Human proximal tubular epithelial cell-derived small extracellular vesicles mediate synchronized tubular ferroptosis in hypoxic kidney injury.

Hypoxia is the key pathobiological trigger of tubular oxidative stress and cell death that drives the transition of acute kidney injury (AKI) to chronic kidney disease (CKD). The mitochondrial-rich proximal tubular epithelial cells (PTEC) are uniquely sensitive to hypoxia and thus, are pivotal in propagating the sustained tubular loss of AKI-to-CKD transition. Here, we examined the role of PTEC-derived small extracellular vesicles (sEV) in propagating the 'wave of tubular death'. Ex vivo patient-derived PTEC were cultured under normoxia (21 % O2) and hypoxia (1 % O2) on Transwell inserts for isolation and analysis of sEV secreted from apical versus basolateral PTEC surfaces. Increased numbers of sEV were secreted from the apical surface of hypoxic PTEC compared with normoxic PTEC. No differences in basolateral sEV numbers were observed between culture conditions. Biological pathway analysis of hypoxic-apical sEV cargo identified distinct miRNAs linked with cellular injury pathways. In functional assays, hypoxic-apical sEV selectively induced ferroptotic cell death (↓glutathione peroxidase-4, ↑lipid peroxidation) in autologous PTEC compared with normoxic-apical sEV. The addition of ferroptosis inhibitors, ferrostatin-1 and baicalein, attenuated PTEC ferroptosis. RNAse A pretreatment of hypoxic-apical sEV also abrogated PTEC ferroptosis, demonstrating a role for sEV RNA in ferroptotic 'wave of death' signalling. In line with these in vitro findings, in situ immunolabelling of diagnostic kidney biopsies from AKI patients with clinical progression to CKD (AKI-to-CKD transition) showed evidence of ferroptosis propagation (increased numbers of ACSL4+ PTEC), while urine-derived sEV (usEV) from these 'AKI-to-CKD transition' patients triggered PTEC ferroptosis (↑lipid peroxidation) in functional studies. Our data establish PTEC-derived apical sEV and their intravesicular RNA as mediators of tubular lipid peroxidation and ferroptosis in hypoxic kidney injury. This concept of how tubular pathology is propagated from the initiating insult into a 'wave of death' provides novel therapeutic check-points for targeting AKI-to-CKD transition.

Hypoxic human proximal tubular epithelial cells undergo ferroptosis and elicit an NLRP3 inflammasome response in CD1c+ dendritic cells.

Inflammasomes are multiprotein platforms responsible for the release of pro-inflammatory cytokines interleukin (IL)-1ß and IL-18. Mouse studies have identified inflammasome activation within dendritic cells (DC) as pivotal for driving tubulointerstitial fibrosis and inflammation, the hallmarks of chronic kidney disease (CKD). However, translation of this work to human CKD remains limited. Here, we examined the complex tubular cell death pathways mediating inflammasome activation in human kidney DC and, thus, CKD progression. Ex vivo patient-derived proximal tubular epithelial cells (PTEC) cultured under hypoxic (1% O2) conditions modelling the CKD microenvironment showed characteristics of ferroptotic cell death, including mitochondrial dysfunction, reductions in the lipid repair enzyme glutathione peroxidase 4 (GPX4) and increases in lipid peroxidation by-product 4-hydroxynonenal (4-HNE) compared with normoxic PTEC. The addition of ferroptosis inhibitor, ferrostatin-1, significantly reduced hypoxic PTEC death. Human CD1c+ DC activated in the presence of hypoxic PTEC displayed significantly increased production of inflammasome-dependent cytokines IL-1ß and IL-18. Treatment of co-cultures with VX-765 (caspase-1/4 inhibitor) and MCC950 (NLRP3 inflammasome inhibitor) significantly attenuated IL-1ß/IL-18 levels, supporting an NLRP3 inflammasome-dependent DC response. In line with these in vitro findings, in situ immunolabelling of human fibrotic kidney tissue revealed a significant accumulation of tubulointerstitial CD1c+ DC containing active inflammasome (ASC) specks adjacent to ferroptotic PTEC. These data establish ferroptosis as the primary pattern of PTEC necrosis under the hypoxic conditions of CKD. Moreover, this study identifies NLRP3 inflammasome signalling driven by complex tubulointerstitial PTEC-DC interactions as a key checkpoint for therapeutic targeting in human CKD.

