A zebrafish model of infection-associated acute kidney injury.

Sepsis-associated acute kidney injury (S-AKI) independently predicts mortality among critically ill patients. The role of innate immunity in this process is unclear, and there is an unmet need for S-AKI models to delineate the pathophysiological response. Mammals and zebrafish ( Danio rerio) share a conserved nephron structure and homologous innate immune systems, making the latter suitable for S-AKI research. We introduced Edwardsiella tarda to the zebrafish. Systemic E. tarda bacteremia resulted in sustained bacterial infection and dose-dependent mortality. A systemic immune reaction was characterized by increased mRNA expressions of il1b, tnfa, tgfb1a, and cxcl8-l1 ( P < 0.0001, P < 0.001, P < 0.001, and P < 0.01, respectively). Increase of host stress response genes ccnd1 and tp53 was observed at 24 h postinjection ( P < 0.0001 and P < 0.05, respectively). Moderate E. tarda infection induced zebrafish mortality of over 50% in larvae and 20% in adults, accompanied by pericardial edema in larvae and renal dysfunction in both larval and adult zebrafish. Expression of AKI markers insulin-like growth factor-binding protein-7 (IGFBP7), tissue inhibitor of metalloproteinases 2 (TIMP-2), and kidney injury molecule-1 (KIM-1) was found to be significantly increased in the septic animals at the transcription level ( P < 0.01, P < 0.05, and P < 0.05) and in nephric tubules compared with noninfected animals. In conclusion, we established a zebrafish model of S-AKI induced by E. tarda injection, with both larval and adult zebrafish showing nephron injury in the setting of infection.

Leukocyte-derived MMP9 is crucial for the recruitment of proinflammatory macrophages in experimental glomerulonephritis.

Matrix metalloproteinase 9 (MMP9) is a conditionally expressed enzyme and is upregulated in glomerulonephritis. Its function in these diseases, however, remains to be fully elucidated. The induction of nephrotoxic serum nephritis (NTN) in wild-type mice resulted in an upregulation of MMP9, followed by leukocyte infiltration, albuminuria, and subsequent renal failure. MMP9 deficiency ameliorated the course of NTN as indicated by reduced histological injury and reduced infiltration of proinflammatory macrophages. The chemotaxis of MMP9-deficient macrophages in vitro was impaired. Intrarenal macrophages isolated from the kidneys of nephritic MMP9 knockout mice still displayed the typical features of a proinflammatory phenotype and were indistinguishable from wild type-derived cells. Bone marrow transplantation restored renal tissue injury and macrophage recruitment when wild type-derived donor cells were transplanted onto MMP9-deficient mice prior to the induction of NTN. Thus, leukocyte-derived MMP9 mediates the recruitment of proinflammatory macrophages into kidneys during experimental crescentic glomerulonephritis.

Mechanisms of light chain injury along the tubular nephron.

The tubular nephron is responsible for reabsorption and catabolism of filtered low molecular weight proteins that include Ig free light chains. In the setting of a plasma cell dyscrasia, significant amounts of free light chains, now monoclonal proteins, present to the tubular nephron for disposal. The result may be clinical renal dysfunction in the form of AKI, progressive CKD, and end-stage kidney disease. Here, I review the mechanisms involved in these processes that result in tubular injury, including proximal tubulopathy and cast nephropathy.

Effect of intra-amniotic lipopolysaccharide on nephron number in preterm fetal sheep.

