Proteomic Analysis Identifies Distinct Glomerular Extracellular Matrix in Collapsing Focal Segmental Glomerulosclerosis.

BACKGROUND: The mechanisms leading to extracellular matrix (ECM) replacement of areas of glomerular capillaries in histologic variants of FSGS are unknown. This study used proteomics to test the hypothesis that glomerular ECM composition in collapsing FSGS (cFSGS) differs from that of other variants. METHODS: ECM proteins in glomeruli from biopsy specimens of patients with FSGS not otherwise specified (FSGS-NOS) or cFSGS and from normal controls were distinguished and quantified using mass spectrometry, verified and localized using immunohistochemistry (IHC) and confocal microscopy, and assessed for gene expression. The analysis also quantified urinary excretion of ECM proteins and peptides. RESULTS: Of 58 ECM proteins that differed in abundance between cFSGS and FSGS-NOS, 41 were more abundant in cFSGS and 17 in FSGS-NOS. IHC showed that glomerular tuft staining for cathepsin B, cathepsin C, and annexin A3 in cFSGS was significantly greater than in other FSGS variants, in minimal change disease, or in membranous nephropathy. Annexin A3 colocalized with cathepsin B and C, claudin-1, phosphorylated ERK1/2, and CD44, but not with synaptopodin, in parietal epithelial cells (PECs) infiltrating cFSGS glomeruli. Transcripts for cathepsins B and C were increased in FSGS glomeruli compared with normal controls, and urinary excretion of both cathepsins was significantly greater in cFSGS compared with FSGS-NOS. Urinary excretion of ECM-derived peptides was enhanced in cFSGS, although in silico analysis did not identify enhanced excretion of peptides derived from cathepsin B or C. CONCLUSIONS: ECM differences suggest that glomerular sclerosis in cFSGS differs from that in other FSGS variants. Infiltration of activated PECs may disrupt ECM remodeling in cFSGS. These cells and their cathepsins may be therapeutic targets.

Characterization of glomerular extracellular matrix by proteomic analysis of laser-captured microdissected glomeruli.

Abnormal extracellular matrix (ECM) remodeling is a prominent feature of many glomerular diseases and is a final common pathway of glomerular injury. However, changes in ECM composition accompanying disease-related remodeling are unknown. The physical properties of ECM create challenges for characterization of composition using standard protein extraction techniques, as the insoluble components of ECM are frequently discarded and many ECM proteins are in low abundance compared to other cell proteins. Prior proteomic studies defining normal ECM composition used a large number of glomeruli isolated from human kidneys retrieved for transplantation or by nephrectomy for cancer. Here we examined the ability to identify ECM proteins by mass spectrometry using glomerular sections compatible with those available from standard renal biopsy specimens. Proteins were classified as ECM by comparison to the Matrisome database and previously identified glomerular ECM proteins. Optimal ECM protein identification resulted from sequential decellularization and protein extraction of 100 human glomerular sections isolated by laser capture microdissection from either frozen or formalin-fixed, paraffin-embedded tissue. In total, 147 ECM proteins were identified, including the majority of structural and GBM proteins previously identified along with a number of matrix and glomerular basement membrane proteins not previously associated with glomeruli. Thus, our study demonstrates the feasibility of proteomic analysis of glomerular ECM from retrieved glomerular sections isolated from renal biopsy tissue and expands the list of known ECM proteins in glomeruli.

Renal thrombotic microangiopathy and podocytopathy associated with the use of carfilzomib in a patient with multiple myeloma.

BACKGROUND: Proteasome inhibitors are a relatively new class of chemotherapeutic agents. Bortezomib is the first agent of this class and is currently being used for the treatment of multiple myeloma. However, recent reports have linked exposure to bortezomib with the development of thrombotic microangiopathy. A new agent in this class, carfilzomib, has been recently introduced as alternative therapy for relapsing and refractory multiple myeloma. We report a case of renal thrombotic microangiopathy associated with the use of carfilzomib in a patient with refractory multiple myeloma. CASE PRESENTATION: A 62 year-old Caucasian man with hypertension and a 4-year history of multiple myeloma, had been previously treated with lenalidomide, bortezomib and two autologous hematopoietic stem cell transplants. After the second hematopoietic stem cell transplant, he developed acute kidney injury secondary to septic shock and required dialysis for 4 weeks. Subsequently, his serum creatinine stabilized at 2.1 mg/dL (185.64 µmol/L). Seventeen months after the second hematopoietic stem cell transplant, he was initiated on carfilzomib for relapse of multiple myeloma. Six weeks later, he developed abrupt worsening of lower extremity edema and hypertension, and new onset proteinuria. His kidney function remained stable. Kidney biopsy findings were consistent with thrombotic microangiopathy. Eight weeks after discontinuation of carfilzomib, proteinuria and hypertension improved. Due to progression of multiple myeloma, he died a few months later. CONCLUSION: In view of the previously reported association of bortezomib with thrombotic microangiopathy, the temporal association of the clinical picture with the initiation of carfilzomib, and the partial resolution of symptoms after discontinuation of the drug, we conclude that carfilzomib may have precipitated a case of clinically evident renal thrombotic microangiopathy in our patient.