Adenine overload induces ferroptosis in human primary proximal tubular epithelial cells.

The pathogenesis of crystal nephropathy involves deposition of intratubular crystals, tubular obstruction and cell death. The deposition of 8-dihydroxyadenine (DHA) crystals within kidney tubules, for instance, is caused by a hereditary deficiency of adenine phosphoribosyl transferase in humans or adenine overload in preclinical models. However, the downstream pathobiological patterns of tubular cell attrition in adenine/DHA-induced nephropathy remain poorly understood. In this study, we investigated: (i) the modes of adenine-induced tubular cell death in an experimental rat model and in human primary proximal tubular epithelial cells (PTEC); and (ii) the therapeutic effect of the flavonoid baicalein as a novel cell death inhibitor. In a rat model of adenine diet-induced crystal nephropathy, significantly elevated levels of tubular iron deposition and lipid peroxidation (4-hydroxynonenal; 4-HNE) were detected. This phenotype is indicative of ferroptosis, a novel form of regulated necrosis. In cultures of human primary PTEC, adenine overload-induced significantly increased mitochondrial superoxide levels, mitochondrial depolarisation, DNA damage and necrotic cell death compared with untreated PTEC. Molecular interrogation of adenine-stimulated PTEC revealed a significant reduction in the lipid repair enzyme glutathione peroxidase 4 (GPX4) and the significant increase in 4-HNE compared with untreated PTEC, supporting the concept of ferroptotic cell death. Moreover, baicalein treatment inhibited ferroptosis in adenine-stimulated PTEC by selectively modulating the mitochondrial antioxidant enzyme superoxide dismutase 2 (SOD2) and thus, suppressing mitochondrial superoxide production and DNA damage. These data identify ferroptosis as the primary pattern of PTEC necrosis in adenine-induced nephropathy and establish baicalein as a potential therapeutic tool for the clinical management of ferroptosis-associated crystal nephropathies (e.g., DHA nephropathy, oxalate nephropathy).

Molecular and functional profiling of apical versus basolateral small extracellular vesicles derived from primary human proximal tubular epithelial cells under inflammatory conditions.

Proximal tubular epithelial cells (PTEC) are central players in inflammatory kidney diseases. However, the complex signalling mechanism/s via which polarized PTEC mediate disease progression are poorly understood. Small extracellular vesicles (sEV), including exosomes, are recognized as fundamental components of cellular communication and signalling courtesy of their molecular cargo (lipids, microRNA, proteins). In this study, we examined the molecular content and function of sEV secreted from the apical versus basolateral surfaces of polarized human primary PTEC under inflammatory diseased conditions. PTEC were cultured under normal and inflammatory conditions on Transwell inserts to enable separate collection and isolation of apical/basolateral sEV. Significantly increased numbers of apical and basolateral sEV were secreted under inflammatory conditions compared with equivalent normal conditions. Multi-omics analysis revealed distinct molecular profiles (lipids, microRNA, proteins) between inflammatory and normal conditions for both apical and basolateral sEV. Biological pathway analyses of significantly differentially expressed molecules associated apical inflammatory sEV with processes of cell survival and immunological disease, while basolateral inflammatory sEV were linked to pathways of immune cell trafficking and cell-to-cell signalling. In line with this mechanistic concept, functional assays demonstrated significantly increased production of chemokines (monocyte chemoattractant protein-1, interleukin-8) and immuno-regulatory cytokine interleukin-10 by peripheral blood mononuclear cells activated with basolateral sEV derived from inflammatory PTEC. We propose that the distinct molecular composition of sEV released from the apical versus basolateral membranes of human inflammatory PTEC may reflect specialized functional roles, with basolateral-derived sEV pivotal in modulating tubulointerstitial inflammatory responses observed in many immune-mediated kidney diseases. These findings provide a rationale to further evaluate these sEV-mediated inflammatory pathways as targets for biomarker and therapeutic development.