Chorioamnionitis is an antecedent of preterm birth. We aimed to determine the effect of experimental chorioamnionitis in fetal sheep during late gestation on 1) nephron number, 2) renal corpuscle volume, and 3) renal inflammation. We hypothesized that exposure to chorioamnionitis would lead to inflammation in fetal kidneys and adversely impact on the development of nephrons, leading to a reduction in nephron number. At ∼121 days of gestation (term ∼147 days), pregnant ewes bearing twin or singleton fetuses received a single intra-amniotic injection of lipopolysaccharide (n = 6; 3 singletons, 3 twins); controls were either untreated or received an intra-amniotic injection of saline (n = 8; 4 singletons, 4 twins). One twin was used from each twin-bearing ewe. At ∼128 days of gestation, fetuses were delivered via Caesarean section. Kidneys were collected and stereologically analyzed to determine nephron number and renal corpuscle volume. Renal inflammation was assessed using immunohistochemistry. Experimental chorioamnionitis did not affect body weight or relative kidney weight. There was a significant reduction in nephron number but no change in renal corpuscle volume in LPS-exposed fetuses relative to controls. On average, nephron number was significantly reduced by 23 and 18% in singleton and twin LPS-exposed fetuses, respectively. The degree of renal inflammation did not differ between groups. Importantly, this study demonstrates that exposure to experimental chorioamnionitis adversely impacts on nephron number in the developing fetus.

Immune compartments of the nephron in relation to the immune system.

The paper presents data regarding the compartmentalization of the nephron related to immune processes taking place at this level. The morphofunctional compartments of the nephron (glomerular, tubulo-interstitial and juxtaglomerular) become immune compartments during immune processes. The paper shows the immune cells located in the morphofunctional compartments of the nephron and the relationship between them. It is considered that the presence of immune cells in these compartments is a dynamic process; the number of infiltrating cells is reduced under physiological conditions and increases during pathological immune processes. The paper presents also the resident cells of the nephron and their immune capabilities. It also presents the professional immune cells originating in the bone marrow, which are involved in immune processes. The complex relationship between these cells by means of the cytokine network, chemokines as well as other mediators, as well as the role of immune receptors, mainly Toll-like receptors is outlined. During an immune aggression immune aggregates defined as tertiary lymphoid organs are formed at the level of the nephron. These lymphoid follicle-like structures might represent an intrarenal immune system. The compartmentalization of the nephron is part of the recently described concept of compartmentalization of the immune system.

In crescentic IgA nephropathy, fractional excretion of IgG in combination with nephron loss is the best predictor of progression and responsiveness to immunosuppression.

BACKGROUND AND OBJECTIVES: The aim of this study was to evaluate the relationship between proteinuric markers (urinary excretion of IgG, alpha2-macroglobulin, alpha1-microglobulin) and serum creatinine (sCr), histologic lesions, progression, and immunosuppression responsiveness in crescentic IgA nephropathy. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Fractional excretion of IgG (FEIgG) and of alpha1-microglobulin and urinary excretion of alpha2-macroglobulin were evaluated in 37 patients, 23 treated with steroids and cyclophosphamide. For assessment of the effective tubular load of proteins in surviving nephrons, new markers that take into account not only the absolute excretion value but also nephron loss were obtained dividing proteinuric markers for percentage of nonobsolescent glomeruli (surviving glomeruli [SG]). For each parameter, low- and high-risk groups were defined according to cutoffs with the highest sensitivity and specificity for progression (ESRD/doubling sCr) assessed by receiver operating characteristic analysis; follow up was 60 +/- 40 mo. RESULTS: FEIgG/SG is the most powerful progression predictor: 5 versus 83% in all patients; in treated patients, 0 versus 89%, increased to 0 versus 100% by sCr and FEIgG/SG in combination (low risk: both markers or only one below cutoff (n = 15); high risk: both markers above cutoff (n = 8). The nonprogressors showed at last observation 65% proteinuria reduction and 10% sCr reduction. CONCLUSIONS: In crescentic IgA nephropathy, FEIgG/SG, which evaluates altered size selectivity in relation to nephron loss, is the best progression predictor. In treated patients, progression prediction was increased by FEIgG/SG and sCr in combination. Treatment may be restricted to low-risk patients.

Characterization of basolateral chloride/bicarbonate exchange in macula densa cells.

