Natalya Seredkina

Anti-dsDNA antibodies promote initiation, and acquired loss of renal Dnase1 promotes progression of lupus nephritis in autoimmune (NZBxNZW)F1 mice.

Background Lupus nephritis is characterized by deposition of chromatin fragment-IgG complexes in the mesangial matrix and glomerular basement membranes (GBM). The latter defines end-stage disease. Methodology/Principals In the present study we determined the impact of antibodies to dsDNA, renal Dnase1 and matrix metalloprotease (MMP) mRNA levels and enzyme activities on early and late events in murine lupus nephritis. The major focus was to analyse if these factors were interrelated, and if changes in their expression explain basic processes accounting for lupus nephritis. Findings Early phases of nephritis were associated with chromatin-IgG complex deposition in the mesangial matrix. A striking observation was that this event correlated with appearance of anti-dsDNA antibodies and mild or clinically silent nephritis. These events preceded down-regulation of renal Dnase1. Later, renal Dnase1 mRNA level and enzyme activity were reduced, while MMP2 mRNA level and enzyme activity increased. Reduced levels of renal Dnase1 were associated in time with deficient fragmentation of chromatin from dead cells. Large fragments were retained and accumulated in GBM. Also, since chromatin fragments are prone to stimulate Toll-like receptors in e.g. dendritic cells, this may in fact explain increased expression of MMPs. Significance These scenarios may explain the basis for deposition of chromatin-IgG complexes in glomeruli in early and late stages of nephritis, loss of glomerular integrity and finally renal failure.

Progression of Murine Lupus Nephritis Is Linked to Acquired Renal Dnase1 Deficiency and Not to Up-Regulated Apoptosis

The accumulation of apoptotic cells has been suggested as a possible mechanism of nucleosome conversion into self-antigens that may both initiate autoimmune responses and participate in immune complex deposition in lupus nephritis. In this study, we analyzed both the rate of transcription of apoptosis-related genes and the presence of activated apoptotic factors within kidneys of lupus-prone (NZBxNZW) F1 mice during disease progression. The results of this study demonstrated no activation of apoptotic pathways in kidneys of these lupus-prone mice at the time of appearance of anti-double standard DNA antibodies in serum, as well as the formation of mesangial immune deposits in glomeruli. In contrast, the transition of mesangial into membranoproliferative lupus nephritis coincided with an accumulation of activated caspase 3-positive cells in kidneys, in addition to a dramatic decrease in Dnase1 gene transcription. Highly reduced expression levels of the Dnase1 gene may be responsible for the accumulation of large chromatin-containing immune complexes in glomerular capillary membranes. Thus, the initiation of lupus nephritis is not linked to increased apoptotic activity in kidneys. The combined down-regulation of Dnase1 and the increased number of apoptotic cells, which is possibly due to their reduced clearance in affected kidneys, may together be responsible for the transformation of mild mesangial lupus nephritis into severe membranoproliferative, end-stage organ disease.

Lupus Nephritis: Enigmas, Conflicting Models and an Emerging Concept

Autoantibodies to components of chromatin, which include double-stranded DNA (dsDNA), histones and nucleosomes, are central in the pathogenesis of lupus nephritis. How anti-chromatin autoantibodies exert their nephritogenic activity, however, is controversial. One model assumes that autoantibodies initiate inflammation when they cross-react with intrinsic glomerular structures such as components of membranes, matrices or exposed nonchromatin ligands released from cells. Another model suggests glomerular deposition of autoantibodies in complex with chromatin, thereby inducing classic immune complex-mediated tissue damage. Recent data suggest acquired error of renal chromatin degradation due to the loss of renal DNasel enzyme activity is an important contributing factor to the development of lupus nephritis in lupus-prone (NZBxNZW)F1 mice and in patients with lupus nephritis. Down-regulation of DNasel expression results in reduced chromatin fragmentation and in deposition of extracellular chromatin-IgG complexes in glomerular basement membranes in individuals who produce IgG anti-chromatin autoantibodies. The main focus of the present review is to discuss whether exposed chromatin fragments in glomeruli are targeted by potentially nephritogenic anti-dsDNA autoantibodies or if the nephritogenic activity of these autoantibodies is explained by cross-reaction with intrinsic glomerular constituents or if both models coexist in diseased kidneys. In addition, the role of silencing of the renal DNasel gene and the biological consequences of reduced chromatin fragmentation in nephritic kidneys are discussed.

