Renal DNase Research Articles

Renal Dnase1 expression is regulated by FGF23 but loss of Dnase1 does not alter renal phosphate handling

Fibroblast growth factor 23 (FGF23) is a bone-derived endocrine hormone that regulates phosphate and vitamin D metabolism. In models of FGF23 excess, renal deoxyribonuclease 1 (Dnase1) mRNA expression is downregulated. Dnase-1 is an endonuclease which binds monomeric actin. We investigated whether FGF23 suppresses renal Dnase-1 expression to facilitate endocytic retrieval of renal sodium dependent phosphate co-transporters (NaPi-IIa/c) from the brush border membrane by promoting actin polymerization. We showed that wild type mice on low phosphate diet and Fgf23−/− mice with hyperphosphatemia have increased renal Dnase1 mRNA expression while in Hyp mice with FGF23 excess and hypophosphatemia, Dnase1 mRNA expression is decreased. Administration of FGF23 in wild type and Fgf23−/− mice lowered Dnase1 expression. Taken together, our data shows that Dnase1 is regulated by FGF23. In 6-week-old Dnase1−/− mice, plasma phosphate and renal NaPi-IIa protein were significantly lower compared to wild-type mice. However, these changes were transient, normalized by 12 weeks of age and had no impact on bone morphology. Adaptation to low and high phosphate diet were similar in Dnase1−/− and Dnase1+/+ mice, and loss of Dnase1 gene expression did not rescue hyperphosphatemia in Fgf23−/− mice. We conclude that Dnase-1 does not mediate FGF23-induced inhibition of renal tubular phosphate reabsorption.

A rationally engineered DNase1-Fc fusion protein ameliorates autoimmune glomerulonephritis

Abstract Loss of B cell tolerance and generation of autoreactive anti-nuclear antibodies are hallmarks of systemic lupus erythematosus and lupus nephritis. The mechanism of anti-nuclear immunoglobulin accumulation and clearance in lupus nephritis pathogenesis remains largely uncharacterized. Here, we show that innate immune activation in the NZB/W F1 mouse model and human lupus nephritis biopsies rapidly reduces expression of DNase1 (also called DNase I) in renal cortex proximal tubular cells. Unexpectedly, kidney DNase1 levels were no longer affected by TLR ligand agonism upon depletion of macrophages, indicating that local macrophage activation is required for the downmodulation of DNase1 in kidney epithelial cells. To overcome the loss of endogenous DNase1, we rationally designed a hyperactive actin-resistant variant of DNase1 with improved catalytic activity and acceptable in vivo pharmacokinetics. The engineered DNase1-Fc fusion protein ameliorates nephritis in a murine model of lupus and reduces immune complex deposition/complement fixation. Our data suggest that the loss of renal DNase1 through TLR signaling or other innate immune activation impairs clearance of autoreactive anti-nuclear immune complex deposits in the kidney to promote nephritis progression. While autoantibodies and autoreactive B cell activation are central to lupus pathogenesis, we hypothesize that renal cortical epithelial cells expressing high levels of DNase1 serve as gatekeepers to prevent detrimental nuclear immune complex deposition. Our findings provide a therapeutic rationale for using an engineered hyperactive actin-resistant DNase1-Fc for lupus nephritis.

A Rationally Engineered Hyperactive Actin‐Resistant DNase1‐Fc Fusion Protein Ameliorates Autoimmune Glomerulonephritis

Loss of B cell tolerance and generation of autoreactive anti‐nuclear antibodies are hallmarks of systemic lupus erythematosus (SLE) and lupus nephritis. Lupus nephritis is characterized by glomerular and tubulointerstitial inflammation often initiated by the renal glomerular deposition of anti‐nuclear immune complexes which trigger subsequent activation of complement, macrophages/monocytes and other innate inflammatory cells. The mechanism of anti‐nuclear immunoglobulin accumulation and clearance in lupus nephritis pathogenesis remains largely uncharacterized. Here, we show that innate immune activation in the NZB/W F1 mouse model and in human lupus nephritis biopsies rapidly reduces DNase1 expression in renal cortex proximal tubular cells. To overcome the loss of endogenous DNase1, we treated lupus‐prone mice with a hyperactive actin resistant variant of DNase1 with improved catalytic activity against nucleic acid‐IgG immune complexes and acceptable in vivo pharmacokinetics. Hyperactive DNase1‐Fc fusion protein ameliorates nephritis in a murine model of lupus nephritis and reduces immune complex deposition/complement fixation. Taken together, our data suggest that the loss of renal DNase1 through TLR signaling or other innate immune activation impairs clearance of autoreactive anti‐nuclear immune complex deposits in the kidney to promote nephritis progression. Our findings provide a therapeutic rationale for using an engineered DNase1‐Fc as a potential therapeutic approach in lupus nephritis.Support or Funding InformationAll authors are current or previous employees of Genentech, Inc.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lupus nephritis: low urinary DNase I levels reflect loss of renal DNase I and may be utilized as a biomarker of disease progression.