Functional and immunohistological studies were performed to identify basolateral chloride/bicarbonate exchange in macula densa cells. Using the isolated, perfused thick ascending limb with attached glomerulus preparation dissected from rabbit kidney, macula densa intracellular pH (pH(i)) was measured with fluorescence microscopy and BCECF. For these experiments, basolateral chloride was reduced, resulting in reversible macula densa cell alkalinization. Anion exchange activity was assessed by measuring the maximal net base efflux on readdition of bath chloride. Anion exchange activity required the presence of bicarbonate, was independent of changes in membrane potential, did not require the presence of sodium, and was inhibited by high concentrations of DIDS. Inhibition of macula densa anion exchange activity by basolateral DIDS increased luminal NaCl concentration-induced elevations in pH(i). Immunohistochemical studies using antibodies against AE2 demonstrated expression of AE2 along the basolateral membrane of macula densa cells of rabbit kidney. These results suggest that macula densa cells functionally and immunologically express a chloride/bicarbonate exchanger at the basolateral membrane. This transporter likely participates in the regulation of pH(i) and might be involved in macula densa signaling.

Existence of a dense reticular meshwork surrounding the nephron inducer in neonatal rabbit kidney.

While more and more humoral factors involved in nephrogenesis are being discovered, there is no detailed knowledge of the morphological structures at the interface of the nephron inducer and the surrounding mesenchyme. For that reason we examined this area in the cortex of neonatal rabbit kidneys by scanning electron-microscopical and transmission electron-microscopical techniques. Our interest was focused on the basal aspect of the collecting duct ampulla and the surrounding competent mesenchyme, where morphogenic signals are to be exchanged during nephron induction. Close contact between these two tissues involved in nephrogenesis is assumed to allow direct cellular contact or diffusion of soluble factors across a short distance. Our data, however, show the presence of a dense fibrillar meshwork around the collecting duct ampulla, spatially separating the inducer and the competent mesenchyme during nephron induction.

Tubulointerstitial nephritis antigen (TIN-ag) is expressed in distinct segments of the developing human nephron.

Tubulointerstitial nephritis antigen (TIN-ag) is a 58 kDa glycoprotein restricted within the kidney to basement membranes underlying the epithelium of Bowman's capsule and proximal and distal tubules. Autoantibody formation against this component has been described in association with primary immune-mediated tubulointerstital nephritis, membranous nephropathy and anti-glomerular basement membrane nephritis. In the present report, the ontogeny of this protein was studied in human fetal kidney tissue by immunohistochemical analysis of immature and developing nephrons using a panel of monoclonal and polyclonal antibodies. TIN-ag is first detected in basement membranes underlying the epithelium of Bowman's capsule of early capillary loop stage glomeruli and the primitive proximal tubule. No detectable expression is observed in the basement membranes of the branching ureteric bud, nephrogenic vesicle, or comma shape and s-shape stages of nephrogenic development. Increased staining of the proximal tubular basement membrane is associated with outgrowth of the primitive tubule from the urinary pole of the developing glomerulus. In more mature fetal tubules, TIN-ag expression closely resembles that of previously reported observations in mature tissue where it is present in high amounts in the basement membranes of proximal tubules, and to a lesser extent in Bowman's capsule and distal tubules. Our results suggest that TIN-ag expression is developmentally regulated in a precise spatial and temporal pattern throughout nephrogenesis.

Immunodissection of the human proximal nephron: flow sorting of S1S2S3, S1S2 and S3 proximal tubular cells.

