Key Points: Neutrophil degranulation participates in glomerular injury in proliferative lupus nephritis. Urine excretion of neutrophil granule proteins is a potential diagnostic for proliferative lupus nephritis. Background: Lupus nephritis (LN) occurs in more than half of patients with systemic lupus erythematosus, but the cellular and molecular events that contribute to LN are not clearly defined. We reported previously that neutrophil degranulation participates in glomerular injury in mouse models of acute LN. This study tests the hypothesis that glomerular recruitment and subsequent activation of neutrophils result in urine excretion of neutrophil granule constituents that are predictive of glomerular inflammation in proliferative LN. Methods: Urine and serum levels of 11 neutrophil granule proteins were measured by antibody-based array in patients with proliferative LN and healthy donors (HDs), and the results were confirmed by ELISA. Glomerular neutrophil accumulation was assessed in biopsies of patients with LN who contributed urine for granule cargo quantitation and normal kidney tissue by microscopy. Degranulation was measured by flow cytometry in neutrophils isolated from patients with LN and HD controls by cell surface granule markers CD63 (azurophilic), CC66b (specific), and CD35 (secretory). Nonparametric statistical analyses were performed and corrected for multiple comparisons. Results: Eight granule proteins (myeloperoxidase, neutrophil elastase, azurocidin, olfactomedin-4, lactoferrin, alpha-1-acid glycoprotein 1, matrix metalloproteinase 9, and cathelicidin) were significantly elevated in urine from patients with active proliferative LN by array and/or ELISA, whereas only neutrophil elastase was increased in LN serum. Urine excretion of alpha-1-acid glycoprotein 1 declined in patients who achieved remission. The majority of LN glomeruli contained ≥3 neutrophils. Basal levels of specific granule markers were increased in neutrophils from patients with LN compared with HD controls. Serum from patients with active LN stimulated specific and secretory, but not azurophilic granule, release by HD neutrophils. Conclusions: Circulating neutrophils in patients with LN are primed for enhanced degranulation. Glomerular recruitment of those primed neutrophils leads to release and urine excretion of neutrophil granule cargo that serves as a urine marker of active glomerular inflammation in proliferative LN.

Background: Lupus nephritis (LN) is a prevalent and severe complication of systemic lupus erythematosus (SLE). Non-invasive diagnostics are limited, and current therapies have inadequate response rates. Expression of the chemokine Interferon-γ-induced protein 10 (IP-10) is regulated by Interferon-γ signaling and NF-κB, and its molecular activity and enhanced urine concentrations are implicated in LN, but its utility as a diagnostic marker and association with demographic, clinical, or pathologic features is not defined. Methods: 38 LN patients and 11 patients with non-LN glomerular diseases (GD) with active disease were included. Eighteen of the LN patients had achieved remission at one follow-up during the study time. Serum and urine were obtained from these samples, and the IP-10 levels were measured. Results: Serum and urine IP-10 levels are significantly enhanced in LN patients with active disease as compared with normal individuals (serum average 179.7 pg/mL vs. 7.2 pg/mL, p < 0.0001; urine average 28.7 pg/mg vs. 1.6 pg/mg, p = 0.0019) and patients with other forms of glomerular disease (serum average 179.7 pg/mL vs. 84.9 pg/mL, p = 0.0176; urine average 28.7 pg/mg vs. 0.18 pg/mg, p = 0.0011). Urine IP-10 levels are significantly higher in patients with proliferative LN (PLN) than those with membranous LN (MLN) (average 32.8 pg/mg vs. 7.6 pg/mg, p = 0.0155). Urine IP-10 levels are also higher in MLN versus primary membranous nephropathy (MN) (average 7.6 pg/mg vs. 0.2 pg/mg, p = 0.0193). Importantly, serum IP-10 levels remain elevated during active LN and LN remission, but urine IP-10 levels are decreased from active LN to remission in 72% of our patients. Lastly, serum, but not urine IP-10 levels are significantly higher in African American than White American LN patients in active LN (average 227.8 pg/mL vs. 103.4 pg/mL, p = 0.0309) and during LN remission (average 254.6 pg/mL vs. 89.2 pg/mL, p = 0.0399). Conclusions: Our findings suggest that serum and urine IP-10 measurements provide promising tests for monitoring LN activity, differentiation between classifications of LN, and differentiation between LN and other forms of glomerular disease. We also conclude that further assessment of elevated IP-10 levels in the serum and urine of high-risk populations (i.e., African American) could be beneficial in determining why many of these patients have worse outcomes and are non-responsive to standard therapeutics.