Reduced fragmentation of apoptotic chromatin is associated with nephritis in lupus-prone (NZB × NZW)F1 mice

OBJECTIVE Antinucleosome autoantibodies are pathogenic factors in lupus nephritis. Defects in apoptotic pathways may result in increased levels of apoptotic nucleosomes. The objectives of this study were 1) to determine whether low molecular weight oligonucleosomes are present in the kidneys of autoimmune (NZB x NZW)F(1) mice, 2) to analyze whether the presence of glomerular membrane-associated TUNEL-positive electron-dense structures reflect the existence of low molecular weight oligonucleosomes, and 3) to determine an eventual temporal relationship between glomerular electron-dense structures, oligonucleosomes, and proteinuria in these mice. METHODS DNA was isolated from mouse 111s34 hybridoma cells and from the kidneys of normal BALB/c mice in which apoptosis was induced by camptothecin and from the kidneys of (NZB x NZW)F(1) mice at ages 4 weeks, 8 weeks, 20 weeks, and > or = 26 weeks (nephritic mice). The DNA fragmentation pattern was determined with an Agilent bioanalyzer. An electron microscopy-based TUNEL assay was performed to detect apoptotic chromatin in glomerular membranes, and immunoelectron microscopy was used to determine antibody binding. Transcription levels for nucleases associated with apoptosis and necrosis were determined by real-time polymerase chain reaction. RESULTS DNA from camptothecin-treated cell lines and BALB/c mouse kidneys, but not that from untreated (NZB x NZW)F(1) mouse kidneys, demonstrated DNA cleavage consistent with apoptotic fragmentation. DNA from (NZB x NZW)F(1) mice was devoid of apoptotic fragmentation, irrespective of the age of the mice, whereas TUNEL-positive chromatin particles were detected in glomerular membranes in nephritic mice. Renal DNase I transcription was reduced in nephritic mice. Nucleosomal DNA fragmentation in response to camptothecin exposure was highly reduced in (NZB x NZW)F(1) mouse kidneys compared with that in their normal counterparts. CONCLUSION The results of this study demonstrate that TUNEL-positive chromatin particles are deposited in the glomeruli of nephritic (NZB x NZW)F(1) mice, due to reduced fragmentation and clearance of chromatin.

Acquired Loss of Renal Nuclease Activity Is Restricted to DNaseI and Is an Organ-Selective Feature in Murine Lupus Nephritis

An acquired loss of renal DNaseI promotes transformation of mild mesangial lupus nephritis into membranoproliferative end-stage organ disease. In this study, we analyzed expression profiles of DNaseI in other organs of lupus-prone (NZB×NZW)F1 mice during disease progression to determine whether silencing of the renal DNaseI gene is an organ-specific feature or whether loss of DNaseI reflects a systemic error in mice with sever lupus nephritis. The present results demonstrate normal or elevated levels of DNaseI mRNA and enzyme activity in liver, spleen, and serum samples from (NZB×NZW)F1 mice throughout all the stages of lupus nephritis. DNaseI activity was dramatically reduced only in kidneys of mice with sever nephritis and was the only nuclease that was down-regulated, whereas six other nucleases (DNaseII1 to 3, caspase-activated DNase, Dnase2a, and endonuclease G) were approximately normally expressed in kidneys, liver, and spleen. Loss of renal DNaseI was not accompanied by changes in serum DNaseI activity, suggesting independent mechanisms of DNaseI regulation in circulation and in kidneys and an absence of compensatory up-regulation of serum DNaseI activity in the case of renal DNaseI deficiency. Thus, silencing of renal DNaseI is a unique renal feature in membranoproliferative lupus nephritis. Determining the mechanism(s) responsible for DNaseI down-regulation might lead to the generation of new therapeutic targets to treat and prevent progressive lupus nephritis.

Review: antinucleosome antibodies: a critical reflection on their specificities and diagnostic impact

There is growing interest in testing for antibodies to nucleosomes as a diagnostic tool. In this critical review, we will discuss whether antibodies against nucleosomes are meaningful (or just meaningless) in a diagnostic context in systemic lupus erythematosus (SLE) and related disorders. In fact, the term antinucleosome antibodies may embrace all types of antibodies that can bind nucleosomes, some of which occur at a higher frequency than others, indicating that the control of tolerance to components of nucleosomes varies considerably. Central problems to resolve derive from the following major questions: What do antibodies against nucleosomes recognize: histones, histone variants, modified histones, nonhistone proteins, different double-stranded DNA (dsDNA) structures, or diverse conformational determinants unique to the complex structure in chromatin? How will we be able to identify autoantibodies to these molecules and structures using analytical assays? In our opinion, this is not possible. Here, we present examples of the diversity of nucleosome-reactive antibodies and conclude that it is principally an irresolvable task to define these antibodies as “nucleosome specific” in a unifying and meaningful way. Considering the diversity of antinucleosome antibody reactivities, the current practice of testing for antibodies against nucleosomes and to confer the results to diagnosis or disease severity is highly questionable.

