Altered thymic selection by overexpressing cellular FLICE inhibitory protein in T cells causes lupus-like syndrome in a BALB/c but not C57BL/6 strain.

The cellular FLICE inhibitory protein (c-FLIP) is an endogenous inhibitor of the caspase-8 proapoptotic signaling pathway downstream of death receptors. Recent evidence indicates that the long form of c-FLIP (c-FLIP(L)) is required for proliferation and effector T-cell development. However, the role of c-FLIP(L) in triggering autoimmunity has not been carefully analyzed. We now report that c-FLIP(L) transgenic (Tg) mice develop splenomegaly, lymphadenopathy, multiorgan infiltration, high titers of auto-antibodies, and proliferative glomerulonephritis with immune complex deposition in a strain-dependent manner. The development of autoimmunity requires CD4(+) T cells and may result from impaired thymic selection. At the molecular level, c-FLIP(L) overexpression inhibits the zeta chain-associated protein tyrosine kinase of 70 kDa (ZAP-70) activation, thus impairing the signaling pathway derived from ZAP-70 required for thymic selection. Therefore, we have identified c-FLIP(L) as a susceptibility factor under the influence of epistatic modifiers for the development of autoimmunity.

Inhibition of NF-kappaB-dependent Bcl-xL expression by clusterin promotes albumin-induced tubular cell apoptosis.

Apoptosis and inflammation, important contributors to the progression of chronic kidney disease, can be influenced by clusterin (a secreted glycoprotein that regulates apoptosis) and nuclear factor-kappaB (NF-kappaB, a transcription factor modifying the expression of inflammatory genes). We studied proteinuria-induced renal disease and its influence on clusterin-mediated apoptosis. Exposure of cultured mouse proximal tubule epithelial cells to bovine serum albumin (BSA) resulted in activation of NF-kappaB and activator protein-1 (AP-1) within hours followed by a decline in their activation, decreased activation of extracellular signal-regulated kinases (ERK1/2), decreased cell-associated antiapoptotic Bcl-xL protein but increased apoptosis. Clusterin progressively increased in the media over a 3 day period. Clusterin siRNA blocked protein production, increased NF-kappaB activation, and significantly increased cellular Bcl-xL protein, thereby reducing spontaneous and BSA-induced apoptosis. An siRNA to the NF-kappaB inhibitor IkappaBalpha had similar results. BSA-stimulated NF-kappaB activation reciprocally decreased AP-1 activity by preventing ERK1/2 phosphorylation. These in vitro studies suggest that clusterin inhibits NF-kappaB-mediated antiapoptotic effects by the apparent stabilization of IkappaBalpha switching from promoting inflammation to apoptosis during proteinuria.

Renoprotective role of the vitamin D receptor in diabetic nephropathy.

1,25-Dihydroxyvitamin D3 negatively regulates the renin-angiotensin system (RAS), which plays a critical role in the development of diabetic nephropathy. We tested if mice lacking the vitamin D receptor (VDR) are more susceptible to hyperglycemia-induced renal injury. Diabetic VDR knockout mice developed more severe albuminuria and glomerulosclerosis due to increased glomerular basement membrane thickening and podocyte effacement. More fibronectin (FN) and less nephrin were expressed in the VDR knockout mice compared to diabetic wild-type mice. In receptor knockout mice, increased renin, angiotensinogen, transforming growth factor-beta (TGF-beta), and connective tissue growth factor accompanied the more severe renal injury. 1,25-Dihydroxyvitmain D3 inhibited high glucose (HG)-induced FN production in cultured mesangial cells and increased nephrin expression in cultured podocytes. 1,25-Dihydroxyvitmain D3 also suppressed HG-induced activation of the RAS and TGF-beta in mesangial and juxtaglomerular cells. Our study suggests that receptor-mediated vitamin D actions are renoprotective in diabetic nephropathy.

Altered vitamin D metabolism in type II diabetic mouse glomeruli may provide protection from diabetic nephropathy.