Chronic kidney disease (CKD) affects 30 million adults, costs ∼$79 billion dollars (2016) in Medicare expenditures, and is the ninth leading cause of death in the USA. The disease is silent or undiagnosed in almost half of people with severely reduced kidney function. The urine provides an ideal biofluid that is accessible to high-sensitivity mass spectrometry-based proteomic interrogation and is an indicator of renal homeostasis. While the accurate and precise diagnosis and better disease management of CKD can be aided using urine biomarkers, their discovery in excessive protein or nephrotic urine samples can present challenges. In this work, we present a mass spectrometry-based method utilizing multiplex tandem mass tag (TMT) quantification and improved protein quantification using reporter ion normalization to urinary creatinine to analyze urinary proteins from patients with a form of nephrotic syndrome (focal segmental glomerulosclerosis [FSGS]). A comparative analysis was performed for urine from patients in remission (RM) versus active disease (DX) flare. Two-dimensional LC-MS/MS TMT quantitative analysis identified over 1,058 urine proteins and 580 proteins with 2 peptides or greater and quantifiable. Normalization of TMT abundance values to creatinine per ml of urine concentrated reduced variability in 2D-TMT-LC-MS/MS experiments. Univariate and multivariate analyses showed that 27 proteins were significantly increased in a proteinuric DX flare. Hierarchical heatmap clustering showed that SERPINA1 and ORM1 were increased >1.5-fold in active DX versus RM urine samples. ELISA validation of SERPINA1 and ORM1 abundance agreed with our quantitative TMT proteomics analysis. These findings provide support for the utility of this method for identification of novel diagnostic markers of CKD and identify SERPINA1 and ORM1 as promising candidate diagnostic markers for FSGS.

Background: Lupus nephritis (LN) is kidney inflammation that commonly occurs from systemic lupus erythematosus. NF-κB activity is increased in LN patients, leading to elevated circulating concentrations of immune modulators that contribute to LN pathophysiology. This study sought to investigate this phenomenon with the aim of discovering novel serum biomarkers for LN. Methods: A multiplex antibody-based assay was performed with serum from 24 LN patients and 7 healthy controls (HC) to determine if 48 NF-κB-regulated proteins were elevated in LN patients. Confirmation ELISAs were performed on stem cell factor (SCF), macrophage colony stimulating factor (M-CSF), and interleukin-2 receptor alpha subunit (IL-2Rα) in a separate sample cohort of 27 LN patients and 10 HC. Follow-up ELISAs were performed on samples obtained from the same patients during LN remission to determine if these candidates were reliable predictors of disease activity. Comparisons of protein levels between LN patients and HC were performed using a series of 2-tailed Mann-Whitney tests. Paired samples were analyzed using a Wilcoxon matched pairs test. Two-tailed Spearman correlation analyses were used to compare serum protein concentrations with LN clinical parameters. All p-values were adjusted for multiple comparisons Results: SCF, M-CSF, and IL-2Rα were significantly elevated in LN serum. Elevated serum SCF and M-CSF were significantly correlated with elevated urine protein to creatinine ratio (UPCR), decreased estimated glomerular filtration rate (eGFR), and elevated serum creatinine. Elevated serum IL-2Rα was significantly correlated with elevated serum creatinine. Serum SCF concentration was significantly decreased during LN remission in paired samples from individuals, but it was not a good predictor at the population level (AUC= 0.6265). Discussion: We identified the NF-κB-regulated proteins SCF, M-CSF, and IL-2Rα as candidate serum biomarkers for consideration in monitoring LN activity. Our findings also indicate the importance for follow-up mechanistic studies pertaining to these inflammatory mediators.

SUMMARYPancreatic islet-like clusters differentiated from induced pluripotent or embryonic stem cells (SC-islets) are a promising cellular therapy for type 1 diabetes. A standardized and easily applied protocol to assess the quality of SC-islet differentiations would be a valuable resource to ensure consistency across cell preparations. We present a workflow for processing SC-islet clusters to single cells and for intracellular flow cytometry stains using commercially available reagents and antibodies to distinguish cell types generated during a 7-stage differentiation protocol.