Oxidative stress and inflammasome activation in human rhabdomyolysis-induced acute kidney injury.

Acute kidney injury (AKI) is a life-threatening complication of rhabdomyolysis. The pathophysiological mechanisms of rhabdomyolysis-induced AKI (RIAKI) have been extensively studied in the murine system, yet clinical translation of this knowledge to humans is lacking. In this study, we investigated the cellular and molecular pathways of human RIAKI. Renal biopsy tissue from a RIAKI patient was examined by quantitative immunohistochemistry (Q-IHC) and compared to healthy kidney cortical tissue. We identified myoglobin casts and uric acid localised to sites of histological tubular injury, consistent with the diagnosis of RIAKI. These pathological features were associated with tubular oxidative stress (4-hydroxynonenal staining), regulated necrosis/necroptosis (phosphorylated mixed-lineage kinase domain-like protein staining) and inflammation (tumour necrosis factor (TNF)-α staining). Expression of these markers was significantly elevated in the RIAKI tissue compared to the healthy control. A tubulointerstitial inflammatory infiltrate accumulated adjacent to these sites of RIAKI oxidative injury, consisting of macrophages (CD68), dendritic cells (CD1c) and T lymphocytes (CD3). Foci of inflammasome activation were co-localised with these immune cell infiltrate, with significantly increased staining for adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase activation and recruitment domain) and active caspase-1 in the RIAKI tissue compared to the healthy control. Our clinical findings identify multiple pathophysiological pathways previously only reported in murine RIAKI, providing first evidence in humans linking deposition of myoglobin and presence of uric acid to tubular oxidative stress/necroptosis, inflammasome activation and necroinflammation.

Role of inflammation and inflammasome activation in human bile cast nephropathy.

Bile cast nephropathy (BCN) is an underdiagnosed cause of acute kidney injury (AKI). The precise pathogenesis of bilirubin tubular toxicity remains unknown. The aim of this study is to explore the cellular and molecular pathophysiology of human BCN. Paraffin-embedded sections of renal biopsy tissue from a BCN patient were stained by immunohistochemistry (IHC) for oxidative stress (4-hydroxynonenal), immune cell subpopulations, including dendritic cells (CD1c), macrophages (CD68) and T cells (CD3), and inflammasome activation by staining for active-caspase-1 and the inflammasome adaptor protein, ASC (apoptosis-associated speck-like protein containing a caspase activation and recruitment domain). Quantitative analyses of IHC staining were compared to healthy renal cortical tissue. We identified yellow to brown granular casts within the BCN case, consistent with the presence of bile pigment. The presence of bile pigment was associated with strong tubular 4-hydroxynonenal staining intensity, a marker of oxidative stress. Diffuse tubulointerstitial inflammatory cell infiltrate was detected, with elevated CD1c, CD68 and CD3 staining. Foci of inflammasome activity were co-localized with this intense immune cell infiltration, with increased active-caspase-1 and ASC staining. Our findings are the first to suggest that bile casts may lead to oxidative stress and trigger the inflammasome signalling cascade, leading to interstitial inflammation and driving AKI pathobiology. SUMMARY AT A GLANCE The report suggests that bile casts may lead to oxidative stress and trigger the inflammasome signalling cascade, leading to interstitial inflammation and driving bile cast nephropathy pathobiology.

Underlying Histopathology Determines Response to Oxidative Stress in Cultured Human Primary Proximal Tubular Epithelial Cells.

Proximal tubular epithelial cells (PTEC) are key players in the progression of kidney diseases. PTEC studies to date have primarily used mouse models and transformed human PTEC lines. However, the translatability of these models to human kidney disease has been questioned. In this study, we investigated the phenotypic and functional response of human primary PTEC to oxidative stress, an established driver of kidney disease. Furthermore, we examined the functional contribution of the underlying histopathology of the cortical tissue used to generate our PTEC. We demonstrated that human primary PTEC from both histologically 'normal' and 'diseased' cortical tissue responded to H2O2-induced oxidative stress with significantly elevated mitochondrial superoxide levels, DNA damage, and significantly decreased proliferation. The functional response of 'normal' PTEC to oxidative stress mirrored the reported pathogenesis of human kidney disease, with significantly attenuated mitochondrial function and increased cell death. In contrast, 'diseased' PTEC were functionally resistant to oxidative stress, with maintenance of mitochondrial function and cell viability. This selective survival of 'diseased' PTEC under oxidizing conditions is reminiscent of the in vivo persistence of maladaptive PTEC following kidney injury. We are now exploring the impact that these differential PTEC responses have in the therapeutic targeting of oxidative stress pathways.

