Sphingosine-1-phosphate (S1P), a potent bioactive lipid, is emerging as a central mediator in inflammation and immune responses. We have previously implicated S1P and its synthetic enzyme sphingosine kinase (SK) in inflammatory and autoimmune disorders, including inflammatory bowel disease and rheumatoid arthritis. Generation of S1P requires phosphorylation of sphingosine by SK, of which there are two isoforms. Numerous studies have implicated SK1 in immune cell trafficking, inflammation and autoimmune disorders. In this study, we set out to determine the role of SK and S1P in lupus nephritis (LN). To this end, we examined S1P and dihydro-S1P (dh-S1P) levels in serum and kidney tissues from a mouse model of LN. Interestingly dh-S1P was significantly elevated in serum and kidney tissue from LN mice, which is more readily phosphorylated by SK2. Therefore, we employed the use of the specific SK2 inhibitor, ABC294640 in our murine model of LN. Treatment with ABC294640 did not improve vascular or interstitial pathology associated with LN. However, mice treated with the SK2 inhibitor did demonstrate decreases in glomerular pathology and accumulation of B and T cells in the spleen these were not statistically different from lpr mice treated with vehicle. LN mice treated with ABC294640 did not have improved urine thromboxane levels or urine proteinuria measurements. Both S1P and dh-S1P levels in circulation were significantly reduced with ABC294640 treatment; however, dh-S1P was actually elevated in kidneys from LN mice treated with ABC294640. Together these data demonstrate a role for SKs in LN; however, they suggest that inhibition of SK1 or perhaps both SK isoforms would better prevent elevations in S1P and dh-S1P and potentially better protect against LN.

Objective

Reactive intermediate production is an essential component of the innate immune response that is induced during disease activity in murine lupus. This study was undertaken to determine whether a marker of systemic nitric oxide (NO) production correlates with prospectively studied disease activity in human systemic lupus erythematosus (SLE) and lupus nephritis patients.

Methods

Eighty-three SLE patients and 40 control subjects were studied longitudinally. The SLE group included 23 patients with lupus nephritis documented by renal biopsy and 26 with a history of lupus nephritis. During each visit, following a 24-hour low-nitrate diet, traditional markers of disease activity and damage were determined. Serum nitrate plus nitrite (NOx) levels were determined by chemiluminescence detection.

Results

NOx levels were higher in SLE patients than in controls during the first visit. In univariate longitudinal analyses, NOx levels were associated with SLE Disease Activity Index scores. In multivariate analyses, NOx levels were associated with serum levels of C3 and creatinine and the urinary protein:creatinine ratio. Among patients with lupus nephritis, those with proliferative lesions had higher NOx levels, and higher NOx levels were associated with accumulation of renal damage and lack of response to therapy.

Conclusion

This is the first study to prospectively demonstrate longitudinal associations between serum NOx levels and markers of SLE and lupus nephritis disease activity. The more pronounced association with proliferative lupus nephritis and with longitudinal response to lupus nephritis therapy provides a rationale for the study of reactive intermediates as biomarkers of disease activity and therapeutic targets in proliferative lupus nephritis.

Background

Proliferative lupus nephritis (LN) is marked by increased renal thromboxane (TXA2) production. Targeting the TXA2 receptor or TXA2 synthase effectively improves renal function in humans with LN and improves glomerular pathology in murine LN. This study was designed to address the following hypotheses: 1) TXA2 production in the MRL/MpJ-Tnfrsf6lpr/J (MRL/lpr) model of proliferative lupus nephritis is COX2-dependent, and 2) COX2 inhibitor therapy improves glomerular filtration rate (GFR), proteinuria, markers of innate immune response, and glomerular pathology.

Methods

20 female MRL/lpr and 20 BALB/cJ mice were divided into two equal treatment groups: 1) SC-236, a moderately selective COX2 inhibitor, or 2) vehicle. After treatment from 10 to 20 weeks of age, the effectiveness of inhibition of TXA2 was determined by measuring urine TXB2. Response endpoints measured at 20 weeks of age were renal function (GFR), proteinuria, urine nitrate + nitrite (NOX), and glomerular histopathology.

Results

SC236 therapy reduced surrogate markers of renal TXA2 production during early, active glomerulonephritis. When this pharmacodynamic endpoint was reached, therapy improved GFR. Parallel reductions in markers of the innate immune response (urine NOX) during therapy were observed. However, the beneficial effect of SC236 therapy on GFR was only transient, and renal histopathology was not improved in late disease.

Conclusions

These data demonstrate that renal TXA2 production is COX2-dependent in murine LN and suggest that NO production is directly or indirectly COX2-dependent. However, COX2 inhibitor therapy in this model failed to improve renal pathology, making COX2 inhibition a less attractive approach for treating LN.

Using multiplex bead assays to measure urine proteins has a great potential for biomarker discovery, but substances in urine (the matrix) can interfere with assay measurements. By comparing the recovery of urine spiked with known quantities of several common analytes, this study demonstrated that the urine matrix variably interfered with the accurate measurement of low abundance proteins. Dilution of the urine permitted a more accurate measure of these proteins, equivalent to the standard dilution technique when the diluted analytes were above the limits of detection of the assay. Therefore, dilution can be used as an effective technique for over-coming urine matrix effects in urine immunoassays. These results may be applicable to other biological fluids in which matrix components interfere with assay performance.

Systemic lupus erythematosus (SLE) is an autoimmune syndrome marked by autoantibody production. Innate immunity is essential to transform humoral autoimmunity into the clinical lupus phenotype. Nitric oxide (NO) is a membrane-permeable signaling molecule involved in a broad array of biologic processes through its ability to modify proteins, lipids, and DNA and alter their function and immunogenicity. The literature regarding mechanisms through which NO regulates inflammation and cell survival is filled with contradictory findings. However, the effects of NO on cellular processes depend on its concentration and its interaction with reactive oxygen. Understanding this interaction will be essential to determine mechanisms through which reactive intermediates induce cellular autoimmunity and contribute to a sustained innate immune response and organ damage in SLE.

