Biology subjects

Limbutara, K.

Publications and source records attributed to Limbutara, K..

3 recordsLinked to original sources

bioRxiv2022

Early Signaling Events in Renal Compensatory Hypertrophy Revealed by Multi-Omics

Loss of a kidney results in compensatory growth of the remaining kidney, a phenomenon of considerable clinical importance. However, the mechanisms involved are largely unknown. Here, we used a multi-omic approach in a mouse unilateral nephrectomy model to identify signaling processes associated with compensatory hypertrophy of the renal proximal tubule. Morphometry applied to microdissected proximal tubules showed that growth of the proximal tubule involves a marked, rapid increase in cell volume rather than cell number. Measurements of DNA accessibility (ATAC-seq), transcriptome (RNA-seq) and proteome (quantitative protein mass spectrometry) independently identified patterns of change that are indicative of activation of the lipid-regulated transcription factor, PPAR. Activation of PPAR by fenofibrate administration increased proximal tubule cell size, while genetic deletion of PPAR in mice decreased it. The results indicate that PPAR is an important determinant of proximal tubule cell size and is a likely mediator of compensatory proximal tubule hypertrophy.

systems biology↗

bioRxiv2020

Protein kinase A catalytic-α and catalytic-β proteins have non-redundant functions

Vasopressin regulates osmotic water transport in the renal collecting duct by PKA-mediated control of the water channel aquaporin-2 (AQP2). Collecting duct principal cells express two seemingly redundant PKA catalytic subunits, PKA catalytic (PKA-C) and PKA catalytic {beta} (PKA-C{beta}). To identify the roles of these two protein kinases, we carried out deep phosphoproteomic analysis in cultured mpkCCD cells in which either PKA-C or PKA-C{beta} was deleted using CRISPR-Cas9-based genome editing. Controls were cells carried through the genome editing procedure, but without deletion of PKA. TMT mass tagging was used for protein mass spectrometric quantification. Of the 4635 phosphopeptides that were quantified 67 were significantly altered in abundance with PKA-C deletion, while 21 were significantly altered in abundance with PKA-C{beta} deletion. However, only four sites were changed in both. The target proteins identified in PKA-C-null cells were largely associated with cell membranes and membrane vesicles, while target proteins in the PKA-C{beta}-null cells were largely associated with the actin cytoskeleton and cell junctions. In contrast, in vitro incubation of mpkCCD proteins with recombinant PKA-C and PKA-C{beta} resulted in virtually identical phosphorylation changes. In addition, analysis of total protein abundances in the in vivo samples showed that PKA-C deletion resulted in a near disappearance of AQP2 protein, while PKA-C{beta} deletion did not decrease AQP2 abundance. We conclude that PKA-C and PKA-C{beta} serve substantially different functions in renal collecting duct cells and that differences in phosphorylation targets may be due to differences in protein interactions, e.g. mediated by AKAP, C-KAP or PDZ binding.

systems biology↗

bioRxiv2018

RNA-Seq and Protein Mass Spectrometry in Microdissected Kidney Tubules Reveal Signaling Processes that Initiate Lithium-Induced Diabetes Insipidus

ABSTRACT1Lithium salts, used for treatment of bipolar disorder, frequently induce nephrogenic diabetes insipidus (NDI), limiting therapeutic success. NDI is associated with loss of expression of the molecular water channel, aquaporin-2, in the renal collecting duct (CD). Here, we use the methods of systems biology in a well-established rat model of lithium-induced NDI to identify signaling pathways activated at the onset of polyuria. Using single-tubule RNA-Seq, full transcriptomes were determined in microdissected cortical CDs of rats 72 hrs after initiation of lithium chloride (LiCl) administration (vs. time-controls without LiCl). Transcriptome-wide changes in mRNA abundances were mapped to gene sets associated with curated canonical signaling pathways, showing evidence for activation of NF-{kappa}B signaling with induction of genes coding for multiple chemokines as well as most components of the Major Histocompatibility Complex (MHC) Class I antigen-presenting complex. Administration of antiinflammatory doses of dexamethasone to LiCl-treated rats countered the loss of aquaporin-2 protein. RNA-Seq also confirmed prior evidence of a shift from quiescence into the cell cycle with arrest. Time course studies demonstrated an early (12 hrs) increase in multiple immediate early genes including several transcription factors. Protein mass spectrometry in microdissected cortical CDs provided corroborative evidence but also identified decreased abundance of several anti-oxidant proteins. Integration of new data with prior data about lithium effects at a molecular level leads to a signaling model in which lithium increases ERK activation leading to induction of NF-{kappa}B signaling and an inflammatory-like response that represses Aqp2 gene transcription.

systems biology↗