The db/db mouse develops features of type II diabetes mellitus as the result of impaired signaling through its abnormal leptin receptor. In spite of accurate metabolic features of diabetes, renal disease manifestations in these mice are not as severe as in humans suggesting the presence of protective genes. There is a growing body of evidence in humans for the relevance of vitamin D in diabetes. Here we followed a large cohort of db/db mice and their non-diabetic db/+ littermates. Transcriptional profiling revealed significant upregulation of 23 genes involved in Ca2+ homeostasis and vitamin D metabolism in db/db glomeruli relative to db/+ glomeruli. Increased glomerular expression of vitamin D3 1alpha-hydroxylase, vitamin D binding protein, calbindins D9K and D28K, and calcyclin mRNA was confirmed by quantitative reverse transcription-polymerase chain reaction in 20-, 36-, and 52-week-old db/db glomeruli. Although vitamin D3 1alpha-hydroxylase protein was primarily expressed and upregulated in db/db renal tubules, it was also expressed in glomerular podocytes in vivo. Serum 1,25-dihydroxyvitamin D3 and urinary Ca2+ excretion were increased >3-fold in db/db mice compared to db/+ mice. Cultured glomerular podocytes had mRNA for vitamin D3 1alpha-hydroxylase, vitamin D receptor, and calbindin D28K, each of which was increased in high glucose conditions. High glucose also led to enhanced production of fibronectin and collagen IV protein, which was blocked by 1,25-dihydroxyvitamin D3. These results show that vitamin D metabolism is altered in db/db mice leading to metabolic and transcriptional effects. The podocyte is affected by paracrine and potentially autocrine effects of vitamin D, which may explain why db/db mice are resistant to progressive diabetic nephropathy.

Glomerular complement regulation is overwhelmed in passive Heymann nephritis.

BACKGROUND: An injection of anti-Fx1A antibodies in rats leads to passive Heymann nephritis (PHN), a model of membranous nephropathy. Fx1A is a crude extract of renal cortex that contains megalin as a principal component. However, when rats are given anti-megalin antibodies, abnormal proteinuria does not occur. Because of the established complement dependence of PHN, we hypothesized that antibodies neutralizing complement regulatory proteins in the rat glomerulus also were required to induce PHN. Two likely targets are Crry and CD59, proteins abundant on the rat podocyte and contained within Fx1A that inhibit the C3 convertase and C5b-9 assembly, respectively. METHODS: Rats were injected with anti-megalin monoclonal antibodies, followed by anti-Crry and/or anti-CD59 F(ab')(2) antibodies five days later. In a second group of experiments, rats were injected with anti-Fx1A or anti-Fx1A immunodepleted of reactivity against Crry and/or CD59. RESULTS: In the setting of podocyte-associated anti-megalin monoclonal antibodies, simultaneous neutralization of Crry and CD59 function led to the development of significant proteinuria (11.0 +/- 2.1 mg/day, P < 0.001 vs. all other groups). In contrast, animals that had neither or only one of these complement regulators inhibited had normal urinary protein excretion (< or =6 mg/day). In animals given anti-Fx1A depleted of anti-Crry and/or anti-CD59, all groups developed typical PHN, characterized by heavy proteinuria and extensive glomerular deposition of C3 and C5b-9. CONCLUSION: Crry and CD59 play an important role in restraining complement-mediated injury following subepithelial immune complex deposition; however, in PHN, their regulatory capacity is overwhelmed.

Transforming growth factor-beta isoforms and glomerular injury in nephrotoxic nephritis.