Formation of reactive nitrogen and oxygen intermediates (RNI and ROI) is an essential part of the innate immune response. Markers of systemic RNI production are increased in the setting of systemic lupus erythematosus (SLE) activity. Several lines of evidence suggest mechanisms through which the activity of inducible nitric oxide synthase (iNOS) is pathogenic in SLE, including the ability of peroxynitrite (ONOO−, a product of iNOS activity) to modify proteins, lipids, and DNA. These modifications can alter enzyme activity and may increase the immunogenicity of self antigens, leading to a break in immune tolerance. In humans, observational data suggest that overexpression of iNOS and increased production of ONOO− lead to glomerular and vascular pathology. Therapies designed to target iNOS activity or scavenge ROI and RNI are in development and may provide the means to reduce the pathogenic consequences of ROI and RNI in SLE.

Several studies have demonstrated the effectiveness of arginine analog nitric oxide synthase (NOS) inhibitor therapy in preventing and treating murine lupus nephritis. However, MRL/MpJ-FASlpr (MRL/lpr) mice lacking a functional NOS2 (inducible NOS [iNOS]) gene (NOS2−/−) develop proliferative glomerulonephritis in a fashion similar to their wild-type (wt) littermates. This finding suggests that the effect of arginine analog NOS inhibitors is through a non-iNOS–mediated mechanism. This study was designed to address this hypothesis.

NOS2−/− mice were given either vehicle or a NOS inhibitor (SD-3651) to determine if pharmacological NOS inhibition prevented glomerulonephritis, using wt mice as positive controls. Urine was collected fortnightly to measure albumin. At the time of full disease expression in wt mice, all mice were killed, and renal tissue was examined for light, immunofluorescence, and electron microscopic evidence of disease. Serum was analyzed for anti–double-stranded DNA antibody production.

NOS2−/− mice had higher serum anti–double-stranded DNA antibody antibody levels than those of wt mice. SD-3651 therapy reduced proteinuria, glomerular immunoglobulin G deposition, and electron microscopic evidence of podocytopathy and endothelial cell swelling without affecting proliferative lesions by light microscopy.

These studies confirm that genetic iNOS deficiency alone is insufficient to prevent proliferative glomerulonephritis and suggest that iNOS activity may inhibit autoantibody production. These results also suggest that SD-3651 therapy acts via a non–iNOS-mediated mechanism to prevent endothelial cell and podocyte pathology. Studies that elucidate this mechanism could provide a useful drug target for the treatment of nephritis.

Diagnosis of the type of glomerular disease that causes the nephrotic syndrome is necessary for appropriate treatment and typically requires a renal biopsy. The goal of this study was to identify candidate protein biomarkers to diagnose glomerular diseases. Proteomic methods and informatic analysis were used to identify patterns of urine proteins that are characteristic of the diseases. Urine proteins were separated by two-dimensional electrophoresis in 32 patients with FSGS, lupus nephritis, membranous nephropathy, or diabetic nephropathy. Protein abundances from 16 patients were used to train an artificial neural network to create a prediction algorithm. The remaining 16 patients were used as an external validation set to test the accuracy of the prediction algorithm. In the validation set, the model predicted the presence of the diseases with sensitivities between 75 and 86% and specificities from 92 to 67%. The probability of obtaining these results in the novel set by chance is 5 × 10−8. Twenty-one gel spots were most important for the differentiation of the diseases. The spots were cut from the gel, and 20 were identified by mass spectrometry as charge forms of 11 plasma proteins: Orosomucoid, transferrin, α-1 microglobulin, zinc α-2 glycoprotein, α-1 antitrypsin, complement factor B, haptoglobin, transthyretin, plasma retinol binding protein, albumin, and hemopexin. These data show that diseases that cause nephrotic syndrome change glomerular protein permeability in characteristic patterns. The fingerprint of urine protein charge forms identifies the glomerular disease. The identified proteins are candidate biomarkers that can be tested in assays that are more amenable to clinical testing.

Background

Lupus nephritis is divided into six classes and scored according to activity and chronicity indices based on histologic findings. Treatment differs based on the pathologic findings. Renal biopsy is currently the only way to accurately predict class and activity and chronicity indices. We propose to use patterns of abundance of urine proteins to identify class and disease indices.

Methods

Urine was collected from 20 consecutive patients immediately prior to biopsy for evaluation of lupus nephritis. The International Society of Nephrology/Renal Pathology Society (ISN/RPS) class of lupus nephritis, activity, and chronicity indices were determined by a renal pathologist. Proteins were separated by two-dimensional gel electrophoresis. Artificial neural networks were trained on normalized spot abundance values.

Results

Biopsy specimens were classified in the database according to ISN/RPS class, activity, and chronicity. Nine samples had characteristics of more than one class present. Receiver operating characteristic (ROC) curves of the trained networks demonstrated areas under the curve ranging from 0.85 to 0.95. The sensitivity and specificity for the ISN/RPS classes were class II 100%, 100%; III 86%, 100%; IV 100%, 92%; and V 92%, 50%. Activity and chronicity indices had r values of 0.77 and 0.87, respectively. A list of spots was obtained that provided diagnostic sensitivity to the analysis.

Conclusion

We have identified a list of protein spots that can be used to develop a clinical assay to predict ISN/RPS class and chronicity for patients with lupus nephritis. An assay based on antibodies against these spots could eliminate the need for renal biopsy, allow frequent evaluation of disease status, and begin specific therapy for patients with lupus nephritis.