David J. Salant - US grants

Recently, a leukocyte-independent form of complement (C')-mediated glomerular injury was defined in passive Heymann nephritis (PHN), a rat model that closely resembles human membranous nephropathy which is a common cause of nephrotic syndrome in adults. While further observations have suggested a role for the C5b-9 membrane attack complex (MAC) in this and other forms of tissue injury, this has not been conclusively shown. Variants of PHN and a recently developed model of antibody (Ab)-directed, C'-mediated glomerular injury in the isolated perfused rat kidney (IPRK) will be used to further study this unique form of tissue injury. Kidneys that contain an antigen planted in the subepithelial space are perfused in vitro with a cell-free perfusate containing C'-fixing Ab and various sources of C'=replete and C'-deficient sera. The effects on glomerular function are oserved for up to two hours. With the aid of this model, definitive evidence of a functional role for the MAC will be sought using sera deficient in C6 and C8. Additional studies will examine: the possible role of intermediary systems; C'-mediated alterations in glomerular sieving properties, GBM charge residues, and glomerular podocyte morphology; and localization of the MAC by immunohistological and ultrastructural techniques. The role of the MAC will also be examined in vivo by depleting rats of C8 with a specific antiserum. Similar observations in the IPRK and in vivo will also be made with antiglomerular basement membrane (GBM) antibody. Also to be examined in the IPRK are: a) a potential relationship between immunopathogenetic events, locally-derived vasoactive hormones and disordered renal hemodynamics in PHN; and b) the influence of hemodynamic factors on the binding of Abs to glomerular antigens. An erythrocyte-perfused IPRK, in which hemodynamic variables approach physiological values, will be used for these studies. In addition, the relationship between the density of glomerular antigens and the abilities of their respective antibodies to fix C' and induce glomerular injury will be explored. Sheep antisera to laminin and rat GBM and quantitative in vivo and in vitro glomerular binding techniques will be used for this purpose. The proposed studies will further the long term goals of Investigator, which are to comprehensively analyze the factors that influence Ab deposition in glomeruli and the ways in which such Abs interact with C' and other mediators to induce glomerular injury.

DESCRIPTION (provided by applicant): The goals of this program are to identify, inspire and train promising candidates for research careers in academic nephrology and to assist qualified trainees in obtaining suitable academic positions. The program is designed to provide physicians heading for careers in academic nephrology the thorough scientific grounding necessary for a successful research career and to attract recent PhD graduates and provide them postdoctoral research training in a scientific discipline relevant to nephrology. Research training will continue for at least two years. On average, three new trainees will enter the program each year. The principal mechanism for training is direct performance of research under the personal supervision of one or more of the training staff comprised of nine nephrologists and seven basic scientists. Experienced, interdisciplinary training staff provide training in immunopathology, autoimmunity, renal cell physiology, ischemic and toxic renal injury, gene regulation related to renal cancer and developmental biology, and clinical research relevant to kidney diseases. Well-established collaborative arrangements allow each trainee to acquire a broad research experience and focus a variety of skills on a specific research protocol. Trainees assume graded responsibility for the design, conduct and interpretation of experiments, progressively becoming more independent but always remaining under the supervision of the preceptor. All trainees and staff meet frequently for seminars, journal clubs and other formal group teaching exercises. Coursework leading to a PhD is available in the Graduate Program in Molecular Medicine, and formal training in clinical research is provided through the BU School of Public Health and Division of Graduate Medical Sciences. Trainees work in modern, well-equipped laboratory space in the Evans Biomedical Research Center and laboratories of basic science faculty. These are in close proximity to core facilities, modern animal care quarters supervised by a full-time veterinarian, and the BUSPH. The General Clinical Research Center at Boston Medical Center is available for clinical studies. PUBLIC HEALTH RELEVANCE: This program is designed to provide research training for physicians and scientists to investigate the causes and consequences of kidney diseases;to identify ways to prevent diseaes of the kidney from progressing to kidney failure and to improve the quality and duration of the lives of those who do develop end stage kidney failure.

