Podocytes use FcRn to clear IgG from the glomerular basement membrane

Abstract

The glomerular filtration barrier prevents large serum proteins from being lost into the urine. It is not known, however, why the filter does not routinely clog with large proteins that enter the glomerular basement membrane (GBM). Here, we provide evidence that an active transport mechanism exists to remove immunoglobulins that accumulate at the filtration barrier. We found that FcRn, an IgG and albumin transport receptor, is expressed in podocytes and functions to internalize IgG from the GBM. Mice lacking FcRn accumulated IgG in the GBM as they aged, and tracer studies showed delayed clearance of IgG from the kidneys of FcRn-deficient mice. Supporting a role for this pathway in disease, saturating the clearance mechanism potentiated the pathogenicity of nephrotoxic sera. These studies support the idea that podocytes play an active role in removing proteins from the GBM and suggest that genetic or acquired impairment of the clearance machinery is likely to be a common mechanism promoting glomerular diseases.

Fig. 1.

FcRn is expressed in murine podocytes. (A) Affymetrix genechip data were analyzed by using dChip software (43). The model-based expression index indicates relative expression levels of the indicated genes across tissues. For comparison, expression data for spleen and skeletal muscle were obtained from a published dataset (43) deposited in the public Gene Expression Omnibus (GEo) database (www.ncbi.nlm.nih.gov/geo/). The gene symbols are as follows: FcγRI, Fcgr1; FcγRIIB, Fcgr2b; FcγRIII, Fcgr3; FcγRIV, Fcgr4; Fcγ chain, Fcer1g; Megalin, Lrp2; and FcRn, Fcgrt. (B) RT-PCR of cDNA from podocytes differentiated for 10 days at 37°C is shown for FcRn (Fcgrt) and its obligate light chain, β2-microglobulin (b2m). cDNA from the mouse macrophage cell line RAW 264.7 is shown as a positive control. RT-PCR for genomic DNA contamination [without reverse transcriptase (RT)] was negative. Results are representative of two different experiments. (C) Two hundred twenty-five micrograms of postnuclear lysis supernatant from FcRn wild-type and knockout podocytes was immunoblotted with affinity-purified rabbit anti-FcRn serum. Liver lysates from FcRn wild-type and knockout mice were immunoblotted as positive and negative controls. Protein loading was detected by immunoblotting for α-tubulin. (D) Two hundred twenty-five micrograms of postnuclear lysis supernatant from FcRn wild-type and primary mesangial cells was immunoblotted with affinity-purified rabbit anti-FcRn serum. Expression and loading controls are as described in Fig. 1C.

Fig. 2.

FcRn-deficient mice have a reduced ability to clear IgG from the GBM. (A) FcRn wild-type and knockout mice were injected with 1 mg of human IgG intravenously. Then, 1 or 16 h after injection, kidneys were harvested and stained for human IgG. (B Upper) One week after the injection of I¹²⁵-labeled IgG, counts in whole decapsulated kidneys were normalized to TCA-precipitable radioactive counts from 50 μl of serum. (B Lower) Raw counts are shown. Blood cpm represents TCA-precipitable radioactivity from 50 μl of serum, and kidney cpm represents counts from whole unperfused, decapsulated kidney. P < 0.0002 by two-tailed Student's t test. Results are the combined results from two independent experiments using age- and sex-matched mice with similar results.

Fig. 3.

FcRn-deficient mice accumulate glomerular IgG with age. Kidneys from FcRn wild-type and knockout mice were stained for mouse IgG retention at 6 months of age. The results are representative of two different experiments with six to eight age-matched mice per group. (A) Low-power (×200) view of IgG staining from a representative wild-type and FcRn-deficient mouse. Arrowheads indicate individual glomeruli. (B) High-power (×630) view of IgG staining. Panels represent typical glomerular IgG staining from six mice in each group (one panel per mouse).

Fig. 4.

Saturating the glomerular clearance mechanism increases the pathogenicity of nephrotoxic serum. (A) Injection of increasing doses of purified mouse IgG i.v. leads to glomerular retention of mouse IgG in 6-month-old wild-type mice only at higher doses. Capillary loop staining is indicated by arrowheads. (Magnification: ×200.) (B) A protein bolus sensitizes mice to nephrotoxic nephritis. HSA alone, mice were injected with two doses of HSA alone; NTS alone, mice were injected with a low dose of nephrotoxic serum (NTS) alone; HAS + NTS, mice were injected with a low dose of NTS after injection of HAS (see Experimental Procedures for injection protocol). The protein content of urine for all mice in HSA alone and NTS alone groups was <30 mg/dl. Six of seven mice in the HAS + NTS group had proteinuria of >3,000 mg/dl, and one mouse had proteinuria of >300 mg/dl. The four remaining mice in this group had <30 mg/dl of protein in their urine. Values are the combined results from two independent experiments with similar results and were significant (P ≤ 0.002) by Fisher's exact test.