We report on the use of several proximal tubular cell (PTC) surface markets and corresponding antibodies in fluorescence-activated cell sorting (FACS), and their ability to identify and flow sort cells of defined proximal tubular origin (S1S2S3) or of defined proximal subsegmental origin (S1S2 only/S3 only). We tested monoclonal/polyclonal antibodies directed against five different surface peptidases [leucine aminopeptidase (LAP), neutral endopeptidase 24.11 (NEP), dipeptidyl peptidase IV (DPPIV), aminopeptidase A (APA) and gamma-glutamyl transferase (gamma-GT)], the S3 segment-specific marker intestinal type alkaline phosphatase (iAP) and an S1S2 marker (TN20-antigen), originally proposed as a surface marker for interstitial fibroblasts. Segmental (proximal tubular vs. distal tubular) and proximal subsegmental (S1S2 vs. S3) expression of all five surface peptidases and TN20 antigen were first assessed by comparing immunohistochemical staining on normal human kidney tissue with staining for well-known segment-specific differentiation markers (intestinal type alkaline phosphatase, Tamm-Horsfall protein) on adjacent sections. All five peptidases were found to be expressed to a certain degree in all subsegments (S1 S2 and S3) of the proximal nephron, whereas expression was never seen in the more distal parts of the nephron. Flow cytometry was performed on cells obtained following gradient purification of collagenase-digested human renal tissue. Labeling cells for expression of LAP, NEP or DPPIV resulted in high yields of specifically labeled PTC (S1S2S3 origin). Labeling with anti-LAP resulted in the clearest distinction between positive and negative cell subpopulations, and therefore LAP was considered the best PTC marker for use in FACS. iAP histochemical staining on sorted cells showed that flow sorting with monoclonal antibody (moAb) 250 (anti-intestinal type alkaline phosphatase) allowed sorting of S3 cells with > 90% purity. Likewise, moAb TN20 enabled us to obtain a highly purified S1S2 population as confirmed by the absence of iAP on sorted cells.

Le(y) antigen expression is correlated with apoptosis (programmed cell death).

Apoptosis (programmed cell death) is a basic physiological process which determines specific patterns of tissue size and shape, and balance of cell number, during morphogenesis, and seems to play an integral role in oncogenic progression. Since dramatic changes of cellular glycosylation pattern are well known to be closely correlated with differentiation, development and oncogenesis, it is likely that similar specific changes are associated with apoptosis. However, this possibility has not been systematically investigated. We therefore carried out histological studies of many tumours and normal tissues for which a high incidence of apoptosis is believed to occur. Sections were stained with monoclonal antibodies (MoAbs) directed to carbohydrate antigens Le(y) and Le(x), proliferating cellular nuclear antigen (PCNA) and Fas (previously claimed to be an apoptosis-inducing antigen). Antibody staining patterns were compared with morphological cell characteristics as revealed by haematoxylin/eosin staining, and DNA fragmentation patterns (a marker of apoptosis) as revealed by 3'-OH nick-end labelling technique. We found that expression of Le(y) (defined by MoAb BM1) is closely correlated with the process of apoptosis, but not with cell proliferation or necrosis. Within Le(y)-positive areas of tissue sections, typical apoptotic morphological changes and DNA fragmentation (as revealed by positive nick-end labelling) were frequently observed in certain loci, although not all Le(y)-positive cells showed such signs of apoptosis. Le(y)-positive areas showed consistent negative staining by MoAb directed to PCNA and negative or weak staining by MoAb directed to Fas antigen, regardless of tissue source. No such trends were observed for Le(x) glycosylation. We conclude that Le(y) expression is a useful phenotypic marker predictive of apoptosis, i.e. some (although not all) Le(y)-positive cells subsequently become apoptotic.

The nephritogenic immune response.

Recent work has improved our understanding of a number of aspects of the nephritogenic immune response. Progress has been made in the understanding of the development of idiotypic networks, and in understanding the structural nature of the targets of self-reactive T cells and the paracrine mediators that are released as part of the local inflammatory response.

Immunogenicity of rat renal allograft nephron components in vivo. Lack of correlation between MHC expression and immunogenic potential.