This is a proposal to study the structural biology of the glomerular visceral epithelial cell (GEC) by analyzing a GEC-specific plasma membrane protein that is the target of a nephritogenic monoclonal antibody, mAb 5- 1-6. In tissue sections, mAb 5-1-6 binds exclusively to rat GECs, especially in the vicinity of GEC slit diaphragms, and it precipitates a 51 kDa protein from normal rat glomeruli. After a single in vivo injection, mAb 5-1-6 localizes to GEC foot processes and causes immediate, massive proteinuria in the absence of complement or inflammatory cells. Simultaneously, the antibody:antigen complex appears to redistribute on the GEC surface and may be endocytosed. It is suggested that the antigen (p51) recognized by mAb 5-1-6 may be an integral transmembrane protein of the slit diaphragm and that binding of antibody to its extracellular domain alters the molecular arrangement that constitutes a normally functioning slit diaphragm. An alternative possibility is that divalent antibody binding causes p5l to shift from the basolateral surface of the podocyte to the slit diaphragm where the complex might interfere with the normal molecular arrangement between cytoplasmic and as yet unknown transmembrane proteins. In specific aim #1, a series of complementary methods are proposed to define the primary amino acid composition and structure of p51, identify putative nephritogenic domains and seek mouse and human homologues. In specific aim #2, anti-p51 antibodies and isolated glomerular cells in short term culture will be used to: examine the normal synthesis, cellular distribution and organization of p51; and explain, in cell biological terms, what happens when p51 is bound by antibody. An attempt will also be made to develop a p51-expressing line of rat GECs to facilitate these studies. Specific aim #3 is designed to: define the precise location of p51 and its relationship to the slit diaphragm in the normal mature and developing GEC; and determine how this is altered by mAb 5-1-6 binding in vivo and in certain proteinuric states in which predictable and well documented changes occur in the slit diaphragm. Immunohistology will be used to define the distribution of p5l and immunoblot analysis the amount of membrane-associated p5l. Metabolic labelling studies and mRNA levels will provide evidence of new p5l synthesis. Changes in p5l oligomerization status will be studied by cell surface labeling of cells isolated from injured and recovering glomeruli. Defining the molecular structure of this protein, together with its cellular organization and response to antibody binding in vitro and in vivo, will give insight into its role in maintaining the integrity of the GEC as a permeability barrier and perhaps shed light on the molecular composition of the slit diaphragm and how it is altered in proteinuric diseases.

DESCRIPTION (Adapted from Investigators Abstract): This application proposes to examine the phenomenon of antibody-induced glomerular injury that can occur in the absence of complement fixation. The investigator has previously determined that sheep nephrotoxic serum induces proteinuria in rats and mice in the absence of complement fixation in the glomerulus, and has further demonstrated that a major target of this polyclonal antibody is beta1 integrins in glomerular epithelial cells. The goal of the present studies is to determine whether the anti-beta1 integrin activity of this nephrotoxic serum is necessary and sufficient to produce the observed proteinuria, or whether there are additional or alternative antigens responsible for this effect. The first specific aim will examine whether the anti beta1 integrin antibody activity is necessary and sufficient to induce the glomerular injury. This will be purified using immunoabsorption techniques and in addition, antibodies will be produced to fusion proteins of external domains of beta1 integrin and alpha3 integrin (the likely alpha partner to the beta1 integrin in glomerular epithelial cells), and the nephrotoxicity of these antibodies will be tested in vivo, and will also be important in initial attempts to map the pathogenic epitope of the nephrotoxic serum, if indeed the beta1 integrin is determined to be the major or only antigenic site. The second specific aim examines the possibility that anti-integrin properties of nephrotoxic serum may be necessary but insufficient to cause renal injury, or is an epiphenomenon. These experiments are designed to determine how other as yet undefined glomerular antigens contribute to the nephrogenic effect. Immunopurification and molecular biological techniques will be used to identify other possible glomerular cell membrane proteins that may contribute to the effects. In addition, the possibility that the noncollagenous domain of the alpha3 chain of type IV collagen will be examined as a possible antigen. The third specific aim will examine the hypothesis that the binding of nephrotoxic serum to glomerular epithelial cells activates events that disrupt glomerular permselectivity by altering the relationship between the cells and basement membrane. This specific aim will be important if the experiments in the first specific aim determine that the anti-integrin effect of nephrotoxic serum is primarily responsible for the glomerular alterations. In this revised specific aim, studies will be performed in a cultured system of rat glomerular epithelial cells grown on permeable supports. Cells exposed to antibodies will be examined for alterations in distribution and phosphorylation status of proteins that are involved in integrin mediated formation of cell and cell extracellular matrix adhesion. The morphology of cytoskeletal elements and the proteins that link them to integrins will also be examined in relation to the altered macromolecular permeability induced by nephrotoxic serum. Integrin-related signaling will be analyzed by phospholipid hydrolysis and second messenger production. Finally, the investigator will examine whether there is antigen shedding after exposure to antibody, or whether the antigen is endocytosed in response to antibody. These studies are designed to investigate further, the role of the glomerular epithelial cell as a primary target of injury in proteinuric renal diseases, and to define the role of integrins in the maintenance of normal glomerular structure and function.