Renal DNase I is lost in advanced stages of lupus nephritis. Here, we determined if loss of renal DNase I reflects a concurrent loss of urinary DNase I, and whether absence of urinary DNase I predicts disease progression. Mouse and human DNase I protein and DNase I endonuclease activity levels were determined by western blot, gel, and radial activity assays at different stages of the murine and human forms of the disease. Cellular localization of DNase I was analyzed by immunohistochemistry, immunofluorescence, confocal microscopy, and immunoelectron microscopy. We further compared DNase I levels in human native and transplanted kidneys to determine if the disease depended on autologous renal genes, or whether the nephritic process proceeded also in transplanted kidneys. The data indicate that reduced renal DNase I expression level relates to serious progression of lupus nephritis in murine, human native, and transplanted kidneys. Notably, silencing of renal DNase I correlated with loss of DNase I endonuclease activity in the urine samples. Thus, urinary DNase I levels may therefore be used as a marker of lupus nephritis disease progression and reduce the need for renal biopsies.

Lupus nephritis progression in FcγRIIB-/-yaa mice is associated with early development of glomerular electron dense deposits and loss of renal DNase I in severe disease.

FcγRIIB-/-yaa mice develop severe lupus glomerulonephritis due to lack of an inhibitory immune cell receptor combined with a Y-chromosome linked autoimmune accelerator mutation. In the present study, we have investigated nephritis development and progression in FcγRIIB-/-yaa mice to find shared features with NZB/NZW F1 lupus prone mice and human disease. We sacrificed 25 male FcγRIIB-/-yaa mice at various disease stages, and grouped them according to activity and chronicity indices for lupus nephritis. Glomerular morphology and localization of electron dense deposits containing IgG were further determined by immune electron microscopy. Renal DNase I and pro-inflammatory cytokine mRNA levels were measured by real-time quantitative PCR. DNase I protein levels was assessed by immunohistochemistry and zymography. Our results demonstrate early development of electron dense deposits containing IgG in FcγRIIB-/-yaa mice, before detectable levels of serum anti-dsDNA antibodies. Similar to NZB/NZW F1, electron dense deposits in FcγRIIB-/-yaa progressed from being confined to the mesangium in the early stage of lupus nephritis to be present also in capillary glomerular basement membranes. In the advanced stage of lupus nephritis, renal DNase I was lost on both transcriptional and protein levels, which has previously been shown in NZB/NZW F1 mice and in human disease. Although lupus nephritis appears on different genetic backgrounds, our findings suggest similar processes when comparing different murine models and human lupus nephritis.

Nephritogenic-antinephritogenic antibody network in lupus glomerulonephritis

Lupus glomerulonephritis (LGN) is one of the most threatening manifestations of systemic lupus erythematosus (SLE) and a major predictor of poor prognosis. The mechanisms leading to kidney inflammation are not completely clear; however, autoantibodies seem to play a pivotal role. Apoptosis dysregulation in SLE is likely to trigger generation of autoantibodies, the released nucleosomes being the driving autoantigen for further epitope amplification and selection of DNA or nucleosome-specific B cells. Growing evidence supports a multistep path to LGN involving initial autoantibody binding to chromatin fragments in the mesangial matrix, where they can induce mesangial inflammation leading to a shut-down of the renal DNase gene, generation and deposition of secondary necrotic chromatin on the glomerular basement membrane favouring antibody binding, complement activation and development of membrano-proliferative glomerular lesions. Anti-DNA IgG antibodies display the major pathogenetic potential in LGN initiation; however, other isotypes (IgA or IgE) as well as autoantibodies targeting other molecules (e.g. anti-C1q, anti-C reactive protein) can perpetuate renal injury. Conversely, protective autoantibodies are also likely in SLE which can contain renal damage targeting either DNA (i.e. IgM anti-DNA) or other molecules (e.g. pentraxin 3). Thus, lupus nephritogenic-antinephritogenic antibodies orchestrate the balance between harm and defence of renal tissue.