Cultured rat nephron components--i.e., tubular cells, glomerular mesangial cells, and glomerular epithelial cells--were compared with cultured rat heart endothelial cells for their in vivo immunogenicity using the primed rejection assay (PRA). Gamma-interferon (gIFN) was used to regulate the class I and class II MHC antigen expression of the cells tested. The heart endothelial cells proved to be the only cell type capable of inducing a maximal rejection response in the native state. This was achieved with a cell number of 10(6) when the survival of a subsequent heart allograft from a relevant donor was reduced from 5 to 3 days. All kidney nephron components proved to be immunogenic in PRA but to a far lesser extent than the endothelial cells. A reduction of graft survival from 5 to 4 days was achieved with a cell number of 10(6) cells, and no immunogenic effect (with the exception of mesangial cells contaminated by a small number of macrophages) was observed in smaller cell numbers. The gIFN-treated endothelial cells were more potent than untreated endothelial cells. They reduced the graft survival to 4 days with a cell number of 10(3) and caused a maximal reduction of the survival to 3 days with a cell number of 10(5). The nephron components failed to increase their immunogenicity after 3-day gIFN treatment, regardless of a high increase in their class I and class II expression rates. The study suggests that, in contrast to the endothelial cells, none of the nephron components are able to act as an antigen-presenting cell on their own.

Developmental modifications of sheep kidney antigens.

We have used monoclonal antibodies to study the changes in the expression of four kidney antigens during organogenesis in the sheep. Two of these antibodies, EE24.6 and EJ30.1, label intensely only the adult kidney, whereas the other two, EK17.1 and EJ15.1, bind to the extracellular matrix of the embryonic kidney. For EJ15.1, the staining of the extracellular matrix decreases temporarily during the second half of intrauterine life, a period during which a light staining appears in the mesangium. For the other, EK17.1, the extracellular matrix staining in the stroma gradually decreases as the embryo grows, while staining of the mesangium and the arterial intima becomes evident. With EK17.1, fibronectin is identified in the extracellular matrix of the embryonic kidney and intracellularly in the mesangial cells after these cells have colonized the glomerulus. The mesonephros staining seems to be the same as that of the metanephros. In the adult, extraglomerular vascular endothelial cells bind EK17.1, whereas intraglomerular endothelial cells do not express fibronectin, which suggests a functional difference between endothelial cells in these two localizations.

Expression of segment-specific antigens in the human nephron and in renal epithelial tumors.

The immunohistological staining patterns of several hundred monoclonal antibodies (Mabs) were studied in normal human kidney tissue. Seven Mabs, 3 hematopoietic (F103.12/CD10, MY7/CD13, 3C4/CD15) and 4 non-hematopoietic (LP34, E29, HEA81, HEA125) revealed a segment-specific or pan-nephron staining. The expression of antigens (Ags) labelled by the 7 selected Mabs was then studied in cryostat sections of a series of 43 renal epithelial tumors (31 renal cell carcinomas, 2 oncocytomas, 10 adenomas) in order to correlate the results with the prevailing hypothesis for the histogenesis of these tumors. The adenomas displayed poor expression of CD10-Ag (proximal nephron marker) compared to carcinomas. The chromophobic type of renal cell carcinoma and the benign oncocytoma did not express CD13-Ag, suggesting a possible histogenetic relationship. More than 95% of all tumors simultaneously expressed a proximal and a distal marker. Our results suggest that CD10-antibody may be of value in the distinction between benign and malignant small-sized renal tumors. We conclude that neoplastic transformation may imply such alterations in the expression of marker-Ags (proximal/distal) that no conclusion can be drawn regarding the tubular segment from which a renal epithelial tumor takes its origin.

Secretory immune responses in human kidneys.

