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Quantitative proteomic analysis of the role of knocking down NRP1 after OGD/R in primary renal tubular epithelial cells

ABSTRACT: Neuropilin-1 (NRP1), a co-receptor for various cytokines, including TGF-β, has been identified as a potential therapeutic target for fibrosis. However, its role and mechanism in renal fibrosis remains elusive. Here, we show that NRP1 is upregulated in distal tubular (DT) cells of patients with transplant renal insufficiency and mice with renal ischemia-reperfusion (I-R) injury. Knockout of Nrp1 reduced multiple endpoints of renal injury and fibrosis. We found that Nrp1 facilitates the binding of TNF-α to its receptor in DT cells after renal injury. This signaling results in a downregulation of lysine crotonylation of the metabolic enzyme Cox4i1, decreased cellular energetics and exacerbation of renal injury. Furthermore, by single-cell RNA-sequencing we found that Nrp1-positive DT cells secrete collagen and communicate with myofibroblasts, exacerbating acute kidney injury (AKI)-induced renal fibrosis by activating Smad3. Dual genetic deletion of Nrp1 and Tgfbr1 in DT cells better improves renal injury and fibrosis than either single knockout. Together, these results reveal that targeting of NRP1 represents a promising strategy for the treatment of AKI and subsequent chronic kidney disease

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Yinzheng Li

LAB HEAD: Rui Zeng

PROVIDER: PXD052244 | Pride | 2024-06-03

REPOSITORIES: Pride

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Project description:Renal hypoxia is widespread in acute kidney injury (AKI) of various aetiologies. Hypoxia adaptation, conferred through the hypoxia-inducible factor (HIF), appears to be insufficient. Here we show that HIF activation in renal tubules through Pax8-rtTA-based inducible knockout of von Hippel-Lindau protein (VHL-KO) protects from rhabdomyolysis-induced AKI. In this model, histological observations indicate that injury mainly affects proximal convoluted tubules, with 5% necrosis at d1 and 40% necrosis at d2. HIF-1alpha up-regulation in distal tubules reflects renal hypoxia. However, lack of HIF in proximal tubules suggests insufficient adaptation by HIF. AKI in VHL-KO mice leads to prominent HIF activation in all nephron segments, as well as to reduced serum creatinine, serum urea, tubular necrosis, and apoptosis marker caspase-3 protein. At d1 after rhabdomyolysis, when tubular injury is potentially reversible, HIF mediated protection in AKI is associated with activated glycolysis, cellular glucose uptake and utilization, autophagy, vasodilation, and proton removal as demonstrated by qPCR, pathway enrichment analysis and immunohistochemistry. Together, our data provide evidence for a HIF-orchestrated multi-level shift towards glycolysis as a major mechanism for protection against acute tubular injury. All experiments were carried out in transgenic mice in which selective renal tubular VHL knockout (VHL-KO) was inducible by doxycycline (Reference: Mathia S, Paliege A, Koesters R, Peters H, Neumayer HH, Bachmann S, Rosenberger C. Action of hypoxia-inducible factor in liver and kidney from mice with Pax8-rtTA-based deletion of von Hippel-Lindau protein. Acta Physiol (Oxf). 2013; 207(3):565-76.). Four groups of animals were used: 1) controls: untreated mice; 2) VHL-KO: injected with doxycycline (0.1 mg per 10 g body weight SC), 4 days prior to sacrifice; 3) AKI: rhabdomyolysis; 4) VHL-KO/AKI: doxycycline plus rhabdomyolysis. To induce AKI, 50% glycerol (0.05 ml per 10 g body weight) was injected IM into the left hind limb under isoflurane narcosis. Drinking water was withdrawn between 20 h prior and 24 h after glycerol injection.

Project description:Sepsis is a severe and dysregulated inflammatory disease that often precedes the development of acute kidney injury (AKI) with consequent worsening outcome. The main characteristics of sepsis-induced AKI include endothelial cell (EC) dysfunction, infiltration of inflammatory cells, glomerular thrombosis, and renal tubular epithelial cells (RTEC) injury. Numerous studies have demonstrated that mammalian target of rapamycin (mTOR) activation has been implicated in the initiation and progression of renal injury in course of sepsis. However, little is known, about the molecular basis of mTOR role in EC and RTEC dysfunction. Here, we evaluate whether mTOR inhibition by Rapamycin (Rp) as potential strategy to ameliorate renal function and dissected the molecular mechanisms involved. In a mouse model of lipopolysaccharide (LPS)-induced AKI , LPS injection led to a time-dependent increase of serum creatinine and significant morphological changes in renal parenchyma associated with increased collagen deposits and endothelial dysfunction. Interestingly, Rp treatment significantly decreased creatine levels and preserved renal parenchyma, counteracting Endothelial-to mesenchymal transition (EndMT) process and early fibrosis through the inhibition of ERK pathway. Next, we examined the effects of LPS-TLR4 interaction in RTECs. Through a whole-genome DNA methylation analysis in cultured RTEC, we found that LPS induced aberrant methylation, particularly in regions involved in premature aging. The most represented genes were CD39 and WFS1. LPS stimulation of RTEC led to up-regulation of SA-β Gal and cell cycle arrest markers such as p21. In accordance, in endotoxemic mice, we found a decreased expression of CD39 concurrent with Klotho down-regulation. Administration of Rp exerted anti-aging effects in endotoxemic mice, preserving CD39 and Klotho expression. In conclusion, we demonstrated that mTOR inhibition could offer novel strategies to protect endothelial and tubular compartment from accelerated aging and fibrosis thus counteracting the progression to chronic kidney disease.

Project description:Microarray analysis of human kidneys with acute kidney injury (AKI) has been limited because such kidneys are seldom biopsied. However, all kidney transplants experience AKI, and early kidney transplants without rejection are an excellent model for human AKI: they are screened to exclude chronic kidney disease, frequently biopsied, and have extensive follow-up. We used histopathology and microarrays to compare indication biopsies from 28 transplants with AKI to 11 pristine protocol biopsies of stable transplants. Kidneys with AKI showed increased expression of 394 injury-repair response associated transcripts, including many known epithelial injury molecules (e.g. ITGB6, LCN2), tissue remodeling molecules (e.g. VCAN), and inflammation molecules (S100A8, ITGB3). Many other genes also predict the phenotype, depending on statistical filtering rules, including AKI biomarkers as HAVCR1 and IL18. Most mouse orthologs of the top injury-repair transcripts were increased in published mouse AKI models. Pathway analysis of the injury-repair transcripts revealed similarities to cancer, development, and cell movement. The injury-repair transcript score AKI kidneys correlated with reduced function, future recovery, brain death, and need for dialysis, but not future graft loss. In contrast, histologic features of "acute tubular injury" did not correlate with function or with the molecular changes. Thus the injury-repair associated transcripts represent a massive coordinate injury-repair response of kidney parenchyma to AKI, similar to mouse AKI models, and provide an objective measure for assessing the severity of AKI in kidney biopsies and validation for the use of many AKI biomarkers. AKI biopsies sample names and CEL files are from GSE21374. All consenting renal transplant patients undergoing biopsies for cause as standard of care between 09/2004 and 10/2007 at the university of Alberta or between 11/2006 and 02/2007 at the University of Illinois were included in the analysis. In addition to the cores required for standard histopathology, we collected one core for gene expression studies. the relationship between gene expression in the biopsy and subsequent graft loss was analyzed. This dataset is part of the TransQST collection.

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Quantitative proteomic analysis of the role of knocking down NRP1 after OGD/R in primary renal tubular epithelial cells

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