BACKGROUND: Transforming growth factor-beta has three main isoforms (TGF-beta1, TGF-beta2, and TGF-beta3) that have distinct but overlapping functions in immunity, inflammation, and tissue repair. TGF-beta1 has been implicated in progressive renal scarring, but the roles of TGF-beta2 and TGF-beta3 are less clear. The purpose of this study was to characterize the expression of all three isoforms in nephrotoxic nephritis (NTN) in rats and to determine the effect of TGF-beta3 infusions on injury because of its reported combined anti-inflammatory and antifibrotic effects. METHODS: TGF-beta1, TGF-beta2, and TGF-beta3 expression was analyzed by immunohistochemistry and RNase protection assays. TGF-beta3 was administered by osmotic minipumps at 2 microg/day, a dose shown to alter glomerular macrophage function in vivo. Injury was assessed morphologically and functionally. RESULTS: The three TGF-beta isoforms showed a different distribution in normal rats and after the induction of nephritis. TGF-beta1 was only detected in glomeruli of the most severely nephritic rats. TGF-beta2 was found in glomerular neutrophils, whereas damaged podocytes expressed TGF-beta3. Infusions of TGF-beta3 did not reduce proteinuria over seven days after the induction of nephritis. They did, however, have a profound effect on glomerular macrophage number (7.76 +/- 4.1 in treated rats vs. 14.4 +/- 4.7 in controls, P < 0.02). The numbers of class II-positive macrophages were similar in the two groups, whereas class II-negative macrophages infiltrating glomeruli were significantly decreased (4.06 +/- 3.1 vs. 9.1 +/- 4.4, P < 0.02). TGF-beta did not influence the amount of glomerular matrix. CONCLUSIONS: TGF-beta isoforms have different expressions and presumptively different roles in NTN. The infusion of pharmacological doses of TGF-beta3 has profound effects on macrophages infiltrating nephritic glomeruli and reveals marked heterogeneity of infiltrating macrophages.

Augmented expression of glomerular basement membrane specific type IV collagen isoforms (alpha3-alpha5) in experimental membranous nephropathy.

In human and experimental membranous nephropathy, new extracellular matrix accumulates between, and eventually surrounds, immune deposits on the subepithelial aspect of the glomerular basement membrane (GBM). To define the nature and source of this newly deposited matrix, we studied by in situ hybridization and immunohistology the production and tissue deposition of the recently defined basement membrane type IV collagen chain isoforms alpha3, alpha4, and alpha5, the mesangium-specific alpha1 and alpha2 isoforms of type IV collagen, and the fibrillar interstitial type I collagen during the development of immunological injury in passive Heymann nephritis (PHN), a rodent model of membranous nephropathy. Our results show that steady-state mRNA levels of alpha3-alpha5 (IV) but not alpha1 (IV) are significantly increased in the glomeruli of rats with PHN at the peak of immunological injury after 14 days. Increased signal for alpha4 (IV) and the new appearance of alpha1 (I) could be clearly localized to glomerular podocytes, the target of injury in this model. In addition, increased levels of immunoreactive alpha3-alpha5 were visible in the peripheral and paramesangial GBM together with de novo deposits of type I collagen. A modest increase in mesangial staining for alpha1/alpha2 (IV) was present in PHN glomeruli. In rats depleted of complement for 5 days after PHN induction, the peak of alpha4 (IV) mRNA expression on day 14 was blunted. In conclusion, we have shown increased production of the intrinsic GBM type IV collagen isoforms alpha3-alpha5 and ectopic production of type I collagen by injured podocytes in PHN. These changes may contribute to the formation of an expanded and disorganized GBM, as seen in experimental and human membranous nephropathy.

RANTES and monocyte chemoattractant protein-1 (MCP-1) play an important role in the inflammatory phase of crescentic nephritis, but only MCP-1 is involved in crescent formation and interstitial fibrosis.

The involvement of chemokines in inflammation is well established, but their functional role in disease progression, and particularly in the development of fibrosis, is not yet understood. To investigate the functional role that the chemokines monocyte chemoattractant protein-1 (MCP-1) and RANTES play in inflammation and the progression to fibrosis during crescentic nephritis we have developed and characterized a murine model for this syndrome. Significant increases in T-lymphocytes and macrophages were observed within glomeruli and interstitium, paralleled by an induction of mRNA expression of MCP-1 and RANTES, early after disease initiation. Blocking the function of MCP-1 or RANTES resulted in significant decreases in proteinuria as well as in numbers of infiltrating leukocytes, indicating that both MCP-1 and RANTES (regulated upon activation in normal T cells expressed and secreted) play an important role in the inflammatory phase of crescentic nephritis. In addition, neutralization of MCP-1 resulted in a dramatic decrease in both glomerular crescent formation and deposition of type I collagen. These results highlight a novel role for MCP-1 in crescent formation and development of interstitial fibrosis, and indicate that in addition to recruiting inflammatory cells this chemokine is critically involved in irreversible tissue damage.