Human Tissue-Resident Mucosal-Associated Invariant T (MAIT) Cells in Renal Fibrosis and CKD.

BACKGROUND: Mucosal-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. METHODS: To 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. RESULTS: We 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. CONCLUSIONS: Our findings indicate that human tissue-resident MAIT cells in the kidney may contribute to the fibrotic process of CKD via complex interactions with PTECs.

Interferon-γ production by tubulointerstitial human CD56bright natural killer cells contributes to renal fibrosis and chronic kidney disease progression.

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 (CD3-CD56+) 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 CD56dim NK cell subset and particularly the CD56bright NK cell subset were elevated in fibrotic kidney tissue. However, only CD56bright NK cells significantly correlated with the loss of kidney function. Expression of the tissue-retention and -activation molecule CD69 on CD56bright 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 CD56bright NK cells. Multi-color immunofluorescent staining of fibrotic kidney tissue localized the accumulation of NK cells within the tubulointerstitium, with CD56bright NK cells (NKp46+ CD117+) identified as the source of pro-inflammatory cytokine interferon-γ within the NK cell compartment. Thus, activated interferon-γ-producing CD56bright NK cells are positioned to play a key role in the fibrotic process and progression to chronic kidney disease.

Human proximal tubule epithelial cells modulate autologous B-cell function.

BACKGROUND: Descriptions of inflammatory cells infiltrating the human kidney rarely mention B cells, other than in the specific scenario of transplantation. In these reports, B cells are localized almost exclusively within the kidney tubulointerstitium where they are ideally placed to interact with proximal tubule epithelial cells (PTEC). We have previously shown that activated PTEC down-modulate autologous T lymphocyte and dendritic cell function. In this report, we extend these prior studies to describe PTEC-B cell interactions. METHODS: Stimulated B cells were cultured in the absence or presence of activated autologous human PTEC and monitored for proliferation, surface antigen expression, cytokine secretion and antibody (Ab) production. RESULTS: PTEC decreased B cell proliferative responses, whilst B cells cultured in the presence of PTEC displayed decreased levels of CD27, a marker of plasma B cells and memory cells. Interestingly, autologous PTEC also significantly decreased the number of B cells secreting both IgG and IgM and overall levels of Ab production. Transwell studies demonstrated that this modulation was primarily contact-dependent, and blocking studies with anti-PD-L1 led to partial restoration in Ab production. Further blocking studies targeting soluble HLA-G (sHLA-G) and IDO, two other immunoinhibitory molecules also up-regulated in our activated PTEC, demonstrated minor restoration of Ab responses. DISCUSSION: We report, for the first time, that PTEC are also able to modulate autologous B-cell phenotype and function via complex contact-dependent (PD-L1), soluble (sHLA-G) and intracellular (IDO) factors. We hypothesize that such mechanisms may have evolved to maintain peripheral immune-homeostasis, especially within the inflammatory milieu that exists within many kidney diseases.

Fractalkine-CX3CR1-dependent recruitment and retention of human CD1c+ myeloid dendritic cells by in vitro-activated proximal tubular epithelial cells.