The primary goal of this proposal is to explore the hypothesis that Notch4 contributes to the integrity of the mature podocyte. This will be accomplished through an analysis of in vitro and in vivo models of antibody-mediated podocyte injury and Notch4-deficient (N4-/-) mice. We found that Notch4 is present in the podocytes of mature rats and mice and that the podocytes of N4-/- mice are focally effaced with increased expression, clustering and dislocation of nephrin. Moreover, Notch4 expression is abundant and total nephrin is reduced coincident with the onset of proteinuria in rats with passive Heymann nephritis, which suggests that Notch4 might regulate podocyte gene transcription. Thus, we will examine the role of Notch4 in the mature podocyte according to four specific aims. In the first specific aim we will use an in vitro model of antibody- and complement-mediated cell injury to determine if Notch4 is activated by gamma- secretase and translocates to the nucleus to bind and activate its nuclear target, CSL. In the second specific aim we will examine N4-/- mice over time to determine if Notch4 deficiency leads to proteinuria and alterations in podocyte morphology and podocyte-associated proteins. The third specific aim is to determine if Notch4 influences the susceptibility to and/or recovery from immune glomerular injury. N4+/+ and N-/- littermates will be studied using various models of antibody-mediated podocyte injury. Their susceptibility to injury and time to recovery will be monitored, and the effect of gamma-secretase inhibition will be examined. The fourth specific aim will utilize a comparative transcriptome analysis of glomerular RNA derived from young male N4+/+ and N-/- littermates before and after antibody-mediated podocyte injury to identify genes that Notch4 regulates in mature glomeruli. Differentially expressed genes will be examined for appropriate location and expression in glomeruli and regulation by Notch4. The results of these studies may disclose mechanisms underlying premature podocyte degeneration and glomerular sclerosis and the variable clinical and pathological response of patients with antibody-mediated podocyte injury.

DESCRIPTION (provided by applicant): In the R21 phase of this project we propose to use proteomic techniques with which we have had previous success in identifying nephritogenic podocyte antigens to isolate and characterize the target antigen/s of membranous nephropathy (MN). Sera from patients with biopsy-proven MN will be screened for reactivity with glomerular antigen/s and positive samples will be used with extracts of normal human glomeruli in a proteomic approach involving immunoprecipitation, gel electrophoresis, and mass spectrometry to isolate and sequence immunoreactive peptides. Peptide sequences will be used to search protein databases to identify known proteins, search EST databases for matching cDNA clones, or to design oligonucleotides to screen cDNA expression libraries. When a putative antigen is identified, a monospecific antibody will be generated and used to determine if the antigen is appropriately located on the podocyte plasma membrane and tested to establish if it is able to produce membranous immune deposits in vivo in experimental animals or in ex vivo perfused human kidneys. A full-length cDNA will be acquired or produced and used to generate a bacterial or mammalian fusion protein for antibody production and for future use in the R33 phase. Having identified a target glomerular antigen that is reactive with sera from patients with MN and established that it has pathogenic relevance, we will embark on the R33 phase. The recombinant antigen will be used to develop a diagnostic enzyme-linked immunoassay (ELISA) for the serological detection of MN as compared with other common causes of nephrotic syndrome, namely focal and segmental glomerulosclerosis (FSGS) and diabetic nephropathy (DN). Serum from normal volunteers will be used to define the normal limits of the ELISA and MN sera that are positive on western blotting with the recombinant antigen will be used to establish the lower limits of detection of the assay. Once the ELISA has been developed, a prospective case-control study will be conducted with serum from patients with newly-diagnosed, biopsy-proven idiopathic and lupus MN and compared to serum from patients with FSGS and DN as well as that of normal volunteers to assess the diagnostic utility of the assay. Finally, the nephritogenic properties of MN autoantibodies will be examined using standard techniques to assess their class and subclass characteristics, as well as their cytopathic effects using cells expressing the MN antigen.

