Human pulmonary microvascular endothelial cells respond to DAMPs from injured renal tubular cells.

Acute kidney injury (AKI) causes distant organ dysfunction through yet unknown mechanisms, leading to multiorgan failure and death. The lungs are one of the most common extrarenal organs affected by AKI, and combined lung and kidney injury has a mortality as high as 60%-80%. One mechanism that has been implicated in lung injury after AKI involves molecules released from injured kidney cells (DAMPs, or damage-associated molecular patterns) that promote a noninfectious inflammatory response by binding to pattern recognition receptors (PRRs) constitutively expressed on the pulmonary endothelium. To date there are limited data investigating the role of PRRs and DAMPs in the pulmonary endothelial response to AKI. Understanding these mechanisms holds great promise for therapeutics aimed at ameliorating the devastating effects of AKI. In this study, we stimulate primary human microvascular endothelial cells with DAMPs derived from injured primary renal tubular epithelial cells (RTECs) as an ex-vivo model of lung injury following AKI. We show that DAMPs derived from injured RTECs cause activation of Toll-Like Receptor and NOD-Like Receptor signaling pathways as well as increase human primary pulmonary microvascular endothelial cell (HMVEC) cytokine production, cell signaling activation, and permeability. We further show that cytokine production in HMVECs in response to DAMPs derived from RTECs is reduced by the inhibition of NOD1 and NOD2, which may have implications for future therapeutics. This paper adds to our understanding of PRR expression and function in pulmonary HMVECs and provides a foundation for future work aimed at developing therapeutic strategies to prevent lung injury following AKI.

DAMPs Released From Injured Renal Tubular Epithelial Cells Activate Innate Immune Signals in Healthy Renal Tubular Epithelial Cells.

BACKGROUND: Renal ischemia-reperfusion injury (IRI) predictably causes acute kidney injury after shock and major cardiovascular procedures in all kidneys procured for transplantation. The earliest events of IRI are triggered by molecules released from injured cells, damage-associated molecular patterns (DAMPs), that bind pattern recognition receptors (PRRs) constitutively expressed on many cells within the kidney. Activation of PRR signaling leads to production of proinflammatory molecules, which incite a cascade of inflammatory events leading to acute kidney injury. Renal tubular epithelial cells (RTECs) are particularly susceptible to ischemic injury, and proximal RTEC injury is pathognomonic of renal IRI. To better understand how injured RTECs contribute to the cycle of deleterious inflammation in the setting of renal IRI, this study asked whether DAMPs released from injured RTECs induced PRR signals in healthy RTECs. METHODS: Human RTECs were necrosed ex vivo to release intracellular DAMPs and resulting necrotic supernatant used to stimulate healthy RTECs, T lymphocytes, and monocytes. RESULTS: DAMPs released from necrosed RTECs upregulated PRRs known to be associated with renal IRI and activated mitogen-activated protein kinase signaling pathways. Proinflammatory cytokines were upregulated in response to necrotic supernatant, and this upregulation was abrogated by MEK-1 inhibition. The RTEC-derived DAMPs were also potent inducers of T-cell activation/proliferation and monocyte migration. CONCLUSIONS: This is the first study to our knowledge to show that endogenous DAMPs released from injured RTECs directly activate PRR signaling in healthy RTECs. These findings provide new insights directed to therapeutics for renal IRI.

Blockade of T cell activation induced by the simultaneous absence of Nod1 and Nod2 is bypassed by TLR2 signals.