Chemokines play pivotal roles in tissue recruitment and retention of leukocytes, with CX3CR1 recently identified as a chemokine receptor that selectively targets mouse kidney dendritic cells (DCs). We have previously demonstrated increased tubulointerstitial recruitment of human transforming growth factor-ß (TGF-ß)-producing DCs in renal fibrosis and chronic kidney disease (CKD). However, little is known about the mechanism of human DC recruitment and retention within the renal interstitium. We identified CD1c+ DCs as the predominant source of profibrotic TGF-ß and highest expressors of the fractalkine receptor CX3CR1 within the renal DC compartment. Immunohistochemical analysis of diseased human kidney biopsies showed colocalization of CD1c+ DCs with fractalkine-positive proximal tubular epithelial cells (PTECs). Human primary PTEC activation with interferon-γ and tumor necrosis factor-α induced both secreted and surface fractalkine expression. In line with this, we found fractalkine-dependent chemotaxis of CD1c+ DCs to supernatant from activated PTECs. Finally, in comparison with unactivated PTECs, we showed significantly increased adhesion of CD1c+ DCs to activated PTECs via a fractalkine-dependent mechanism. Thus, TGF-ß-producing CD1c+ DCs are recruited and retained in the renal tubulointerstitium by PTEC-derived fractalkine. These cells are then positioned to play a role in the development of fibrosis and progression of chronic kidney disease.

Laser capture microdissection and multiplex-tandem PCR analysis of proximal tubular epithelial cell signaling in human kidney disease.

Interstitial fibrosis, a histological process common to many kidney diseases, is the precursor state to end stage kidney disease, a devastating and costly outcome for the patient and the health system. Fibrosis is historically associated with chronic kidney disease (CKD) but emerging evidence is now linking many forms of acute kidney disease (AKD) with the development of CKD. Indeed, we and others have observed at least some degree of fibrosis in up to 50% of clinically defined cases of AKD. Epithelial cells of the proximal tubule (PTEC) are central in the development of kidney interstitial fibrosis. We combine the novel techniques of laser capture microdissection and multiplex-tandem PCR to identify and quantitate "real time" gene transcription profiles of purified PTEC isolated from human kidney biopsies that describe signaling pathways associated with this pathological fibrotic process. Our results: (i) confirm previous in-vitro and animal model studies; kidney injury molecule-1 is up-regulated in patients with acute tubular injury, inflammation, neutrophil infiltration and a range of chronic disease diagnoses, (ii) provide data to inform treatment; complement component 3 expression correlates with inflammation and acute tubular injury, (iii) identify potential new biomarkers; proline 4-hydroxylase transcription is down-regulated and vimentin is up-regulated across kidney diseases, (iv) describe previously unrecognized feedback mechanisms within PTEC; Smad-3 is down-regulated in many kidney diseases suggesting a possible negative feedback loop for TGF-ß in the disease state, whilst tight junction protein-1 is up-regulated in many kidney diseases, suggesting feedback interactions with vimentin expression. These data demonstrate that the combined techniques of laser capture microdissection and multiplex-tandem PCR have the power to study molecular signaling within single cell populations derived from clinically sourced tissue.

Increased tubulointerstitial recruitment of human CD141(hi) CLEC9A(+) and CD1c(+) myeloid dendritic cell subsets in renal fibrosis and chronic kidney disease.

Dendritic cells (DCs) play critical roles in immune-mediated kidney diseases. Little is known, however, about DC subsets in human chronic kidney disease, with previous studies restricted to a limited set of pathologies and to using immunohistochemical methods. In this study, we developed novel protocols for extracting renal DC subsets from diseased human kidneys and identified, enumerated, and phenotyped them by multicolor flow cytometry. We detected significantly greater numbers of total DCs as well as CD141(hi) and CD1c(+) myeloid DC (mDCs) subsets in diseased biopsies with interstitial fibrosis than diseased biopsies without fibrosis or healthy kidney tissue. In contrast, plasmacytoid DC numbers were significantly higher in the fibrotic group compared with healthy tissue only. Numbers of all DC subsets correlated with loss of kidney function, recorded as estimated glomerular filtration rate. CD141(hi) DCs expressed C-type lectin domain family 9 member A (CLEC9A), whereas the majority of CD1c(+) DCs lacked the expression of CD1a and DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), suggesting these mDC subsets may be circulating CD141(hi) and CD1c(+) blood DCs infiltrating kidney tissue. Our analysis revealed CLEC9A(+) and CD1c(+) cells were restricted to the tubulointerstitium. Notably, DC expression of the costimulatory and maturation molecule CD86 was significantly increased in both diseased cohorts compared with healthy tissue. Transforming growth factor-ß levels in dissociated tissue supernatants were significantly elevated in diseased biopsies with fibrosis compared with nonfibrotic biopsies, with mDCs identified as a major source of this profibrotic cytokine. Collectively, our data indicate that activated mDC subsets, likely recruited into the tubulointerstitium, are positioned to play a role in the development of fibrosis and, thus, progression to chronic kidney disease.

