Angiopoietin-like-4 and minimal change disease

<div><p>Background</p><p>Minimal Change Disease (MCD) is the most common type of nephrotic syndrome in children. Angiopoietin-like-4 (Angplt4) has been proposed as mediator of proteinuria in MCD. The aim of this study was to evaluate the role of Angptl4 as a biomarker in MCD.</p><p>Methods</p><p>Patients with biopsy-proven primary MCD, focal segmental glomerulosclerosis, membranous nephropathy (60, 52 and 52 respectively) and 18 control subjects had urinary and serum Angptl4 measured by Elisa. Frozen kidney tissue sections were stained for Angptl4.</p><p>Results</p><p>Angptl4 was not identified in glomeruli of MCD patients in relapse. Urinary Angptl4 levels were elevated in MCD in relapse as well as in patients with massive proteinuria due to other glomerular diseases.</p><p>Conclusion</p><p>Neither serum nor urine Angptl4 appear to be good biomarkers in MCD. Elevated urinary Angptl4 n glomerular disease appears to reflect the degree of proteinuria rather than any specific disease.</p></div

Demographic characteristics of patients and control subjects.

<p>Demographic characteristics of patients and control subjects.</p

Glomerular Angplt4 staining in patients with MCD and other glomerulopathies.

<p>Angptl4 angiopoietin-like-4, MCD minimal change disease, FSGS focal segmental glomerulosclerosis, LN lupus nephritis, MPGN membranoproliferative glomerulonephritis, n refers to the number of patients, Green, blue and red stains represent Angptl4, DAPI and synaptopodin respectively. Yellow lines represent scale bars. Magnification x200.</p

Urinary Angptl4 in MCD, FSGS, MN patients and control subjects.

<p>(A) Urinary Angptl4 in MCD, FSGS, MN patients and control subjects. (B) Urinary Angptl4 in FSGS and MN patients during relapse and remission. MCD minimal change disease, FSGS focal segmental glomerulosclerosis, MN membranous nephropathy, n refers to the number of patients, data expressed as mean±SD.</p

Serum Angptl4 in MCD, FSGS, MN during relapse and control subjects.

<p>Angptl4 angiopoietin-like-4, MCD minimal change disease, FSGS focal segmental glomerulosclerosis, MN membranous nephropathy, n refers to the number of patients, data expressed as mean±SD.</p

Nephrin is necessary for podocyte recovery following injury in an adult mature glomerulus

<div><p>Nephrin (<i>Nphs1</i>) is an adhesion protein that is expressed at the podocyte intercellular junction in the glomerulus. <i>Nphs1</i> mutations in humans or deletion in animal genetic models results in a developmental failure of foot process formation. A number of studies have shown decrease in expression of nephrin in various proteinuric kidney diseases as well as in animal models of glomerular disease. Decrease in nephrin expression has been suggested to precede podocyte loss and linked to the progression of kidney disease. Whether the decrease in expression of nephrin is related to loss of podocytes or lead to podocyte detachment is unclear. To answer this central question we generated an inducible model of nephrin deletion (<i>Nphs1</i>^Tam-Cre) in order to lower nephrin expression in healthy adult mice. Following tamoxifen-induction there was a 75% decrease in nephrin expression by 14 days. The <i>Nphs1</i>^Tam-Cre mice had normal foot process ultrastructure and intact filtration barriers up to 4–6 weeks post-induction. Despite the loss of nephrin expression, the podocyte number and density remained unchanged during the initial period. Unexpectedly, nephrin expression, albeit at low levels persisted at the slit diaphragm up to 16–20 weeks post-tamoxifen induction. The mice became progressively proteinuric with glomerular hypertrophy and scarring reminiscent of focal and segmental glomerulosclerosis at 20 weeks. Four week-old <i>Nphs1</i> knockout mice subjected to protamine sulfate model of podocyte injury demonstrated failure to recover from foot process effacement following heparin sulfate. Similarly, <i>Nphs1</i> knockout mice failed to recover following nephrotoxic serum (NTS) with persistence of proteinuria and foot process effacement. Our results suggest that as in development, nephrin is necessary for maintenance of a healthy glomerular filter. In contrast to the developmental phenotype, lowering nephrin expression in a mature glomerulus resulted in a slowly progressive disease that histologically resembles FSGS a disease linked closely with podocyte depletion. Podocytes with low levels of nephrin expression are both susceptible and unable to recover following perturbation. Our results suggest that decreased nephrin expression independent of podocyte loss occurring as an early event in proteinuric kidney diseases might play a role in disease progression.</p></div