Pattern recognition receptors (PRRs) trigger adaptive inflammatory responses and as such are attractive targets for therapeutic manipulation of inflammation. In order to develop effective therapies however we need to understand the complexities of PRR signaling and clarify how individual PRRs contribute to an inflammatory response in a given cell type. Data from our lab and others have shown that cross-talk occurs between different PRR family members that directs T cell responses to a particular stimuli. It is well-established that the cell surface toll-like receptor 2 (TLR2) provides a potent costimulatory signal for TCR-stimulated T cell activation. We have shown that signaling through the intracellular nucleotide-binding oligomerization domain-containing proteins 1 and 2 (Nod1 and Nod2) also provides important signals for T cell activation, and that when both Nod1 and Nod 2 are deleted stimulated T cells undergo activation-induced cell death. This study found that TLR2 costimulation could bypass the defect induced by the simultaneous absence of Nods1 and 2 in both antibody- and antigen-stimulated T cells. Since blocking one set of PRR-mediated responses can be overcome by signaling through another PRR family member, then effective therapeutic immune blockade strategies will likely require a multi-pronged approach in order to be effective.

TLR2 and NODs1 and 2 cooperate in inflammatory responses associated with renal ischemia reperfusion injury.

Pattern recognition receptors (PRRs) are potent triggers of tissue injury following renal ischemia/reperfusion injury (IRI). Specific PRRs, such as the toll-like receptor 2 (TLR2) and the nucleotide-binding oligomerization domain-like receptors (NLRs) NOD1 and NOD2 are promising targets to abrogate inflammatory injury associated with renal IRI. Several recent reports have shown there is crosstalk between TLRs and NODs, which might boost inflammatory responses to tissue injury. This study examined the relative roles of TLR2 and NODs 1 and 2 in activation of myeloid cells that contribute to inflammation after renal IRI. We found that TLR2 and NOD1 and 2 signaling induces neutrophil, macrophage and dendritic cell migration in vitro, however their blockade only decreases neutrophil infiltration into ischemic kidneys. The results of this study suggest that future therapies targeted to innate immune blockade should consider that either TLR2 or NOD1/2 blockade could decrease neutrophil inflammation following an ischemic insult to the kidney, however blockade of these PRRs would not likely impact infiltration of dendritic cells or macrophages. Developing rational approaches that target innate immunity in IRI-induced acute kidney injury requires an understanding of the relative role of PRRs in directing inflammation in the kidney.

Overcoming Translational Barriers in Acute Kidney Injury: A Report from an NIDDK Workshop.

AKI is a complex clinical condition associated with high mortality, morbidity, and health care costs. Despite improvements in methodology and design of clinical trials, and advances in understanding the underlying pathophysiology of rodent AKI, no pharmacologic agent exists for the prevention or treatment of AKI in humans. To address the barriers that affect successful clinical translation of drug targets identified and validated in preclinical animal models of AKI in this patient population, the National Institute of Diabetes and Digestive and Kidney Diseases convened the "AKI Outcomes: Overcoming Barriers in AKI" workshop on February 10-12, 2015. The workshop used a reverse translational medicine approach to identify steps necessary to achieve clinical success. During the workshop, breakout groups were charged first to design feasible, phase 2, proof-of-concept clinical trials for delayed transplant graft function, prevention of AKI (primary prevention), and treatment of AKI (secondary prevention and recovery). Breakout groups then were responsible for identification of preclinical animal models that would replicate the pathophysiology of the phase 2 proof-of-concept patient population, including primary and secondary end points. Breakout groups identified considerable gaps in knowledge regarding human AKI, our understanding of the pathophysiology of AKI in preclinical animal models, and the fidelity of cellular and molecular targets that have been evaluated preclinically to provide information regarding human AKI of various etiologies. The workshop concluded with attendees defining a new path forward to a better understanding of the etiology, pathology, and pathophysiology of human AKI.

Expression of TLR2, NOD1, and NOD2 and the NLRP3 Inflammasome in Renal Tubular Epithelial Cells of Male versus Female Mice.

