TGF expression and macrophage accumulation in atherosclerotic renal artery stenosis.

BACKGROUND AND OBJECTIVES: Atherosclerotic renal artery stenosis (ARAS) reduces renal blood flow and is a potential cause of chronic kidney injury, yet little is known regarding inflammatory pathways in this disorder in human participants. This study aimed to examine the hypothesis that reduced renal blood flow (RBF) in ARAS would be associated with tissue TGF-ß activation and inflammatory cell accumulation. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This cross-sectional study of ARAS of varying severity compared transjugular biopsy specimens in patients with ARAS (n=12, recruited between 2008 and 2012) with tissue from healthy kidney donors (n=15) and nephrectomy specimens from individuals with total vascular occlusion (n=65). ARAS patients were studied under controlled conditions to measure RBF by multidetector computed tomography and tissue oxygenation by blood oxygen level-dependent magnetic resonance imaging. RESULTS: Compared with the nonstenotic contralateral kidneys, RBF was reduced in poststenotic kidneys (242±149 versus 365+174 ml/min; P<0.01) as was single-kidney GFR (28±17 versus 41±19 ml/min; P<0.01), whereas cortical and medullary oxygenation were relatively preserved. Tissue TGF-ß immunoreactivity was higher in ARAS patients compared with those with both normal kidneys and those with total occlusion (mean score 2.4±0.7 versus 1.5+1.1 in the nephrectomy group and versus 0±0 in donors; P<0.01). By contrast, the number of CD68+ macrophages was higher with greater disease severity (from 2.2±2.7 in normal to 22.4±18 cells/high-power field in nephrectomy samples; P<0.001). CONCLUSIONS: The results of this study indicate robust stimulation of TGF-ß associated with macrophage infiltration within the human kidney with vascular occlusive disease.

Interpreting NK cell transcripts versus T cell transcripts in renal transplant biopsies.

NK cell transcripts are increased in biopsies with antibody-mediated rejection, whereas T cell transcripts are increased in T cell-mediated rejection. However, NK and T cells share many features, creating potential ambiguity. Therefore to estimate the NK- versus T cell transcript burdens separately, we defined nonoverlapping transcripts selective for NK cells (N = 4) or T cells (N = 5). We compared NK- versus T cell transcript burdens in microarrays from 403 kidney transplant biopsies (182 early, 221 late). In late biopsies, high NK-cell transcript expression was associated with antibody-mediated rejection, correlating with microvascular inflammation and donor specific HLA antibody. However, some early biopsies with T cell-mediated rejection had high NK-cell transcript expression, as well as T cell transcripts, without evidence of antibody-mediated rejection or DSA, correlating with interstitial inflammation and tubulitis. Both NK-cell and T cell transcripts were moderately increased in many kidneys with inflammation secondary to injury or atrophy scarring. These results support the distinct role of NK cells in late antibody-mediated rejection, but indicate a role for NK-transcript expressing cells (NK cells or T cells with NK features) both in T cell-mediated rejection and in inflammation associated with injury and atrophy scarring.

Immunotherapy for acute kidney injury.

Acute renal failure, now referred to as acute kidney injury, is a common and clinically important problem. Acute kidney injury frequently occurs as a result of acute tubular necrosis (ATN), which is often caused by a reduction in systemic blood pressure or renal blood flow (e.g., as observed in severe sepsis or during renal transplantation). The disease course in ATN is variable, including prolonged dialysis-dependence and chronic renal dysfunction, but there is currently no specific therapy for ATN. There is increasing evidence that the inflammatory response in ATN significantly contributes to disease severity and outcome. In this review, we summarize recent developments in the understanding of how the immune system responds to dying cells, and the relevance of these discoveries to ATN. In particular, NLRP3 inflammasome activation and IL-1ß-mediated neutrophil recruitment are likely to play a key role and may provide novel therapeutic targets for immunotherapy in ATN.

A new diagnostic algorithm for antibody-mediated microcirculation inflammation in kidney transplants.

