Natural killer (NK) cells are a population of lymphoid cells that play a significant role in mediating innate immune responses. Studies in mice suggest a pathological role for NK cells in models of kidney disease. In this study, we characterized the NK cell subsets present in native kidneys of patients with tubulointerstitial fibrosis, the pathological hallmark of chronic kidney disease. Significantly higher numbers of total NK cells (CD3CD56) were detected in renal biopsies with tubulointerstitial fibrosis compared with diseased biopsies without fibrosis and healthy kidney tissue using multi-color flow cytometry. At a subset level, both the CD56 NK cell subset and particularly the CD56 NK cell subset were elevated in fibrotic kidney tissue. However, only CD56 NK cells significantly correlated with the loss of kidney function. Expression of the tissue-retention and -activation molecule CD69 on CD56 NK cells was significantly increased in fibrotic biopsy specimens compared with non-fibrotic kidney tissue, indicative of a pathogenic phenotype. Further flow cytometric phenotyping revealed selective co-expression of activating receptor CD335 (NKp46) and differentiation marker CD117 (c-kit) on CD56 NK cells. Multi-color immunofluorescent staining of fibrotic kidney tissue localized the accumulation of NK cells within the tubulointerstitium, with CD56 NK cells (NKp46 CD117) identified as the source of pro-inflammatory cytokine interferon-γ within the NK cell compartment. Thus, activated interferon-γ-producing CD56 NK cells are positioned to play a key role in the fibrotic process and progression to chronic kidney disease.

Human proximal tubular epithelial cells (PTEC) of the kidney are known to respond to and mediate the disease process in a wide range of kidney diseases, yet their exosomal production and exosome molecular cargo remain a mystery. Here we investigate, for the first time, the production and molecular content of exosomes derived from primary human PTEC cultured under normal and diseased conditions representing a spectrum of in vivo disease severity from early inflammation, experienced in multiple initial kidney disease states, through to hypoxia, frequently seen in late stage chronic kidney disease (CKD) due to fibrosis and vascular compromise. We demonstrate a rapid reproducible methodology for the purification of PTEC-derived exosomes, identify increased numbers of exosomes from disease-state cultures and identify differential expression levels of both known and unique miRNA and protein species from exosomes derived from different disease-culture conditions. The validity of our approach is supported by the identification of miRNA, proteins and pathways with known CKD associations, providing a rationale to further evaluate these novel and known pathways as targets for therapeutic intervention.

BackgroundMucosal-associated invariant T (MAIT) cells represent a specialized lymphocyte population associated with chronic inflammatory disorders. Little is known, however, about MAIT cells in diseases of the kidney, including CKD.MethodsTo evaluate MAIT cells in human native kidneys with tubulointerstitial fibrosis, the hallmark of CKD, we used multicolor flow cytometry to identify, enumerate, and phenotype such cells from human kidney tissue biopsy samples, and immunofluorescence microscopy to localize these cells. We cocultured MAIT cells and human primary proximal tubular epithelial cells (PTECs) under hypoxic (1% oxygen) conditions to enable examination of mechanistic tubulointerstitial interactions.ResultsWe identified MAIT cells (CD3+ TCR Vα7.2+ CD161hi) in healthy and diseased kidney tissues, detecting expression of tissue-resident markers (CD103/CD69) on MAIT cells in both states. Tissue samples from kidneys with tubulointerstitial fibrosis had significantly elevated numbers of MAIT cells compared with either nonfibrotic samples from diseased kidneys or tissue samples from healthy kidneys. Furthermore, CD69 expression levels, also an established marker of lymphocyte activation, were significantly increased on MAIT cells from fibrotic tissue samples. Immunofluorescent analyses of fibrotic kidney tissue identified MAIT cells accumulating adjacent to PTECs. Notably, MAIT cells activated in the presence of human PTECs under hypoxic conditions (modeling the fibrotic microenvironment) displayed significantly upregulated expression of CD69 and cytotoxic molecules perforin and granzyme B; we also observed a corresponding significant increase in PTEC necrosis in these cocultures.ConclusionsOur findings indicate that human tissue-resident MAIT cells in the kidney may contribute to the fibrotic process of CKD via complex interactions with PTECs.

Platinum-based chemotherapy remains the cornerstone of treatment for most non-small cell lung cancer (NSCLC) cases either as maintenance therapy or in combination with immunotherapy. However, resistance remains a primary issue. Our findings point to the possibility of exploiting levels of cell division cycle associated protein-3 (CDCA3) to improve response of NSCLC tumours to therapy. We demonstrate that in patients and in vitro analyses, CDCA3 levels correlate with measures of genome instability and platinum sensitivity, whereby CDCA3high tumours are sensitive to cisplatin and carboplatin. In NSCLC, CDCA3 protein levels are regulated by the ubiquitin ligase APC/C and cofactor Cdh1. Here, we identified that the degradation of CDCA3 is modulated by activity of casein kinase 2 (CK2) which promotes an interaction between CDCA3 and Cdh1. Supporting this, pharmacological inhibition of CK2 with CX-4945 disrupts CDCA3 degradation, elevating CDCA3 levels and increasing sensitivity to platinum agents. We propose that combining CK2 inhibitors with platinum-based chemotherapy could enhance platinum efficacy in CDCA3low NSCLC tumours and benefit patients.