BACKGROUND: Gender-biased outcomes are associated with acute kidney injury (AKI) and human and animal studies have shown that females are preferentially protected from renal ischemia. However, the reason for this is not known. One clue might lie with pattern recognition receptors (PRRs), which are triggers of ischemic injury when ligated by molecules in the ischemic milieu. Several PRR families are expressed by renal tubular epithelial cells (RTEs) and incite cell death signaling and production of pro-inflammatory molecules. Blockade of specific PRRs (e.g., TLR2, NOD1, NOD2, and NLRP3) provides highly significant protection from ischemic RTE injury. As a first step to understand gender-biased outcomes of AKI, we tested whether constitutive gender-based differences exist in expression of these PRRS in RTEs. METHODS: To determine whether PRR expression differences exist, primary RTEs isolated from male and female WT kidneys were examined by FACS, qPCR, and Western Blot for expression of TLR2, NOD1, NOD2, and NLRP3 inflammasome components. RESULTS: No RTE gender-based differences in TLR2, NOD1, NOD2, NLRP3, or ASC were found. RTEs from female kidneys had approximately half the mRNA, but the same protein concentration of pro-caspase-1 compared to RTEs isolated from male kidneys. CONCLUSIONS: Our findings indicate that intrinsic gender differences in RTE expression of TLR2, NOD1, NOD2, NLRP3, and ASC are not responsible for the gender-biased outcomes observed in ischemia/reperfusion injury. The lower caspase-1 mRNA expression in RTEs from females warrants further exploration of additional upstream signals that might differentially regulate caspase-1 in male vs. female RTEs.

Lack of Both Nucleotide-Binding Oligomerization Domain-Containing Proteins 1 and 2 Primes T Cells for Activation-Induced Cell Death.

Nucleotide-binding oligomerization domain (Nod)-containing proteins Nod1 and Nod2 play important roles in the innate immune response to pathogenic microbes, but mounting data suggest these pattern recognition receptors might also play key roles in adaptive immune responses. Targeting Nod1 and Nod2 signaling pathways in T cells is likely to provide a new strategy to modify inflammation in a variety of disease states, particularly those that depend on Ag-induced T cell activation. To better understand how Nod1 and Nod2 proteins contribute to adaptive immunity, this study investigated their role in alloantigen-induced T cell activation and asked whether their absence might impact in vivo alloresponses using a severe acute graft versus host disease model. The study provided several important observations. We found that the simultaneous absence of Nod1 and Nod2 primed T cells for activation-induced cell death. T cells from Nod1 × 2-/- mice rapidly underwent cell death upon exposure to alloantigen. The Nod1 × 2-/- T cells had sustained p53 expression that was associated with downregulation of its negative regulator MDM2. In vivo, mice transplanted with an inoculum containing Nod1 × 2-/- T cells were protected from severe graft versus host disease. The results show that the simultaneous absence of Nod1 and Nod2 is associated with accelerated T cell death upon alloantigen encounter, suggesting these proteins might provide new targets to ameliorate T cell responses in a variety of inflammatory states, including those associated with bone marrow or solid organ transplantation.

mTOR Inhibition by Everolimus Does Not Impair Closure of Punch Biopsy Wounds in Renal Transplant Patients.

BACKGROUND: Mammalian target of rapamycin (mTOR) inhibitors are approved to prevent allograft rejection and control malignancy. Unfortunately, they are associated with adverse effects, such as wound healing complications that detract from more extensive use. There is a lack of prospective wound healing studies to monitor patients treated with mTOR inhibitors, such as everolimus or sirolimus, especially in nondiabetics. METHODS: Patients receiving everolimus with standard immunosuppressant therapy or standard immunosuppressant therapy without everolimus were administered 3-mm skin biopsy punch wounds in the left scapular region. Homeostatic gene expression was examined in the skin obtained from the biopsy and wound surface area was examined on day 7. Peripheral blood mononuclear cells were examined for cytokine production. RESULTS: There are no significant changes in autophagy related 13, epidermal growth factor, insulin-like growth factor binding protein 3, IL-2, kruppel-like factor 4, and TGFB1 gene expression in the skin suggesting that there is little impact of everolimus on these genes within nonwounded skin. Peripheral blood T cells are more sensitive to cell death in everolimus-treated patients, but they retain the ability to produce proinflammatory cytokines required for efficient wound repair. Importantly, there is no delay in the closure of biopsy wounds in patients receiving everolimus as compared to those not receiving mTOR inhibition. CONCLUSIONS: Everolimus treatment is not associated with impaired closure of skin biopsy wounds in kidney transplant recipients. These data highlight the importance of exploring whether larger surgical wounds would show a similar result and how other factors, such as diabetes, impact wound healing complications associated with mTOR suppression.