We studied the significance of microcirculation inflammation in kidney transplants, including 329 indication biopsies from 251 renal allograft recipients, who were mostly nonpresensitized (crossmatch negative). Glomerulitis (g) and peritubular capillaritis (ptc) were often associated with antibody-mediated rejection (65% and 75%, respectively), but were also found in other diseases in the absence of donor-specific antibody (DSA): T-cell-mediated rejection (ptc, g), glomerulonephritis (g) and acute tubular necrosis (ptc). To develop rules for reducing the nonspecificity of microcirculation inflammation and defining the best grading thresholds associated with DSA, we built and validated a decision tree to predict DSA. The decision tree revealed that g + ptc sum (addition of g-score plus ptc-score) was the best predictor of DSA, followed by time posttransplant, then C4d, which had a small role. Late biopsies with g + ptc > 0 showed higher frequency of DSA compared to early biopsies with g + ptc > 0 (79% vs. 27%). Microcirculation inflammation in early biopsies was often false positive (antibody-independent). The decision tree predicted DSA with higher sensitivity and accuracy than C4d staining. Microcirculation inflammation sum score predicted graft failure independently of time, C4d and transplant glomerulopathy. Thus any degree of microcirculation inflammation in late kidney transplant biopsies strongly indicates presence of DSA and predicts progression to graft failure.

Autoantibody against caspase-3, an executioner of apoptosis, in patients with systemic sclerosis.

The objective of the study was to determine the presence or levels of antibodies (Abs) against caspase-3 and their clinical relevance in systemic sclerosis (SSc). Anti-caspase-3 Ab was examined by enzyme-linked immunosorbent assay and immunoblotting. IgG anti-caspase-3 Ab levels in SSc patients were higher than in normal controls. SSc patients positive for IgG anti-caspase-3 Ab had significantly longer disease duration, more frequent presence of decreased %VC and %DLco, and elevated levels of serum immunoglobulin and erythrocyte sedimentation rates. IgG anti-caspase-3 Ab levels correlated positively with serum IgG levels, renal vascular resistance, and serum levels of 8-isoprostane. Immunoblotting analysis confirmed the presence of anti-caspase-3 Ab in sera from SSc patients. Caspase-3 enzymatic activity was inhibited by IgG isolated from SSc sera containing IgG anti-caspase-3 Ab. These results suggest that autoantibody against caspase-3 is generated in SSc and that this Ab is related to the severity of pulmonary fibrosis, vascular damage, and inflammation.

Antibody-mediated microcirculation injury is the major cause of late kidney transplant failure.

We studied the phenotype of late kidney graft failure in a prospective study of unselected kidney transplant biopsies taken for clinical indications. We analyzed histopathology, HLA antibodies and death-censored graft survival in 234 consecutive biopsies from 173 patients, taken 6 days to 31 years posttransplant. Patients with late biopsies (>1 year) frequently displayed donor-specific HLA antibody (particularly class II) and microcirculation changes, including glomerulitis, glomerulopathy, capillaritis, capillary multilayering and C4d staining. Grafts biopsied early rarely failed (1/68), whereas grafts biopsied late often progressed to failure (27/105) within 3 years. T-cell-mediated rejection and its lesions were not associated with an increased risk of failure after biopsy. In multivariable analysis, graft failure correlated with microcirculation inflammation and scarring, but C4d staining was not significant. When microcirculation changes and HLA antibody were used to define antibody-mediated rejection, 17/27 (63%) of late kidney failures after biopsy were attributable to antibody-mediated rejection, but many were C4d negative and missed by current diagnostic criteria. Glomerulonephritis accounted for 6/27 late losses, whereas T-cell-mediated rejection, drug toxicity and unexplained scarring were uncommon. The major cause of late kidney transplant failure is antibody-mediated microcirculation injury, but detection of this phenotype requires new diagnostic criteria.

De novo donor-specific antibody at the time of kidney transplant biopsy associates with microvascular pathology and late graft failure.