Silencing of Renal DNaseI in Murine Lupus Nephritis Imposes Exposure of Large Chromatin Fragments and Activation of Toll Like Receptors and the Clec4e

Recent studies demonstrate that transformation of mild lupus nephritis into end-stage disease is imposed by silencing of renal DNaseI gene expression in (NZBxNZW)F1 mice. Down-regulation of DNaseI results in reduced chromatin fragmentation, and in deposition of extracellular chromatin-IgG complexes in glomerular basement membranes in individuals that produce IgG anti-chromatin antibodies. The main focus of the present study is to describe the biological consequences of renal DNaseI shut-down and reduced chromatin fragmentation with a particular focus on whether exposed large chromatin fragments activate Toll like receptors and the necrosis-related Clec4e receptor in murine and human lupus nephritis. Furthermore, analyses where performed to determine if matrix metalloproteases are up-regulated as a consequence of chromatin-mediated Toll like receptors/Clec4e stimulation. Mouse and human mRNA expression levels of DNaseI, Toll like receptors 7–9, Clec4e, pro-inflammatory cytokines and MMP2/MMP9 were determined and compared with in situ protein expression profiles and clinical data. We demonstrate that exposure of chromatin significantly up-regulate Toll like receptors and Clec4e in mice, and also but less pronounced in patients with lupus nephritis treated with immunosuppresants. In conclusion, silencing of renal DNaseI gene expression initiates a cascade of inflammatory signals leading to progression of both murine and human lupus nephritis. Principal component analyses biplot of data from murine and human lupus nephrits demonstrate the importance of DNaseI gene shut down for progression of the organ disease.

Impact of the tumor necrosis factor receptor-associated protein 1 (Trap1) on renal DNaseI shutdown and on progression of murine and human lupus nephritis.

Recent findings show that transformation of mild glomerulonephritis into end-stage disease coincides with shutdown of renal DNaseI expression in (NZBxNZW)F1 mice. Down-regulation of DNaseI results in reduced chromatin fragmentation and deposition of extracellular chromatin fragments in glomerular basement membranes where they appear in complex with IgG antibodies. Here, we implicate the anti-apoptotic and survival protein, tumor necrosis factor receptor-associated protein 1 (Trap1) in the disease process, based on the observation that annotated transcripts from this gene overlap with transcripts from the DNaseI gene. Furthermore, we translate these observations to human lupus nephritis. In this study, mouse and human DNaseI and Trap1 mRNA levels were determined by real-time quantitative PCR and compared with protein expression levels and clinical data. Cellular localization was analyzed by immune electron microscopy, IHC, and in situ hybridization. Data indicate that silencing of DNaseI gene expression correlates inversely with expression of the Trap1 gene. Our observations suggest that the mouse model is relevant for the aspects of disease progression in human lupus nephritis. Acquired silencing of the renal DNaseI gene has been shown to be important for progression of disease in both the murine and human forms of lupus nephritis. Early mesangial nephritis initiates a cascade of inflammatory signals that lead to up-regulation of Trap1 and a consequent down-regulation of renal DNaseI by transcriptional interference.

Glomerular Targets for Autoantibodies in Lupus Nephritis—An Apoptotic Origin

Abstract:  Systemic lupus erythematosus (SLE) is an autoimmune syndrome where different organs may individually or simultaneously be affected. Whether SLE is one disease entity or represents a variety of intrinsically unrelated organ manifestations is unknown. Variability of clinical presentations of SLE argues against the former. This does not, however, exclude that certain organ manifestations may be pathogenetically linked. It is believed that in situ binding of anti‐dsDNA antibodies by nucleosomes is involved in organ manifestations in SLE. This review will focus on nature and origin of target structures for anti‐dsDNA and anti‐nucleosome antibodies in glomerular capillary and mesangial matrix membranes. We will particularly discuss the potential role of apoptosis and release of apoptotic chromatin in terms of their putative impact in SLE.

Murine and Human Lupus Nephritis: Pathogenic Mechanisms and Theoretical Strategies for Therapy

Lupus nephritis is one of the most serious manifestations of systemic lupus erythematosus, and represents one of the criteria implemented to classify systemic lupus erythematosus. Although studied for decades, no consensus has been reached related to the basic cellular, molecular, and immunologic mechanism(s) responsible for lupus nephritis. No causal treatments have been developed; therapy is approached mainly with nonspecific immunosuppressive medications. More detailed insight into disease mechanisms therefore is indispensable to develop new therapeutic strategies. In this review, contemporary knowledge on the pathogenic mechanisms of lupus nephritis is discussed based on recent data in murine and human lupus nephritis. Specific focus is given to the effect of anti-double-stranded DNA/antinucleosome antibodies in the kidneys and whether they bind exposed chromatin fragments in glomeruli or whether they bind inherent glomerular structures by cross-recognition. Overall, the data presented here favor the exposed chromatin model because we did not find any indication to substantiate the anti-double-stranded DNA antibody cross-reacting model. At the end of this review we present data on why chromatin fragments are expressed in the glomeruli of patients with lupus nephritis, and discuss how this knowledge can be used to direct the development of future therapies.