Conditional deletion of nephrin in podocytes.

<p>(A) Schematic of the targeting vector containing a neomycin cassette, Frt sites and loxP sites were engineered to flank exon 5 of mouse nephrin. Successful recombination was verified using Southern blot with probes 1 and 2 flanking the nephrin gene. (B) Immunofluorescence images showing absence of nephrin in paraffin embedded mouse kidney section. Synaptopodin staining was used to identify podocytes. C) Coomassie blue stained SDS-PAGE gel showing severe albuminuria/proteinuria in the Nphs1^fl/fl,Cre+ animals at 2 weeks following birth. BSA standards (0.5–20 μg/dl). (D) Transmission and Scanning electron microscopy images showing podocyte foot process developmental abnormalities following nephrin deletion.</p

Induced model of nephrin deletion in podocytes.

<p>(A) Western blot showing decrease in nephrin in glomerular lysates from Nphs1^{fl/fl,iCre(ER)T2} mice kidneys 14 weeks post-induction with tamoxifen. Decreasing amounts of lysate volume were loaded in each well to avoid reaching the threshold of the x-ray film. Actin is used as loading control. (B) Densitometry showing quantification of the decrease in total nephrin following deletion. Results are representative of 4 separate experiments. (C) Immunoperoxidase staining for nephrin at various time points following tamoxifen-induction. Arrowheads show loss of cytoplasmic nephrin staining in podocytes at 2 weeks. (D) Light microscopy images of kidney tissue sections following silver enhancement of immunogold particles. (E) Immunogold electron microscopy of mouse kidney sections at 2 weeks and 10 weeks showing gold particles (arrowheads) at the slit diaphragm and within the cell body. Gold particles are seen primarily at the slit diaphragm in Nphs1^fl/fl,Cre+ at 10 weeks. Right panel shows enlarged images of the boxed areas in the left panel. Images were taken at X12,200 and cropped to emphasize the immunogold particles.</p

Deletion of nephrin in adult mature glomerulus.

<p>(A) Immunofluorescence images showing nephrin and synaptopodin staining at 2 weeks following Cre-induction with tamoxifen. There is abrogation of peri-nuclear nephrin staining following induction (arrowheads). (B) Immunofluorescence images showing nephrin and synaptopodin staining at various time points following tamoxifen. Scale bars, 20 μm.</p

Nephrin-deleted mature podocyte fail to recover following protamine sulfate injury.

<p>(A) SEM images of kidneys from 8 weeks old Nphs1^{fl/fl,iCre(ER)T2} mice following protamine sulfate injury. Nphs1^{fl/fl,iCre(ER)T2} mice that received tamoxifen show failure of foot process recovery in response to heparin sulfate following injury with protamine sulfate. (B) Analysis of junction frequency in TEM images show failure of recovery following heparin sulfate in Nphs1^{fl/fl,iCre(ER)T2} mice that received tamoxifen. (C) Immunofluorescence images of wild type animals (Nphs1^{fl/fl,iCre(ER)T2,} tamoxifen^-) show change in nephrin distribution from the membrane to punctate structures following protamine sulfate injury (middle panel). Nephrin staining is reversed back to the membrane on perfusion with heparin sulfate (lower panel). Synaptopodin (synpo) was used to for double staining the podocytes. All mice used in these experiments were 8–10 weeks old prior to receiving tamoxifen.</p