Sex differences in transplantation.

Sex plays a role in the incidence and progression of a wide variety of diseases and conditions related to transplantation. Additionally, a growing body of clinical and experimental evidence suggests that sex can impact the pharmacokinetics and pharmacodynamics of several commonly used immunosuppressive and anti-infective drugs in transplant recipients. A better understanding of these sex differences will facilitate advances in individualizing treatment for patients and improve outcomes of solid organ transplantation. Here, we provide a review of sex-related differences in transplantation and highlight opportunities for future research directions.

The Bacterial Peptidoglycan-Sensing Molecules NOD1 and NOD2 Promote CD8+ Thymocyte Selection.

Nucleotide-binding and oligomerization domain (NOD)-like receptors NOD1 and NOD2 are cytosolic innate immune receptors that recognize microbial peptidoglycans. Although studies have addressed the role of NOD proteins in innate immune responses, little attention has been given to their impact on the developing adaptive immune system. We have assessed the roles of NOD1 and NOD2 deficiency on T cell development in mice. Our results demonstrate that NOD1 and NOD2 promote the positive selection/maturation of CD8 single-positive thymocytes in a thymocyte-intrinsic manner. TCR-mediated ERK phosphorylation is significantly reduced in the absence of NOD proteins, but receptor-interacting protein 2 is not involved in CD8 single-positive thymocyte selection or ERK signaling. Commensal bacteria-free animals have thymocyte maturation defects, and exogenous NOD ligands can enhance thymocyte maturation in culture. These results raise the intriguing possibility that abnormal lymphocyte responses observed in NOD-dependent inflammatory diseases are not driven solely by microbial signals in the gut, but may also involve intrinsic lymphocyte defects resulting from impaired CD8 T cell thymic development.

Pregnancy in chronic kidney disease and kidney transplantation.

Chronic kidney disease (CKD) affects up to 6% of women of childbearing age in high income countries, and is estimated to affect 3% of pregnant women. Advanced renal dysfunction, proteinuria, hypertension, and poorly controlled underlying primary renal disease are all significant risks for adverse maternal, fetal, and renal outcomes. In order to achieve the best outcomes, it is therefore of paramount importance that these pregnancies are planned, where possible, to allow the opportunity to counsel women and their partners in advance and to optimize these risks. These pregnancies should be deemed high risk and they require close antenatal monitoring from an expert multidisciplinary team. We discuss the effect of pregnancy on CKD, and also current guidelines and literature with specific reference to transplantation, autoimmune disease, and medication use in pregnancy. We also discuss the benefits of prepregnancy counseling and give practical recommendations to advise pregnant women with renal disease.

Hematopoietic stem cells and solid organ transplantation.

Solid organ transplantation provides lifesaving therapy for patients with end stage organ disease. In order for the transplanted organ to survive, the recipient must take a lifelong cocktail of immunosuppressive medications that increase the risk for infections, malignancies and drug toxicities. Data from many animal studies have shown that recipients can be made tolerant of their transplanted organ by infusing stem cells, particularly hematopoietic stem cells, prior to the transplant. The animal data have been translated into humans and now several clinical trials have demonstrated that infusion of hematopoietic stem cells, along with specialized conditioning regimens, can permit solid organ allograft survival without immunosuppressive medications. This important therapeutic advance has been made possible by understanding the immunologic mechanisms by which stem cells modify the host immune system, although it must be cautioned that the conditioning regimens are often severe and associated with significant morbidity. This review discusses the role of hematopoietic stem cells in solid organ transplantation, provides an understanding of how these stem cells modify the host immune system and describes how newer information about adaptive and innate immunity might lead to improvements in the use of hematopoietic stem cells to induce tolerance to transplanted organs.