We studied whether de novo donor-specific antibodies (DSA) in sera from patients undergoing kidney transplant biopsies associate with specific histologic lesions in the biopsy and prognosis. DSA were assessed in 145 patients at the time of biopsy between 7 days to 31 years posttransplant. DSA was detected in 54 patients (37%), of which 32 represented de novo DSA. De novo DSA was more frequent in patients having late biopsies (34%) versus early biopsies (4%), and was usually either against class II alone or class I and II but rarely against class I alone. Microcirculation inflammation (glomerulitis, capillaritis) and damage (glomuerulopathy, capillary basement membrane multilayering), and C4d staining were associated with de novo DSA. However, the degree of scarring, arterial fibrosis and tubulo-interstitial inflammation did not correlate with the presence of de novo DSA. De novo DSA correlated with reduced graft survival after the biopsy. Thus, de novo DSA at the time of a late biopsy for clinical indication is primarily against class II, and associates with microcirculation changes in the biopsy and subsequent graft failure. We propose careful assessment of de novo DSA, particularly against class II, be performed in all late kidney transplant biopsies.

Identification and characterization of major proteins carrying ABO blood group antigens in the human kidney.

BACKGROUND: It is generally admitted that ABO(H) blood group antigens are linked to lipids and proteins. Although glycolipids carrying ABO antigens have been well characterized in human kidneys, glycoproteins carrying ABO antigens are largely unknown, and their molecular properties remain to be elucidated. METHODS: All the blood group A antigen-linked proteins in human kidney could be solubilized and captured on immobilized Helix pomatia lectin that recognizes A antigens. These proteins were separated on SDS-PAGE gels. The gel pieces containing protein bands immunoreactive with anti-A antibody were excised, in-gel digested with trypsin, and analyzed by nanoLC tandem mass spectrometer. Protein candidates that carry ABO antigens were confirmed by immunoprecipitation and double-labeled immunofluorescense microscopy. RESULTS: All the glycoproteins carrying ABO antigens were found to be Asn-linked glycoproteins, and presented as multiple bands on SDS-PAGE with molecular masses ranging from 60 to 270 kDa. The protein bands were subjected for mass spectrometric analysis, which identified 121 distinct proteins with high confidence. Of the identified proteins, 55 N-glycosylated, membrane proteins were selected as glycoprotein candidates that carry ABO antigens. Among them, most abundantly expressed proteins as estimated by the number of peptide matches in the MS spectrometric analysis, such as platelet endothelial cell adhesion molecule 1, plasmalemmal vesicle-associated protein, and von Willebrand factor, were further characterized. CONCLUSIONS: Several glycoproteins were identified that represented major glycoproteins carrying ABO antigens in the human kidney, which exhibited distinct features in localization to most of vascular endothelial cells.

Mechanisms of ANCA-mediated leukocyte-endothelial cell interactions in vivo.

Anti-myeloperoxidase (anti-MPO) antibodies have been implicated in the pathogenesis of small-vessel vasculitis, but the molecular mechanisms by which these antibodies contribute to disease are unknown. For determination of how anti-MPO antibodies affect inflammatory cell recruitment in small-vessel vasculitis, intravital microscopy was used to monitor leukocyte behavior in the accessible cremasteric microvessels under various experimental conditions. After local pretreatment of the cremaster muscle with cytokines (TNF-alpha, IL-1beta, or keratinocyte-derived chemokine), administration of anti-MPO IgG to wild-type mice reduced leukocyte rolling in favor of augmented adhesion to and transmigration across the endothelium. This led to a decrease in the number of systemic circulating leukocytes and, similar to the early events in the development of vasculitic lesions, an increase in leukocyte recruitment to renal and pulmonary tissue. TNF-alpha led to the greatest recruitment of inflammatory cells, and IL-1beta led to the least. When anti-CD18 was co-administered, anti-MPO IgG did not affect leukocyte rolling, adhesion, or transmigration; similarly, anti-MPO IgG did not produce these effects in Fc receptor gamma chain-/- mice. This study provides direct in vivo evidence of enhanced leukocyte-endothelial cell interactions in the presence of anti-MPO IgG and highlights the critical roles of Fcgamma receptors and beta2 integrins in mediating these interactions. In addition, it suggests that neutrophils primed by cytokines in the presence of anti-MPO IgG can have systemic effects and target specific vascular beds.