The effect of VH residues 6 and 23 on IgG3 cryoprecipitation and glomerular deposition.

MRL/lpr mice spontaneously develop a lupus-like autoimmune syndrome characterized by immunopathologic manifestations such as necrotizing vasculitis of the skin and glomerulonephritis. A feature of this autoimmune syndrome is the production of extremely large amounts of monoclonal IgG3 cryoglobulins. The structural basis of IgG3 cryoprecipitation is not well understood. Although the IgG3 isotype is necessary for cryoprecipitation, not all IgG3 antibodies cryoprecipitate. It has been postulated that electrostatic charge may be influential in cryoprecipitation. To investigate this problem, the VH and VL sequences of a panel of IgG3 cryoglobulins and non-cryoglobulins were compared, with particular attention to charged amino acid differences. At VH residues 6 and 23 the cryoglobulins were more positively charged than their non-cryoglobulin counterparts. To analyze further the effect of charge on cryoprecipitation, the sequence of an IgG3 monoclonal cryoprecipitating rheumatoid factor was modified by site-directed mutagenesis. The more positive residues at VH 6 and 23 present in some of the cryoglobulin antibodies were mutated to the more negative residues found in the non-cryoglobulins. The results show that VH residue 6 affects cryoprecipitation while residue 23 does not. When injected into normal BALB/c mice, the unmutated antibody produced glomerular immune deposits and focal glomerulonephritis, whereas loss of cryoprecipitability by mutating residue 6 completely abrogated glomerular immune deposition and glomerular injury. In contrast, the mutation at residue 23 which retains cryoprecipitability reduced glomerular immune deposition and prevented glomerular injury.

Fish oil has protective and therapeutic effects on proteinuria in passive Heymann nephritis.

Passive Heymann nephritis (PHN) is a rat model of membranous nephropathy induced by injecting anti-Fx1A. The onset of proteinuria in PHN is caused by complement-mediated injury to glomerular epithelial cells (GEC) accompanied by enhanced glomerular eicosanoid production. In addition, sublethal injury by complement of rat GECs in culture leads to phospholipase activation, phospholipid hydrolysis and release of arachidonic acid and dienoic prostanoids. Based on these findings, we undertook to determine if substituting arachidonic acid (omega-6) in GEC membrane phospholipids with omega-3 fatty acids derived from fish oil would alter the development and course of proteinuria in PHN. We found that rats fed a diet containing 10% fish oil for four weeks prior to antibody injection developed 50 to 60% less proteinuria between two and six weeks after anti-Fx1A than rats fed an equivalent diet containing 10% safflower oil, and had substantial enrichment of glomerular phospholipids with omega-3 fatty acids and displacement of arachidonic acid. This outcome was associated with a 50% reduction in release of glomerular thromboxane B2 (stable metabolite of thromboxane A2) in the fish oil group. More importantly, when PHN rats with well established proteinuria while on regular chow were randomized to three dietary groups, those fed fish oil had a 25 to 50% decline in proteinuria as compared to those fed lard or safflower oil. This difference was evident within two weeks of randomization and persisted until the end of the study after eight weeks. In neither study could the differences in urine protein excretion be accounted for by protein or calorie deprivation, or by differences in blood pressure, renal function, immune response to sheep IgG, or glomerular deposition of IgG or complement. Thus, our results indicate that dietary fish oil has protective and therapeutic effects with regard to proteinuria in PHN. These benefits may relate to alterations in membrane phospholipid composition in favor of omega-3 fatty acids and release of less reactive trienoic eicosanoids.

Expression of type I collagen mRNA in glomeruli of rats with passive Heymann nephritis.