Inflammation in AKI: Current Understanding, Key Questions, and Knowledge Gaps.

Inflammation is a complex biologic response that is essential for eliminating microbial pathogens and repairing tissue after injury. AKI associates with intrarenal and systemic inflammation; thus, improved understanding of the cellular and molecular mechanisms underlying the inflammatory response has high potential for identifying effective therapies to prevent or ameliorate AKI. In the past decade, much knowledge has been generated about the fundamental mechanisms of inflammation. Experimental work in small animal models has revealed many details of the inflammatory response that occurs within the kidney after typical causes of AKI, including insights into the molecular signals released by dying cells, the role of pattern recognition receptors, the diverse subtypes of resident and recruited immune cells, and the phased transition from destructive to reparative inflammation. Although this expansion of the basic knowledge base has increased the number of mechanistically relevant targets of intervention, progress in developing therapies that improve AKI outcomes by modulation of inflammation remains slow. In this article, we summarize the most important recent developments in understanding the inflammatory mechanisms of AKI, highlight key limitations of the commonly used animal models and clinical trial designs that may prevent successful clinical application, and suggest priority approaches for research toward clinical translation in this area.

Ischemia as a factor affecting innate immune responses in kidney transplantation.

PURPOSE OF REVIEW: Ischemic injury inevitably occurs during the procurement of organs for transplantation, and the injury is worsened by inflammation following reperfusion. The purpose of this review is to describe the role of the innate immune system in ischemia-induced renal injury in kidneys procured for transplantation. The key role of pattern recognition receptors in immune responses to ischemia is described. Innate immune receptors are emerging novel targets for the amelioration of ischemic injury of donor kidneys. RECENT FINDINGS: Several families of pattern recognition receptors are direct mediators of early injurious events during kidney procurement, and also innate and adaptive immune responses after transplantation. The deleterious events associated with the activation of the innate immune system in donor kidneys significantly contribute to short and long-term allograft outcomes. SUMMARY: Although a number of therapies have been proposed to decrease ischemic donor kidney injury, targeting the innate immune system is an exciting new area that is gaining significant interest in transplantation. As we learn more about how these important receptors are regulated by ischemia, strategies will likely evolve to allow their modulation in ischemic renal injury.

Exploring principles of hibernation for organ preservation.

Interest in mimicking hibernating states has led investigators to explore the biological mechanisms that permit hibernating mammals to survive for months at extremely low ambient temperatures, with no food or water, and awaken from their hibernation without apparent organ injury. Hibernators have evolved mechanisms to adapt to dramatic reductions in core body temperature and metabolic rate, accompanied by prolonged periods without nutritional intake and at the same time tolerate the metabolic demands of arousal. This review discusses the inherent resilience of hibernators to kidney injury and provides a potential framework for new therapies targeting ex vivo preservation of kidneys for transplantation.

Simultaneous deletion of NOD1 and NOD2 inhibits in vitro alloresponses but does not prevent allograft rejection.