Renal Dnase1 Enzyme Activity and Protein Expression Is Selectively Shut Down in Murine and Human Membranoproliferative Lupus Nephritis

BackgroundDeposition of chromatin-IgG complexes within glomerular membranes is a key event in the pathogenesis of lupus nephritis. We recently reported an acquired loss of renal Dnase1 expression linked to transformation from mild to severe membranoproliferative lupus nephritis in (NZBxNZW)F1 mice. As this may represent a basic mechanism in the progression of lupus nephritis, several aspects of Dnase1 expression in lupus nephritis were analyzed.Methodology/Principal FindingsTotal nuclease activity and Dnase1 expression and activity was evaluated using in situ and in vitro analyses of kidneys and sera from (NZBxNZW)F1 mice of different ages, and from age-matched healthy controls. Immunofluorescence staining for Dnase1 was performed on kidney biopsies from (NZBxNZW)F1 mice as well as from human SLE patients and controls. Reduced serum Dnase1 activity was observed in both mesangial and end-stage lupus nephritis. A selective reduction in renal Dnase1 activity was seen in mice with massive deposition of chromatin-containing immune complexes in glomerular capillary walls. Mice with mild mesangial nephritis showed normal renal Dnase1 activity. Similar differences were seen when comparing human kidneys with severe and mild lupus nephritis. Dnase1 was diffusely expressed within the kidney in normal and mildly affected kidneys, whereas upon progression towards end-stage renal disease, Dnase1 was down-regulated in all renal compartments. This demonstrates that the changes associated with development of severe nephritis in the murine model are also relevant to human lupus nephritis.Conclusions/SignificanceReduction in renal Dnase1 expression and activity is limited to mice and SLE patients with signs of membranoproliferative nephritis, and may be a critical event in the development of severe forms of lupus nephritis. Reduced Dnase1 activity reflects loss in the expression of the protein and not inhibition of enzyme activity.

Deposition of chromatin‐IgG complexes in skin of nephritic MRL‐lpr/lpr mice is associated with increased local matrix metalloprotease activities

Chromatin-IgG complexes appear as electron dense structures (EDS) in glomerular basement membranes in lupus nephritis. Here, we present results of comparative analyses of the composition of EDS in murine lupus dermatitis and nephritis. One focus was to perform an analytical approach to understand why such complex structures bind skin basement membrane components. Transcription of skin membrane-encoding genes was analysed to see if expression of such genes was increased, eventually indicating that binding capacity of immune complexes increased when dermatitis developed. Variations in matrix metalloprotease 2 (MMP2), MMP9 and Dnase1 mRNA levels and enzymatic activities were correlated with circulatory chromatin-IgG complexes and deposition in skin. We also examined if glomerular deposits of EDS predicted similar deposits in skin of (NZB x NZW)F1 or MRL-lpr/lpr mice, as we observed chromatin-IgG complexes in capillary lumina in skin and glomeruli in both strains. EDS consisting of chromatin fragments and IgG were found sub-epidermally in skin with LE-like lesions of end-stage nephritic MRL-lpr/lpr mice. Dermal MMP-encoding genes were up-regulated during disease progression, and gelatinolytic activity was increased in affected skin. Dnase1 mRNA level and total nuclease activity remained stable in skin during the disease, in contrast to progressive loss of renal Dnase1 mRNA and total renal nuclease activity during development of nephritis. Loss of renal Dnase1 may explain release of chromatin fragments, while increased MMP activity may disrupt membranes making them accessible for chromatin fragment-IgG complexes. Circulatory chromatin-IgG complexes, and up-regulated intradermal MMP activity may be crucial for deposition of immune complexes in skin of lupus-prone mice.

