degradation profiles of cytoskeletal proteins in deceased and living donor kidney biopsies. We found that ...[more]

PMID: 34878723

Similar Datasets

Project description:We compared gene expression profiles from six donor kidneys prior to surgical manipulation to six kidneys removed after laparoscopic donor nephrectomy (LDN) and several hours of CO2 pneumoperitoneum. Biopsies were obtained from renal cortex and hybridized to Affymetrix HG-U133A GeneChips. For control kidneys we identified 1380 genes present on all 6 samples that had a signal intensity greater than 1000. Functional classification of these revealed genes for cellular signaling (201; 15%), regulation of transcription (156; 11%), cellular transport (144; 10%) and cellular metabolism (111; 8%). A class comparison between the controls and LDN kidneys yielded 865 differentially expressed genes. Functional classification of the 502 genes differentially up-regulated in LDN kidneys identified associations with apoptosis, cell adhesion, cell signaling, regulation of cell growth/proliferation, immune/inflammation, ischemia/stress response and proteolysis/peptidolysis. These data demonstrate an altered renal transcriptome induced by several hours of CO2 pneumoperitoneum and laparoscopic surgery characterized by up-regulation of ischemia and injury associated genes. Keywords: Comparative Expression Twelve healthy adult kidney donors signed a consent form, and were approved for donation according to the usual evaluation and practice of the Cleveland Clinic Renal Transplant Program. To avoid potentially significant differences in gene expression based on race and/or ethnicity, only white recipients were selected. The patients were divided into two groups; six controls from open donor nephrectomy and six from LDN. Demographic information included age, gender, race, HLA type, and renal function data including serum creatinine (mg/dL), GFR measured by iothalamate clearance (cc/min.), and urine protein excretion (mg/24 hr). Each patient was given general anesthesia and had a diuresis induced with saline, mannitol, and furosemide. The controls had an extraperitoneal flank incision made, and with only minimal manipulation of the kidney two subcapsular core renal biopsies were obtained using a 15-gauge spring-loaded needle. For the LDN cases two core renal biopsies were obtained using the same needles after the kidneys underwent 2 to 3 hours of C02 pneumoperitoneum. Study biopsies went immediately into 1.5 mL of RNALater (Ambion, Austin, TX), and were stored at –80 0C until they were processed. Biopsy specimens were homogenized in 1 mL of Trizol (Invitrogen, Carlsbad, CA); total RNA was purified using an RNeasy column (Qiagen, Valencia, CA); and quality confirmed with an Agilent 2100 BioAnalyzer (Palo Alto, CA). Standard Affymetrix GeneChip (Santa Clara, CA) protocols were used, and labelled samples were hybridized to HG-U133A GeneChip arrays containing 22,283 probe sets representing over 14,500 human genes. Data was analyzed using Signal intensities generated using GeneChip Operating System version 1.0 and were subsequently analysed by Robust Multichip Average Express (RMA Express) using all Affymetrix .CEL files as a training set. BRB ArrayTools (http://linus.nci.nih.gov/BRB-ArrayTools.html) was used to perform class comparisons (significance level of p < 0.005), and create dendrograms. We used NetAffyx (www.affymetrix.com) to annotate the differentially expressed genes according to biological function based on the Gene Ontology database. A Heatmap was created using Cluster and TreeView software. This dataset is part of the TransQST collection.

Project description:Glomerular podocytes are highly differentiated cells that are key components of the kidney filtration units. The podocyte cytoskeleton builds the basis for the dynamic podocyte cytoarchitecture and plays a central role for proper podocyte function. Recent studies implicate that immunosuppressive agents including the mTOR-inhibitor everolimus have a protective role directly on the stability of the podocyte cytoskeleton. To elucidate mechanisms underlying mTOR-inhibitor mediated cytoskeletal rearrangements, we carried out microarray gene expression studies to identify target genes and corresponding pathways in response to everolimus. We analyzed the effect of everolimus in a puromycin aminonucleoside experimental in vitro model of podocyte injury. Upon treatment with puromycin aminonucleoside, microarray analysis revealed gene clusters involving cytoskeletal-associated pathways, adhesion, migration and extracellular matrix composition to be affected. Everolimus is capable of protecting podocytes from injury, both on the transcriptome and protein level. Rescued genes included TUBB2B and DCDC2, both involved in microtubule structure formation in neuronal cells but not identified in podocytes so far. Confirming gene expression data, Western-blot analysis in cultured podocytes showed an increase of TUBB2B and DCDC2 protein after everolimus treatment, and immunohistochemistry in healthy control kidneys confirmed a podocyte-specific expression. Microtubule-inhibitor experiments led to a maldistribution of TUBB2B and DCDC2 as well as an aberrant reorganization of the actin cytoskeleton. Tubb2bbrdp/brdp mice showed a delay in glomerular podocyte and capillary development. Taken together, our study suggests that off-target, non-immune mediated effects of the mTOR-inhibitor everolimus on the podocyte cytoskeleton might involve regulation of microtubules, revealing a potential novel role of TUBB2B and DCDC2 in glomerular podocyte development We analyzed the effect of everolimus on gene expression in a puromycin aminonucleoside experimental in vitro model of podocyte injury using microarrays