In passive Heymann nephritis (PHN) glomeruli exhibit marked basement membrane expansion around subepithelial immune deposits but they fail to show any change in mRNA levels of type IV collagen, laminin or fibronectin by Northern and slot-blot analysis, or in the amount or distribution of type IV collagen or laminin by immunohistology for up to 12 weeks after disease onset. On the other hand, in situ hybridization (ISH) revealed the appearance of positive cells exhibiting mRNA for the alpha 1 chain of rat type I collagen two to three weeks after the onset of PHN in all glomeruli of all rats. Positive cells persisted for at least eight weeks. In many glomeruli, the location of the clusters of silver grains suggested that they were in visceral epithelial cells. In controls injected with normal sheep IgG, and in early PHN (< 11 days after sheep anti-Fx1A), glomeruli were negative but cells in the renal capsule and adventitia of vessels showed strong ISH and served as positive controls. RNAse pre-treatment and the "sense" probe gave appropriately negative results. RNA from PHN glomeruli contained an alpha 1 type I collagen transcript of the same size as that from rat fibroblasts. These results show that the evolution of glomerular basement membrane expansion in rat membranous nephropathy coincides with the induction of a matrix gene that is not normally expressed in glomerular cells. Further, they suggest that the intercalation of ectopically-expressed matrix molecules may contribute to the production of a disorganized basement membrane.

Nephrotoxic antiserum identifies a beta 1-integrin on rat glomerular epithelial cells.

A postulated mechanism of immune glomerular injury is a direct interaction between antibody and glomerular epithelial cell (GEC) surface antigens. To explore this hypothesis, we examined the interaction of the noncomplement-fixing gamma 2-subclass of sheep anti-rat nephrotoxic serum (NTS), which causes immediate complement- and neutrophil-independent proteinuria in vivo, with rat GECs in culture. Reactivity of NTS with GEC surface antigens was determined by positive immunofluorescence of GEC plasma membranes and by the ability of NTS-coated tissue culture wells to provide an adhesive substrate for GECs. NTS immunoprecipitated two proteins (135 and 118 kDa) from surface-labeled GECs. Proteins of similar molecular mass were precipitated by a polyclonal rabbit antibody that identifies the beta 1-integrin chain of the mouse fibronectin receptor (anti-FnR). In addition, NTS identified similarly sized bands on Western blot analysis of cell membranes from isolated rat glomeruli. Similar reactivity was eluted from the glomeruli of proteinuric rats injected with NTS. NTS significantly inhibited GEC adhesion to laminin, types I and IV collagen, and fibronectin and prevented GEC spreading on types I and IV collagen. Anti-FnR similarly inhibited GEC adhesion. Cell viability was not affected. These results show that NTS recognizes a pair of GEC surface proteins that have the characteristics of an alpha- and beta 1-integrin and, at low concentrations, disrupt cell-matrix interactions.

Production and polarized secretion of basement membrane components by glomerular epithelial cells.

To study the formation of basement membrane by glomerular epithelial cells (GECs), production and secretion of type IV collagen and laminin by rat GECs in culture were evaluated. GECs produced two chains of type IV collagen (180 and 170 kDa) in the ratio of approximately 2 to 1, when immunoprecipitated with antibody to type IV collagen of mouse Engelbreth-Holm-Swarm (EHS) sarcoma. GECs also produced proteins that were precipitated by antibody to EHS laminin, i.e., two bands each in the positions of the A and B chains of mouse laminin. On enzyme-linked immunosorbent assay (ELISA), type IV collagen and laminin were found mainly in the cell-associated fraction and in the subepithelial culture medium. Confluent GECs on membrane filters formed a tight barrier against the flux of macromolecules. Under these conditions, 80% of newly synthesized and secreted matrix proteins were detected in the basolateral medium. Moreover, treatment with ammonium chloride, which is known to affect polarized secretion, caused both type IV collagen and laminin to be secreted via the basolateral and apical surfaces in similar amounts. These results indicate that cultured GECs are polarized and that they produce and secrete basement membrane components via the basolateral side.

Glomerular basement membrane expansion in passive Heymann nephritis. Absence of increased synthesis of type IV collagen, laminin, or fibronectin.