Moving Forward With Biologics in Lupus Nephritis.

The majority of patients with systemic lupus erythematosus develop lupus nephritis (LN) which significantly contributes to increased risks of hospitalizations, ESRD, and death. Unfortunately, treatments for LN have not changed over the past 15 years. Despite continued efforts to elucidate the pathogenesis of LN, no new drugs have yet replaced the standard-of-care regimens of cyclophosphamide or mycophenolate mofetil plus high-dose corticosteroids. The significant limitations of standard-of-care are low complete response rates, risk of flares, and ongoing inflammation in the kidney leading to progressive renal dysfunction. Repeat and prolonged treatments are often needed to control disease, leading to a high level of severe side effects. The development of targeted drugs with better efficacy and safety are desperately needed. The rationale for targeting key immunologic pathways in LN continues to be strongly supported by basic and translational research and has generated the hope and excitement of testing these therapies in human LN. This review provides an overview of biologics studied to date in clinical trials of LN, discusses the potential reasons for their failure, and addresses the challenges moving forward.

Comparison of the Rate of Renal Function Decline in NonProteinuric Patients With and Without Diabetes.

BACKGROUND: Patients with diabetes and chronic kidney disease (CKD) without proteinuria are often believed to have a cause of CKD other than diabetes. It was hypothesized that if this is true, the rate of renal function decline should be similar among nonproteinuric patients with and without diabetes. METHODS: Patients seen in the nephrology, endocrinology and general internal medicine clinics at the Medical University of South Carolina (MUSC) between 2008 and 2012 with hypertension and diabetes were identified by ICD9 diagnosis codes. Patients with less than 2 measures of serum creatinine, without urine studies over the study period and with proteinuria were excluded. Four hundred seventy-two patients met the inclusion and exclusion criteria and had an initial estimated glomerular filtration rate (eGFR) between 35 and 80 mL/min per 1.73 m2. The annual rate of decline in eGFR was estimated for each patient from the lowest eGFR in each year by fitting a regression model with random intercept and slope. RESULTS: In unadjusted analyses, the rate of eGFR decline was greater in patients with diabetes than without diabetes (-0.71 versus -0.30 mL · min(-1) · yr(-1), P = 0.03). After adjusting for age, race, sex, baseline eGFR and use of renin-angiotensin-aldosterone system blockade, the rate of decline was still greater among patients with diabetes than among those without diabetes (-0.68 versus -0.36 mL · min(-1) · yr(-1), P = 0.03). CONCLUSIONS: Patients with diabetes had more rapid decline in kidney function compared with individuals without diabetes, despite the absence of proteinuria. These results suggest that even in the absence of proteinuria, diabetes may be associated with CKD.

Changing the concepts of immune-mediated glomerular diseases through proteomics.

Standard classification of glomerular diseases is based on histopathologic abnormalities. The recent application of proteomic technologies has resulted in paradigm changes in the understanding and classification of idiopathic membranous nephropathy and membranoproliferative glomerulonephritis. Those examples provide evidence that proteomics will lead to advances in understanding of the molecular basis of other glomerular diseases, such as lupus nephritis. Proof of principle experiments show that proteomics can be applied to patient renal biopsy specimens. This viewpoint summarizes the advances in immune-mediated glomerular diseases that have relied on proteomics, and potential future applications are discussed.

Lupus-like glomerulonephritis: an autoimmune complication of hepatitis C infection.

Lupus-like glomerulnephritis in patients with negative lupus serologies and no extra-renal manifestations of lupus can create a diagnostic dilemma. We describe a 53-year-old gentleman with chronic hepatitis C virus (HCV) infection who presented with dialysis-requiring renal failure, renal histologic findings of "full-house" immunofluorescence label and tubuloreticular inclusions on electronic microscopy, but no extra-renal or laboratory signs of systemic lupus erythematosis. Attempted treatment with cyclophosphamide and corticosteroids was limited by cyclophosphamide hypersensitivity. The patient remained dialysis-dependent over 18 months of observation and did not develop extra-renal clinical or biological manifestations of lupus. Mimics of seronegative lupus with isolated renal involvement can include HCV-related autoimmunity. Treatment of acute glomerulonephritis may be similar initially, but other concerns in patients with HCV infection include excluding cryoglobulinemia, the potential impact of immunosuppression therapy on liver disease and consideration of subsequent use of antiviral therapy. Given the increasing prevalence of HCV globally, the recognition of extra-hepatic autoimmune manifestations of HCV infection will likely increase.