Steroid resistant nephrotic syndrome and focal segmental glomerulosclerosis (FSGS) pose especially difficult clinical problems because the disease recurs in about 30% of such patients after renal transplantation and leads to transplant loss in many patients. The molecular basis for recurrent FSGS is unknown although abnormal podocyte proteins associated with familial FSGS have been identified. We have documented that serum or plasma from FSGS patients increases glomerular albumin permeability (Palb) and that the specific activity of certain protein fractions of FSGS plasma is more than 10,000-fold greater than that of the initial plasma. Intravenous injection of this fraction causes proteinuria in rats. We have recently used state-of-the-art proteomics techniques to study the proteins in the active plasma fraction from a FSGS patient. We identified a single cytokine, cardiotrophin-like cytokine 1 (CLC-1). CLC-1 is a candidate for the FSGS permeability factor because of this unique presence in active fractions of FSGS plasma. CLC-1 is also overexpressed in peripheral blood cells of patients with recurrent FSGS. CLC-1 is a cytokine of the IL-6 family that is synthesized by cells in many tissues. CLC-1 rapidly increases Palb in a concentration-dependent fashion. The magnitude and time-course of this increase are comparable to that induced by FSGS plasma. The permeability-effect of FSGS plasma as well as of recombinant CLC-1 itself is blocked by anti-CLC-1 antibody. We have found that receptor proteins for CLC-1 are present on glomeruli. The proposed studies are based on a.) the novel discovery of CLC-1 in the active fraction of FSGS plasma, b.) the overexpression CLC-1 in blood cells of patients with FSGS, c.) the expression of receptor proteins in glomeruli, d.) the vigorous activity of CLC-1 in the Palb assay and finally, e.) the striking ability of anti-CLC-1 monoclonal antibodies to block the permeability effect of FSGS plasma. Our central hypothesis is that CLC-1 is the elusive FSGS permeability factor. We will pursue the following Specific Aims. SA1: Confirm the relevance of CLC-1 to activity of the FSGS factor. We will test the sera of 4 patients with FSGS for the presence of CLC-1 using LC-MS/MS and Western blotting for CLC-1 using polyclonal antibodies. Patients with FSGS and rapid progression and/or prior post-transplant recurrence will be studied. Plasma from normal persons and from non-FSGS patients undergoing plasmapheresis prior to renal transplantation will be used as a controls. We will confirm that CLC-1 accounts for permeability activity by showing that antibodies to CLC-1 block permeability activity of FSGS plasma fractions of at least 10 patients. SA2: Confirm that CLC-1 increases permeability and study the mechanism by which this occurs. We will define the effect of CLC-1 on permeability using concentrations of CLC-1 from 0.05 to 50 ng/ml in established Palb and GEC monolayer protocols. Specificity of effects will be tested using blocking antibodies to CLA-1 and known blockers of FSGS plasma effects. Preliminary studies show that CLC-1 increases glomerular permeability in a concentration dependent manner and that anti- CLC-1 blocks this effect. We will determine if the addition of CLC-1 binding-partners cytokine-like factor-1 (CLF-1) or soluble ciliary neurotrophic factor receptor (sCNTFR) enhances the CLC-1 permeability effect. We will study the expression of each of the components of the receptors for CLC- 1 (gp 130, CNTF receptor-1 (CNTFR-1), and leukemia inhibitory factor receptor (LIFR)) by glomeruli and cultured using RT-PCR and Western blotting. These components will be localized in glomeruli and cultured GEC using immunohistochemistry. Preliminary studies show that anti-CLC-1 blocks the effect of FSGS serum and that glomeruli express RNA for receptor components. These studies will verify that CLC-1 is a candidate for the """"""""FSGS permeability factor"""""""" and will identify the potential receptor responsible for its permeability effect on GEC. Future studies will permit characterization of the signaling pathways that control the permeability barrier and design of specific and effective therapies for proteinuria in FSGS, and potentially, for proteinuria in other renal diseases.
Kidney disease that is identified by the appearance of large amounts of protein in the urine, edema or swelling of tissues, low plasma proteins and increased cholesterol is termed nephrotic syndrome. In many cases, nephrotic syndrome can be treated effectively with the use of artificial steroid hormones such as prednisone. In some patients with nephrotic syndrome that does not respond to treatment, microscopic examination of kidney tissue shows patchy or segmental scarring of some of the filters, which are called glomeruli. This pattern of damage is called focal segmental glomerulosclerosis or FSGS. Glomeruli are responsible for making urine and for preventing protein leak. When they are damaged, protein leaks into the urine and, if damage and protein leak continue, further scarring of the entire kidney leads to kidney failure. FSGS a leading cause of kidney failure in children and causes kidney failure in many adults, as well. Genetic abnormalities are present in some children and adults with FSGS, but there is no known genetic basis for FSGS in most patients. FSGS recurs in some patients with FSGS who have kidney failure and receive kidney transplants. The recurrence appears to be related to a small protein in the plasma that injures the glomeruli and leads to protein leak and scarring. The nature and the source of this protein are not known. During the past 20 years, we have studied serum samples from more than 500 patients with FSGS. We have used a variety of biochemical techniques for identifying proteins and determining their characteristics. These techniques include gel chromatography to determine the protein size and electrical charge, affinity chromatography to determine binding properties, and mass spectrometry. We have recently identified a protein, cardiotrophin-like cytokine 1 (CLC- 1), as a candidate for the active substance. This protein is best known for its role in nerve development and maturation. It is also important in the development of the kidney. We have tested this substance and found that even very brief exposure of glomeruli leads to severe increase in permeability in a manner identical to the reaction to FSGS proteins. We have also shown that blood cells of affected patients make more than normal amounts of CLC-1, and that antibodies to CLC-1 prevent the injury caused by patients'plasma. Glomeruli have the proteins that bind to CLC-1 and permit it to act on cells. The presence of CLC-1 in the plasma of a patient with FSGS, its overexpression by patients'blood cells, its activity in injuring glomeruli, and that fact that antibody to CLC blocks the effect of FSGS plasma lead us to the proposed studies to define its role in this serious kidney disease. We will perform studies to verify the presence of CLC-1 in samples from several patients with FSGS and recurrence after transplantation. We will use mass spectrometry and other advanced techniques of protein analysis for these studies. We will also test the effect of purified CLC-1 on glomeruli and on glomerular cells in culture. The results will verify our preliminary finding that CLC-1 is a component of the plasma of FSGS patients and that it injures glomeruli in a manner that is comparable to the injury caused by FSGS proteins. We will do further studies to identify the cell receptor responsible for CLC-1 activity on the glomerulus and to determine the cell responses that are required for its actions. These studies will point to new ways to treat FSGS and to prevent it from progressing to kidney failure or from recurring in newly transplanted kidneys.
|Sharma, Mukut; Sharma, Ram; McCarthy, Ellen T et al. (2017) Hyperfiltration-associated biomechanical forces in glomerular injury and response: Potential role for eicosanoids. Prostaglandins Other Lipid Mediat 132:59-68|
|Sharma, Mukut; Zhou, Jianping; Gauchat, Jean-François et al. (2015) Janus kinase 2/signal transducer and activator of transcription 3 inhibitors attenuate the effect of cardiotrophin-like cytokine factor 1 and human focal segmental glomerulosclerosis serum on glomerular filtration barrier. Transl Res 166:384-98|