The secretory immune system has been well studied in the intestinal, bronchial, and biliary systems and breast. Tissue studies of secretory immunoglobulins in the kidney are scanty, mostly related to nephropathies with IgA. Renal tissues from 37 autopsies selected for any history of renal dysfunction were processed for immunohistologic studies on frozen sections with several antisera, including a purified rabbit anti-human secretory component (SC). By immunohistology, gel diffusion, and immunoblotting, the anti-SC antibody reacted appropriately with purified human SC, saliva, intestinal epithelium, and breast milk and did not cross-react with immunoglobulin heavy or light chains, lactoferrin, and other tissue proteins. IgA and SC were seen in tubular casts in 70% of patients, whereas less impressive staining with IgM, IgG, and albumin was seen, respectively, in 24%, 13%, and 22% of the patients. SC was present in the cytoplasm of distal tubule and Henle's loop cells in 78% of specimens. A control group of 10 healthy individuals who died suddenly showed minimal staining of casts and tubules in 2 specimens. Renal pathology in the group with IgA-SC+ casts included acute tubular necrosis (54%), severe chronic renal disease (61%), and mild chronic renal injury (38%). The group with negative IgA-SC casts included acute tubular necrosis (64%), infectious interstitial nephritis (36%), and negligible renal disease (36%). This study suggests that discrete distal segments of the nephron may have the capability of secreting SC, which is probably coupled with serum-derived IgA and incorporated into luminal tubular secretions. The low level of immunosecretions in kidneys which are normal or minimally damaged suggests that this system may need to be turned on by unknown, probably pathogenic stimulating factors.

Immunohistologic dissection of the human kidney using monoclonal antibodies.

Nine murine monoclonal antibodies which detect differentiation antigens of the human kidney are described. Immunofluorescence and immunoperoxidase studies demonstrate that these antigens are expressed by different cell types comprising the nephron. Monoclonal antibody MA99 detects a glycoprotein complex of the glomerular basement membrane. Monoclonal antibody S4 detects a glycoprotein of 160,000 daltons (gp160) expressed by glomerular and proximal tubular epithelial cells. Monoclonal antibodies S23, S27 and S6 immunoprecipitate a glycoprotein of 120,000 daltons (gp120) found on cells of the proximal tubule and portions of Henle's loop. Monoclonal antibody C26 identifies a glycoprotein of 40,000 daltons (gp40) expressed by cells of the distal and collecting tubules. Monoclonal antibodies M2 and S8 are specific for A and B blood group antigens, respectively, found on cells of the collecting tubule in individuals of the respective blood type. This panel of antibodies is useful in the study of normal renal embryogenesis, microanatomy and physiology as well as pathological processes including tumors.

Development and heterogeneity of antigens in the immature nephron. Reactivity with human antiglomerular basement membrane autoantibodies.

Indirect immunofluorescence microscopy was performed with 15 human anti-glomerular basement membrane (GBM) antibodies and mouse monoclonal antibodies to Type IV collagen (MBM4) and renal basement membranes (MBM15) on renal tissue from 6 fetuses (gestational age, 15-23 weeks), 8 infants (age, 1-21 days), and 8 children and adults (ages, 3-27 years). Of the 15 human anti-GBM antibodies that react with GBM in adult glomeruli, only 4 identified antigens in the GBM of fetal and infant glomeruli. In contrast, the monoclonal antibodies bound to basement membranes in the uninduced nephron and the GBM throughout all development stages of the fetal kidney. These studies demonstrate that the reactivity of human autoantibodies with GBM is developmentally and gestationally related--some identifying an antigen(s) in fetal glomeruli with early capillary loop formation and others reacting only with GBM in fully mature kidneys.

[Monoclonal antibodies against surface antigens of lymphoblasts and blood cells or bone marrow recognize constituents of the human nephron]. / Des anticorps monoclonaux dirigés contre des antigènes de surface de lymphoblastes et de cellules sanguines ou médullaires reconnaissent des constituants du néphron humain.

By immunizing mice with bone marrow cells of a patient with acute lymphoblastic leukemia, monoclonal antibodies have been produced against differentiation antigens of hemopoietic cells. These antibodies also recognize cells in different parts of the normal human kidney. Two antibodies, ALB1 et ALB2, which recognize the common acute lymphoblastic leukemia antigen, (CALLA), label the podocytes and the epithelial cells of the proximal tubules of the kidney; ALB6, which recognizes the "p 24" antigen of lymphocytes, labels the distal tubules; ALB9, which recognizes lymphoblasts and granulocytes, labels the thick ascending loop of Henle and the collecting tubules; PM1, which recognizes immature granulocytic cells, labels the thick ascending loop of Henle.