Probes for INS

Acute kidney injury (AKI), commonly caused by ischemia, sepsis, or nephrotoxic insult, is associated with increased mortality and a heightened risk of chronic kidney disease (CKD). AKI results in the dysfunction or death of proximal tubule cells (PTCs), triggering a poorly understood autologous cellular repair program. Defective repair associates with a long-term transition to CKD. We performed a mild-to-moderate ischemia-reperfusion injury (IRI) to model injury responses reflective of kidney injury in a variety of clinical settings, including kidney transplant surgery. Single-nucleus RNA sequencing of genetically labeled injured PTCs at 7-d ("early") and 28-d ("late") time points post-IRI identified specific gene and pathway activity in the injury-repair transition. In particular, we identified Vcam1 +/Ccl2 + PTCs at a late injury stage distinguished by marked activation of NF-κB-, TNF-, and AP-1-signaling pathways. This population of PTCs showed features of a senescence-associated secretory phenotype but did not exhibit G2/M cell cycle arrest, distinct from other reports of maladaptive PTCs following kidney injury. Fate-mapping experiments identified spatially and temporally distinct origins for these cells. At the cortico-medullary boundary (CMB), where injury initiates, the majority of Vcam1 +/Ccl2 + PTCs arose from early replicating PTCs. In contrast, in cortical regions, only a subset of Vcam1 +/Ccl2 + PTCs could be traced to early repairing cells, suggesting late-arising sites of secondary PTC injury. Together, these data indicate even moderate IRI is associated with a lasting injury, which spreads from the CMB to cortical regions. Remaining failed-repair PTCs are likely triggers for chronic disease progression.

Purpose: The synovial joint exhibits extraordinary biotribological properties allowing the articular cartilage layers to slide past each other at very low friction even under local pressures of up to 18 MPa (~180 atm). Articular cartilage is exquisitely mechanical sensitive. Compressive mechanical load contributes to articular cartilage homeostasis; however, overuse or destabilizing the joint increases surface shear stress, which promotes cartilage degradation. Our previous Results show that shear stress, induced by joint destabilization, regulates a number of inflammatory genes 6h post surgery, including Mmp3, Il1b, Arg1, Ccl2, and Il6. Immobilizing the joint by prolonged anesthesia or sciatic neurectomy abrogates the regulation of inflammatory genes and prevents development of OA. In this study, we use RNA Scope to identify which cells of the cartilage are activated by surface shear after joint destabilisation, and test whether this is modifiable by injection of a biocompatible phospholipid-based lubricant. Methods: Destabilization of the medial meniscus (DMM) or sham surgery was performed on the right knee of 10-week-old male C57BL/6 mice. 30 ml of lubricant (PMPC: poly(methacryloylphosphsphorylcholine)-functionalized lipid vesicles) or vehicle control (PBS) solution was injected in the joint two days before and at the time of surgery. Cartilage from naïve (no surgery) and DMM-operated knees of four mice per data point was collected by microdissection for bulk mRNA extraction. Expression levels of selected genes including shear-responsive genes Il1b and Mmp3 were tested by RT-PCR using TaqMan Low Density Arrays (TLDA) microfluidic cards. In addition, whole joints were collected and processed following the standard protocol for RNAscope (Advanced Cell Diagnostics). Coronal sections in the middle of the joints were sliced by a cryostat. Consecutive sections were used for Safranin O staining and RNAscope to identify anatomical tissues and detect the expression of genes of interest. Gene expression signals were collated from 11 stacks by confocal microscopy (Zeiss Confocal 880) focusing on the medial tibia cartilage, and were quantified by counting individual mRNA dots in the sham, DMM, vehicle and lubricant groups. Results: We observed the upregulation of injury-responsive genes Il1b, Mmp3, Ccl2, Adamts 4, Nos2, and Timp1 in the articular cartilage of DMM operated joints compared to Naïve (non-operated) animals. The injection of the lubricant in the joint significantly suppressed the expression of shear-responsive genes Il1b and Mmp3 after DMM, but did not influence the increase of other injury-induced inflammatory genes, such as Timp1, Adamts 4, Ccl2, Nos2. For RNAscope, focusing on Mmp3 expression, the number of Mmp3 positive cells increased two-fold in the DMM-vehicle group compared with the sham-vehicle group. Most of Mmp3 signal was expressed in the superficial region of the cartilage. DMM-PMPC groups showed a reduced number of Mmp3 positive cells compared with DMM-vehicle, with levels similar to sham-vehicle and sham-PMPC groups. Conclusions: Our data demonstrate that shear stress-induced inflammatory genes are regulated in the superficial layer of cartilage after joint destabilisation and can be suppressed by joint injection of a biocompatible engineered lubricant. As these lubricants have long retention times in the joint (data not presented), we believe that they may provide a potential novel therapeutic strategy for preventing the development of post-trauma OA. These studies are underway

Aging, a universal process that affects all cells in an organism, is a major risk factor for a group of neuropathies called glaucoma, where elevated intraocular pressure is one of the known stresses affecting the tissue. Our understanding of molecular impact of aging on response to stress in retina is very limited; therefore, we developed a new mouse model to approach this question experimentally. Here we show that susceptibility to response to stress increases with age and is primed on chromatin level. We demonstrate that ocular hypertension activates a stress response that is similar to natural aging and involves activation of inflammation and senescence. We show that multiple instances of pressure elevation cause aging of young retina as measured on transcriptional and DNA methylation level and are accompanied by local histone modification changes. Our data show that repeated stress accelerates appearance of aging features in tissues and suggest chromatin modifications as the key molecular components of aging. Lastly, our work further emphasizes the importance of early diagnosis and prevention as well as age-specific management of age-related diseases, including glaucoma.

The mechanisms explaining progression to severe COVID-19 remain poorly understood. It has been proposed that immune system dysregulation/over-stimulation may be implicated, but it is not clear how such processes would lead to respiratory failure. We performed comprehensive multiparameter immune monitoring in a tightly controlled cohort of 128 COVID-19 patients, and used the ratio of oxygen saturation to fraction of inspired oxygen (SpO2 / FiO2) as a physiologic measure of disease severity. Machine learning algorithms integrating 139 parameters identified IL-6 and CCL2 as two factors predictive of severe disease, consistent with the therapeutic benefit observed with anti-IL6-R antibody treatment. However, transcripts encoding these cytokines were not detected among circulating immune cells. Rather, in situ analysis of lung specimens using RNAscope and immunofluorescent staining revealed that elevated IL-6 and CCL2 were dominantly produced by infected lung type II pneumocytes. Severe disease was not associated with higher viral load, deficient antibody responses, or dysfunctional T cell responses. These results refine our understanding of severe COVID-19 pathophysiology, indicating that aberrant cytokine production by infected lung epithelial cells is a major driver of immunopathology. We propose that these factors cause local immune regulation towards the benefit of the virus.