DESCRIPTION (provided by applicant): The primary goal of this exploratory proposal is to develop a tool to determine if circulating autoantibodies directed at the M-type phospholipase receptor (PLA2R) from patients with idiopathic membranous nephropathy (MN) are pathogenic. We have established that about 75% of patients with idiopathic MN have circulating anti-PLA2R autoantibodies whereas none of the disease or normal controls has tested positive, thus indicating a high degree of specificity. The anti-PLA2R antibodies are IgG4, the subclass that predominates in idiopathic but not secondary MN, the antigen PLA2R is expressed on podocytes and colocalizes with IgG4 in the glomerular immune deposits of patients with MN, and the antibodies can be eluted from the glomerular immune deposits of MN renal biopsies. Moreover, the presence of circulating anti-PLA2R antibodies correlates with disease activity in MN. Although these findings strongly suggest that anti-PLA2R antibodies are responsible for, or at least contribute to disease development, proof of pathogenicity has yet to be established. Whereas simple transfer of the disease to laboratory animals with human anti-PLA2R would readily answer this question, human anti-PLA2R does not recognize PLA2R in the glomeruli of any small laboratory animal tested to date. Therefore, we plan to take advantage of both the lack of immunoreactive PLA2R in mouse glomeruli and the refinement of podocyte- specific transgenic techniques to develop a mouse model in which the human PLA2R is expressed in podocytes. To that end we propose the following specific aims: Specific Aim 1: Produce a transgenic mouse utilizing the NPHS1 (nephrin) promoter to drive expression of human PLA2R exclusively in mouse podocytes. Specific Aim 2: Verify that human PLA2R is expressed on mouse podocytes in vivo. Specific Aim 3: a) Determine if injected human anti-PLA2R autoantibodies bind to human PLA2R and form immune deposits typical of MN in the transgenic mouse glomeruli. b) Establish if injected human anti-PLA2R antibodies are able to cause proteinuria in the human PLA2R transgenic mouse. Specific Aim 4: Determine if the human PLA2R transgenic mice develop anti-PLA2R antibodies and MN when actively immunized with human PLA2R. Successful development of this model will pave the way for studies to both establish the pathogenic role of human anti-PLA2R autoantibodies and define the mechanisms of injury. PUBLIC HEALTH RELEVANCE: Having demonstrated that the M-type phospholipase receptor (PLA2R) is a specific target antigen of circulating autoantibodies in a high proportion of patients with idiopathic membranous nephropathy, we propose to develop a mouse model to determine if the antibodies are responsible for the clinical (proteinuria) and pathological manifestations of the disease. To that end, we will produce transgenic mice in which the human PLA2R is expressed on mouse podocytes, the location of the antigen in human kidneys.

DESCRIPTION (provided by applicant): This program will address three questions in order to better understand the role of the M-type phospholipase A2 receptor (PLA2R) as a major target antigen in idiopathic membranous nephropathy (MN) and the properties of the anti-PLA2R antibodies that are specifically detected in patients with the disease. 1. Do variants in the PLA2R1 gene account for susceptibility to idiopathic MN? This aim is based on the finding that the PLA2R epitope identified by anti-PLA2R autoantibodies is conformation dependent; the known propensity for other members of the mannose receptor/PLA2R family to exist in bent or extended configurations; differences in immunoreactivity between PLA2R in normal and MN kidney tissues; and the presence of several non-synonymous SNPs in PLA2R1, including eight within the N- terminal region that contains the epitope, at least three of which are predicted to affect PLA2R structure. Hypothesis: Variants in PLA2R1 may explain the unique properties of the pathogenic epitope in MN. Approach: Genomic variants in PLA2R1 from patients with idiopathic MN will be compared to matched controls to determine if there are unique SNPs that co-segregate with the disease. Particular attention will be paid to those variants that are predicted to affect PLA2R structure or function. 2. Do anti-PLA2R autoantibodies activate complement and, if so, which IgG subclass is responsible and which pathway is activated? This aim will address the apparent paradox that IgG4, the major IgG subclass in idiopathic MN and predominant anti-PLA2R subclass, is incapable of activating the classical complement pathway, yet complement components are commonly present in the glomerular immune deposits in idiopathic MN. The presence of mannan-binding lectin (MBL) and activated C4 but absent C1q in the immune deposits suggests that the lectin pathway may be involved. It is noteworthy that immunoglobulins lacking terminal galactose on Fc N-linked glycans have been shown to activate MBL. Hypothesis: IgG4 anti-PLA2R autoantibodies may activate complement via the lectin pathway. Approach: The ability of PLA2R IgG subclasses to activate complement will be assessed. If IgG4 activates complement, its ability to bind and activate MBL will be determined and its glycosylation state examined. 3. Is recurrent MN in transplanted human kidneys associated with circulating anti-PLA2R? Idiopathic MN frequently recurs in the transplanted kidney and is associated with a high risk of allograft loss. There is presently no way to predict which patients are likely to recur. Hypothesis: The presence of circulating anti-PLA2R will predict the recurrence of MN. Approach: Pretransplant and serial post-transplant sera from patients with idiopathic MN will be tested to determine if the presence of anti-PLA2R antibodies predates and presages the recurrence of MN.