Genomic profiling of kidney ischemia-reperfusion reveals expression of specific alloimmunity-associated genes: Linking "immune" and "nonimmune" injury events.

Increased organ ischemia time leads to delayed graft function (DGF), increased acute rejection (AR), enhanced chronic allograft nephropathy (CAN), and reduced long-term allograft survival. The mechanisms by which IRI predisposes to AR and CAN are unknown. We hypothesized that gene expression profiling of ischemia-reperfusion injury (IRI)-affected kidney would identify how IRI predisposes to AR and CAN. Furthermore, we examined how current immunosuppressive drug molecular targets are altered by IRI. C57BL/6J mice were exposed to 30 (n = 3) or 60 (n = 3) minutes of bilateral kidney ischemia or sham surgery (n = 5). At 36 hour kidney tissue was collected and analyzed using Affymetrix 430MOEA (22626 genes) array and GC-RMA-SAM pipeline. Genes with the false discovery rate (q < 1%) and +/-50% fold change (FC) were considered affected by IRI. Genes coding for histocompatibility and antigen-presenting factors, calcineurin, and mammalian target of rapamycin (mTOR) pathway-associated proteins were selected using Gene Ontology (GO) analysis. GO analysis identified 10 and 17 alloimmunity-related genes affected by IRI induced by 30 and 60 minutes of ischemia, respectively, including Traf6 (FC = 2.99) and H2-D1 (FC = 2.58). We also detected significant IRI genomic responses in calcineurin and mTOR pathways represented by Fkbp5 (FC = 4.18) and Fkbp1a (FC = 2.0), and Eif4ebp1 (FC = 16.8) and Akt1 (FC = 3.64), respectively. These data demonstrated that IRI up-regulates expression of several alloimmunity-associated genes, which can in turn enhance alloimune responses. Our discovery of IRI-induced up-regulation of genes associated with calcineurin and mTOR pathways are consistent with clinical observations that FK506 and Rapamycin can alter the course of DGF. Further validation and dissection of these pathways can lead to novel approaches by which improved management of early "nonimmune" transplant events can decrease susceptibility to more classic "immune" changes and CAN.

Deficiency of 5-lipoxygenase abolishes sex-related survival differences in MRL-lpr/lpr mice.

Leukotrienes, the 5-lipoxygenase (5LO) products of arachidonic acid metabolism, have many proinflammatory actions that have been implicated in the pathogenesis of a variety of inflammatory diseases. To investigate the role of LTs in autoimmune disease, we generated an MRL-lpr/lpr mouse line with a targeted disruption of the 5lo gene. MRL-lpr/lpr mice spontaneously develop autoimmune disease that has many features resembling human systemic lupus erythematosus, including sex-related survival differences; female MRL-lpr/lpr mice experience significant early mortality compared with males. Unexpectedly, we found that mortality was accelerated in male 5LO-deficient MRL-lpr/lpr mice compared with male wild-type MRL-lpr/lpr animals. In contrast, the 5lo mutation had no effect on survival in females. Mortality was also accelerated in male MRL-lpr/lpr mice that were treated chronically with a pharmacological inhibitor of LT synthesis. Furthermore, LT-dependent inflammatory responses are enhanced in male MRL-lpr/lpr mice compared with females, and the 5lo mutation has greater impact on these responses in males. Because immune complex-mediated glomerulonephritis is the major cause of death in MRL-lpr/lpr mice and has been related to arachidonic acid metabolites, we also assessed kidney function and histopathology. In male MRL-lpr/lpr mice, renal plasma flow was significantly reduced in the 5lo-/- compared with the 5lo+/+ group, although there were no differences in the severity of renal histopathology, lymphoid hyperplasia, or arthritis between the groups. These findings suggest that the presence of a functional 5lo gene confers a survival advantage on male MRL-lpr/lpr mice and that, when 5LO function is inhibited, either genetically or pharmacologically, this advantage is abolished.

Expression of the polymeric immunoglobulin receptor and excretion of secretory IgA in the postischemic kidney.