Pattern recognition receptors (PRRs) play an important role in host anti-donor responses to transplanted tissue. A key trigger of the host alloresponse involves recognition of foreign antigen presented on activated antigen presenting cells by the host T cells. Emerging data suggest that PRR blockade can abrogate host anti-donor responses by interfering with activation of antigen presenting cells, particularly activation of dendritic cells. Our study asked whether blockade of a well-characterized family of intracellular PRRs, the NOD family, interfered with alloantigen recognition and allograft rejection. We found that deletion of either NOD1 or NOD2 in antigen presenting cells (APCs) had no effect on induction of T cell proliferation to alloantigen, but that simultaneous deletion of NOD1 and NOD2 significantly inhibited T cell responses. There was however no effect of the NOD deletion on skin graft rejection when NOD1×NOD2 skin was transplanted onto allogeneic hosts or when WT skin was transplanted onto NOD1×NOD2 deficient recipients. The conclusion of this study is that in vitro alloresponses are negatively impacted by the simultaneous deletion of NOD1 and NOD2, but that allograft rejection across a stringent allo barrier is not affected. Our results suggest that the NOD family members, NOD1 and NOD2, play a collaborative role in T cell activation by alloantigen and that their blockade in vitro can inhibit T cell responses.

Regulation of TLR2 and NLRP3 in primary murine renal tubular epithelial cells.

Pattern recognition receptors (PRRs) are now recognized to be key triggers of injury in a variety of renal diseases. Several families of these receptors are present in the kidney, and recent data suggest that they are differentially expressed and regulated in the kidney. This study evaluated the interaction between two distinct PRRs that are expressed in the kidney, i.e. TLR2 (Toll-like receptor 2) and the NLRP3 inflammasome. The regulation and activation of these receptors in primary renal tubular epithelial (RTE) cells from murine kidneys were evaluated. RTE cells were extracted from WT and NLRP3-mutant mice and treated ex vivo with ligands specific for TLR2 or NLRP3. We found that TLR2 upregulated NLRP3 as well as its substrate IL-1ß, and that signaling through the NLRP3 inflammasome induced RTE cell necrosis. The results of this study suggest a previously unknown interaction between TLR2 and NLRP3 in primary RTE cells and highlight the importance of the cross talk that occurs in kidney-related PRRs. Understanding how PRRs are regulated is important for the design of rationale therapeutic strategies to modulate these receptors in renal disease.

Women and transplantation: fertility, sexuality, pregnancy, contraception.

Since 1958, thousands of women with kidney transplants have become pregnant. Although most pregnancies in kidney transplant recipients are successful, they are high-risk endeavors. This seems more a function of the associated issues and comorbidities that often affect individuals with kidney transplants (eg, hypertension) or immunosuppression side effects rather than the kidney transplant per se. Regardless of the underlying pathophysiology, these pregnancies are associated with a high rate of preeclampsia diagnoses, preterm deliveries, Cesarean sections, and small-for-gestational-age babies. Given these risks, it is critical to counsel and inform transplant recipients and prospective transplant recipients of childbearing age and their partners regarding many aspects of pregnancy, including the need for contraception to prevent pregnancy after transplant, immunosuppression concerns, and the potential effect of pregnancy on the outcome of the mother, baby, and kidney transplant.

Innate immunity in donor procurement.

PURPOSE OF REVIEW: Ischaemic kidney injury occurs during organ procurement and can lead to delayed graft function or nonviable grafts. The innate immune system is a key trigger of inflammation in renal ischaemia. This review discusses the components of innate immunity known to be involved in renal ischaemic reperfusion injury (IRI). Understanding how inflammatory damage is initiated in renal IRI is important for the development of targeted therapies aimed at preserving the donor organ. RECENT FINDINGS: Much remains to be determined about the role of innate immune signalling in renal ischaemia/reperfusion injury. Recently, discoveries about complement receptors, Toll-like receptors (TLRs), NOD-like receptors (NLRs) and inflammasomes have opened new avenues of exploration. We are also now learning that macrophages, complement and TLR activation may have additional roles in renal repair following IRI. SUMMARY: A greater understanding of the mechanisms that contribute to innate immune-mediated renal ischaemic damage will allow for the development of therapeutics targeted to the donor organ. New data suggest that treatment limited to specific receptors on specific cells, or localized to specific regions within the kidney, may provide novel approaches to maximize our use of donor organs, particularly those that may have been discarded due to prolonged preimplantation ischaemia.