The distribution and synthetic rate of glomerular basement membrane components was examined in the Passive Heymann Nephritis model of experimental membranous nephropathy. The extensive tissue injury that developed included subepithelial electron-dense deposits, podocyte foot process effacement, and expansion of the glomerular basement membrane. Levels of mRNA for type IV collagen, laminin, and fibronectin from isolated glomeruli was quantitated by slot-blot analysis and showed no change in experimental animals as compared to controls at either 1 week, 3 weeks, or 3 months after disease induction. Immunoelectron microscopy with gold-labeled anti-laminin IgG revealed no difference in the number of particles bound to the glomerular basement membrane of experimental animals and controls. Immunofluorescence with both type IV collagen antisera and anti-laminin antibody showed no difference in the intensity or pattern of staining. Despite extensive glomerular damage and glomerular basement membrane thickening, no evidence was found for either an increase in the synthetic rate of type IV collagen, laminin, or fibronectin or for an accumulation of basement membrane laminin within the damaged glomeruli. Alternate processes, such as diminished density of matrix components or accumulation of other unmeasured matrix constituents, presumably account for the expansion of the glomerular basement membrane seen in experimental membranous nephropathy.

Studies with antibodies to cultured rat glomerular epithelial cells. Subepithelial immune deposit formation after in vivo injection.

To investigate the role of glomerular epithelial cell (GEC) membrane proteins in the in situ formation of subepithelial immune deposits, the authors raised a rabbit antiserum against GEC that had been grown in culture (anti-GEC). By indirect immunofluorescence (IF) on normal rat kidney, anti-GEC stained proximal tubular brush border (BB). After intravenous injection into animals, granular glomerular capillary wall staining for IgG was present by IE and subepithelial immune deposits were identified by standard transmission and immunoelectron microscopy. Using the latter technique, injected anti-GEC IgG was identified beneath slit diaphragms and in endocytic-coated pits and intracellular vesicles of podocytes. Anti-GEC immunoprecipitated gp330 and two other proteins from radiolabeled BB. These proteins also were identified by sheep anti-rat Fx1A, the antiserum responsible for passive Heymann nephritis. Anti-GEC and anti-Fx1A also immunoprecipitated five identical proteins from surface-labeled GEC. Biosynthetically-labeled but not surface-labeled GEC contained immunoprecipitable gp330. Thus, injection into rats of antibodies raised against cultured GEC can produce subepithelial immune deposits, a disease process classically induced by antibodies to BB or its purified components. In addition to gp330, GEC and BB share other antigenic determinants that may contribute to the formation of these immune deposits.

Renal biopsy in the normal cat: examination of the effects of repeated needle biopsy.

Three healthy cats were subjected to percutaneous renal biopsy of the left kidney on three occasions at monthly intervals. Three other cats were subjected to three consecutive biopsy attempts on one occasion using the left kidney. Thereafter the six cats were monitored clinically and by means of laboratory analyses of blood and urine until euthanasia four weeks after the last biopsy. Cautious insertion of the biopsy needle in an attempt to avoid over penetration of the kidney resulted in failure to obtain renal tissue on six occasions but in all 12 specimens which did contain renal tissue, glomeruli were present. Major blood vessels were present in two biopsy specimens. At necropsy, radiographic and histological studies demonstrated renal parenchymal and vascular changes in the biopsied kidneys which were similar to but less severe than those produced by a single biopsy attempt. This confirmed that avoidance of damage to major renal vessels is important and suggested that, with care, repeated biopsies need be no more harmful to the kidney than a single biopsy.

Renal biopsy in the normal cat: an examination of the effects of a single needle biopsy.

Eleven healthy cats were subjected to a single, percutaneous renal biopsy with a disposable biopsy needle and thereafter monitored clinically and by means of laboratory analyses of blood and urine until euthanasia at intervals up to two months after biopsy. Biopsy specimens were obtained at each attempt and the specimen length and numbers of glomeruli compared favourably with results from normal dogs biopsied with Franklin-Silverman needles. At necropsy radiographic studies demonstrated renal vascular changes and histological examinations of the biopsied kidneys revealed lesions varying from barely discernible linear scars to extensive haemorrhage and wedge-shaped infarcts. A direct relationship was established between the severe renal lesions in seven cats and biopsy specimens containing medullary tissue and major renal blood vessels.