The humoral mucosal immune response of the kidney involves the transport of secretory IgA (S-IgA) through renal epithelial cells by the polymeric immunoglobulin receptor (pIgR). The pIgR is cleaved and released as free secretory component (FSC) or attached to IgA (S-IgA). We examined the effects of an ischemic model of acute renal failure (ARF) on the expression of pIgR and the secretion of FSC and S-IgA in the urine. Kidney pIgR mRNA levels decreased in ischemic animals by 55% at 4 h and by 85% at 72 h compared with controls. pIgR protein expression in the medullary thick ascending limb (TAL) decreased within 24 h and was nearly undetectable by 72 h. Urinary S-IgA and FSC concentrations decreased by 60% between days 3 and 6. pIgR mRNA and pIgR protein in the kidney returned to approximately 90% of control levels and urinary FSC and S-IgA concentrations returned to approximately 55% of control levels by day 7. We demonstrate that ischemic ARF decreases renal mucosal S-IgA transport in vivo and may contribute to the increased incidence of urinary tract infections.

Influence of complement on the allospecific antibody response to a primary vascularized organ graft.

The induction of antibody responses against T cell-dependent antigens has been reported to be influenced by complement. We therefore asked if the primary induction of alloantibodies against transplantation antigens, an important determinant of transplant outcome, is complement sensitive and whether this has functional implications. We transplanted rat kidney allografts into fully major histocompatibility complex-mismatched recipients, in which complement activation was inhibited by daily injection of soluble recombinant human complement receptor type 1 (sCR1). Control allograft recipients were injected with saline. Animals in the control group showed a marked antibody response against donor-specific antigens and an increase in the proportion of activated B and T splenocytes by day 5 after transplantation. Complement-inhibited rats showed a reduced level of antibody binding on target cells sharing the same histocompatibility antigens as the donor strain (p < 0.001), and a reduced level of activated splenic B (p < 0.01) and T (p < 0.01) cells. In a functional assay, the plasma of complement-inhibited rats showed reduced cytotoxic activity against donor-specific cells, and their grafts contained less bound antibody than controls. Analysis beyond 6 days was obscured due to the development of antibodies against sCR1. We conclude that complement activation facilitates the induction of the alloantibody response. Sparing of vascular injury and prolongation of graft survival, previously reported in complement-inhibited rats (Pratt J. R. et al., Am. J. Path. 1996, 149: 2055), could therefore be due to down-regulation of the B cell response as well as reduced complement-dependent cytotoxicity. Inhibition of complement may provide an ancillary approach to the prevention of allospecific antibody formation and the prolongation of allograft survival in primary kidney grafting.

Multivariate and Boolean factor analysis of immune complex/complement deposits and their effects on renal blood flow during allograft rejection.

The role of humoral immune factors in graft destruction is not fully understood. With immunofluorescence techniques the possibility of a specific pattern and/or clustering of immune complex or complement deposits was analyzed in 140 percutaneous kidney needle biopsies performed in 73 patients with renal allograft dysfunction. The results were correlated with concomitant alterations in renal blood flow as measured by cortical and global perfusion indexes and graft survival. The deposition of IgG, IgM, C3 and C4 correlated significantly with acute rejection confirmed by biopsy (p less than 0.05, less than 0.001, less than 0.02 and less than 0.001, respectively). Subsequent graft survival was compromised when IgA, IgG, IgM, C3, C4 and properdin were present together in biopsy specimens (p less than 0.05). There was a significant clustering of IgA with C3, of IgG with C3 and C4, and of IgM with C1, C3 and C4 (p less than 0.001). There also was a significant association among alterations in renal blood flow, deposition of IgA (p less than 0.05) and C4 (p less than 0.02), and graft outcome. Higher perfusion indexes, indicative of decreased blood flow, showed significant associations (p less than 0.007 and less than 0.04 for the cortical and global perfusion indexes, respectively) with a greater risk of graft loss. Although it primarily is a cellular event, the data suggest that acute rejection is associated with a deposition of various humoral factors that may mediate alterations in renal blood flow. The latter may affect graft function and structural integrity, and, thus, may show a direct correlation with the outcome of a graft.