Human proximal tubule epithelial cells modulate autologous dendritic cell function.

BACKGROUND: We have previously demonstrated that human kidney proximal tubule epithelial cells (PTEC) are able to modulate autologous T and B lymphocyte responses. It is well established that dendritic cells (DC) are responsible for the initiation and direction of adaptive immune responses and that these cells occur in the renal interstitium in close apposition to PTEC under inflammatory disease settings. However, there is no information regarding the interaction of PTEC with DC in an autologous human context. METHODS: Human monocytes were differentiated into monocyte-derived DC (MoDC) in the absence or presence of primary autologous activated PTEC and matured with polyinosinic:polycytidylic acid [poly(I:C)], while purified, pre-formed myeloid blood DC (CD1c(+) BDC) were cultured with autologous activated PTEC in the absence or presence of poly(I:C) stimulation. DC responses were monitored by surface antigen expression, cytokine secretion, antigen uptake capacity and allogeneic T-cell-stimulatory ability. RESULTS: The presence of autologous activated PTEC inhibited the differentiation of monocytes to MoDC. Furthermore, MoDC differentiated in the presence of PTEC displayed an immature surface phenotype, efficient phagocytic capacity and, upon poly(I:C) stimulation, secreted low levels of pro-inflammatory cytokine interleukin (IL)-12p70, high levels of anti-inflammatory cytokine IL-10 and induced weak Th1 responses. Similarly, pre-formed CD1c(+) BDC matured in the presence of PTEC exhibited an immature tolerogenic surface phenotype, strong endocytic and phagocytic ability and stimulated significantly attenuated T-cell proliferative responses. CONCLUSIONS: Our data suggest that activated PTEC regulate human autologous immunity via complex interactions with DC. The ability of PTEC to modulate autologous DC function has important implications for the dampening of pro-inflammatory immune responses within the tubulointerstitium in renal injuries. Further dissection of the mechanisms of PTEC modulation of autologous immune responses may offer targets for therapeutic intervention in renal medicine.

Activated human renal tubular cells inhibit autologous immune responses.

BACKGROUND: Renal proximal tubule epithelial cells (PTEC) respond and contribute to the pathological process in a range of kidney diseases. Within this disease setting, PTEC up-regulate surface antigens which may enable them to act as non-professional antigen-presenting cells and become targets for infiltrating T cells in the context of disease and allograft rejection. In order to define, for the first time, whether PTEC modulate immune responses within the autologous human system, we monitored their interaction with T and B cells in the presence of stimuli which mimic immunological signalling. METHODS: The expression of PTEC surface antigen in response to inflammatory mediators was monitored by flow cytometry. Purified T and B lymphocyte subsets and peripheral blood mononuclear cells were cultured in the presence or absence of autologous activated PTEC, and their responses to specific activators were monitored by proliferation, cytokine secretion and surface antigen expression. Some experiments were performed in the presence of blocking antibodies to PD-L1. RESULTS: The presence of activated primary autologous PTEC resulted in significantly decreased T- and B-cell proliferative responses, which were only partly mediated by programmed death ligand 1. This modulation was not induced by a decrease in activation markers or an increase in T regulatory cells but was accompanied by strong significant skewing of cytokine profiles. Significant decreases in gamma-interferon, interleukin-2 and tumour necrosis factor and increases in interleukin-4 were detected in the presence of PTEC, indicating that these cells induce a shift away from an inflammatory Th1 effector profile to a Th2 type profile. CONCLUSION: Human PTEC do modulate autologous immune responses. We hypothesize that such mechanisms may have developed to help dampen inflammatory responses and macrophage activation seen within kidney interstitium in many immune-mediated kidney diseases.