Nuclease deficiencies promote end‐stage lupus nephritis but not nephritogenic autoimmunity in (NZB × NZW) F1 mice

New information has profoundly improved our insight into the processes that account for lupus nephritis. This review summarizes the data proving that secondary necrotic chromatin fragments are generated and retained in kidneys at time-points when the major renal nuclease Dnase-1 is selectively and severely downregulated. Second, we discuss data, which may indicate that nuclease deficiencies are not associated with autoimmunity to chromatin. Secondary to downregulation of renal Dnase-1, large chromatin fragment-immunoglobulin G complexes are accumulated in glomerular basement membranes of patients producing anti-chromatin autoantibodies. Exposure of chromatin in situ in glomeruli is the factor that renders anti-chromatin (anti-dsDNA and anti-nucleosome) antibodies nephritogenic. Without exposed chromatin, they circulate as non-pathogenic antibodies. This shows that acquired loss of renal Dnase-1 enzyme activity is a dominant event responsible for the progression of lupus nephritis into end-stage disease. Before the loss of Dnase-1, lupus-prone (NZB × NZW) F1 mice develop mild or silent nephritis with mesangial immune complex deposits, which correlates solely with onset of anti-dsDNA antibody production. The principal cellular and molecular requirements needed to produce these autoantibodies have been explained experimentally, but the mechanism(s) accounting for them in vivo in context of lupus nephritis have not yet been determined. However, published data show that defects in nucleases operational in apoptotic or necrotic cell death are not associated with the induction of nephritogenic anti-dsDNA autoantibodies. The data discussed in this study explain how an unusual exposure of chromatin may be a central factor in the evolution of lupus nephritis in (NZB x NZW) F1 mice, but not in promoting nephritogenic chromatin-specific autoimmunity.

Anti-dsDNA Antibodies Promote Initiation, and Acquired Loss of Renal Dnase1 Promotes Progression of Lupus Nephritis in Autoimmune (NZBxNZW)F1 Mice

BackgroundLupus 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/PrincipalsIn 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.FindingsEarly 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.SignificanceThese 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.

Nephritogenic Potential of Anti-DNA Antibodies against Necrotic Nucleosomes

Systemic lupus erythematosus is an inflammatory autoimmune syndrome of unknown cause. Kidney disease is a central and serious complication in this syndrome. Deposition of chromatin-containing immune complexes within glomerular membranes is considered a key event in the pathogenesis of lupus nephritis. One set of autoantibodies that participate in these complexes is directed against components of chromatin, particularly against double-stranded DNA (dsDNA). Matzinger's danger model implicates chromatin fragments as both inducers and glomerular targets for nephritogenic anti-dsDNA and anti-nucleosome antibodies. In context of this model, apoptosis, secondary necrosis, and exposure of chromatin fragments may causally trigger autoimmunity and subsequent lupus nephritis. The exposure of glomerular basement membrane-associated extracellular chromatin depends on an observed acquired downregulation of renal DNase1 transcription and loss of nuclease activity preceding development of severe nephritis; this downregulation would result in reduced fragmentation and clearance of chromatin fragments. These fragments bind glomerular basement membrane structures with high affinity. In addition, exposed chromatin fragments contain structures that stimulate the innate immune system through Toll-like receptors and the adaptive immune system to produce affinity-maturated pathogenic anti-chromatin and anti-dsDNA antibodies that are central to the development of lupus nephritis.

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

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. 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. 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. 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.

Tissue distribution of deoxyribonuclease I (DNase I) activity level in mice and its sexual dimorphism.

The distribution of deoxyribonuclease I (DNase I) in mouse tissue and body fluids, the effects of sex on the enzyme activity, and the postnatal developmental regulation of the enzyme were examined in C3H/He mice. DNase I activity was high in the salivary gland and urine; and moderate in the liver, kidney, spleen, preputial gland and seminal vesicle. Weak activity was detected in the pancreas, heart, lung, brain, small intestine, thymus, coagulating gland and serum. Sexual dimorphism, of which males had significantly higher activity than females, was observed in the kidney, urine and liver. This sexual dimorphism in renal DNase I activity was eliminated by gonadectomy to males and testosterone treatment to females. These results reveal sex hormonal control of DNase I production in the kidney. DNase I activity in the liver of infant mice was almost equivalent to that of adult mice while the enzyme activity in the salivary gland, kidney and spleen remained at a low level; less than 5% at one week old and less than 20% at four weeks old. These results suggest tissue specific control of DNase I activity levels during postnatal development.