Megalin contributes to the early injury of proximal tubule cells during nonselective proteinuria

Abstract

Megalin, a member of the LDL receptor family, is expressed on the apical membrane of proximal tubules and serves as an endocytic scavenger of filtered proteins and hence might contribute to the tubule injury as a consequence of glomerular disease. To study its role, we crossed megalin knockout mosaic mice (lacking megalin expression in 60% of proximal tubule cells) with NEP25 mice (a transgenic line expressing human CD25 in the podocyte). Treatment of this transgenic mouse with the immunotoxin causes nephrotic syndrome, focal segmental glomerulosclerosis and tubule-interstitial injury. Following this treatment, the double transgenic mice had massive non-selective proteinuria and mild glomerular and tubular injury. Comparison of megalin-containing to megalin-deficient proximal tubule cells within each kidney showed that albumin, immunoglobulin light chain, IgA and IgG were preferentially accumulated in proximal tubule cells expressing megalin. Tubule injury markers such as heme-oxygenase-1, monocyte chemoattractant protein-1 and cellular apoptosis were also preferentially found in these megalin-expressing cells. These results show that megalin plays a pivotal role in the reabsorption of small to large molecular size proteins and provides direct in vivo evidence that reabsorption of filtered proteins triggers events leading to tubule injury.

Figure 1. Western blotting of megalin in megalin-KO/NEP25 (KO) and megalin –intact/NEP25 mice (intact)

The amount of megalin expression in megalin-KO/NEP25 mice was decreased, on average, to 40% of that of megalin-intact/NEP25 mice.

Figure 2. SDS-PAGE analysis of urine collected from megalin-KO/NEP25 (KO) and megalin-intact/NEP25 mice (intact)

The analysis confirmed that megalin-KO/NEP25 mice excreted predominantly low molecular weight proteins before LMB2 injection. The analysis also confirmed that proteinuria after LMB2 injection was non-selective, i.e., not only low but also intermediate and high molecular-weight proteins were excreted in large quantities. The intense bands at ~20 kDa of mouse major urinary protein before LMB2 injection diminished after LMB2 injection by a mechanism beyond the scope of the current investigation.

Figure 3. Histology of the kidney of a megalin-KO/NEP25 mouse given LMB2

The histology shows mild to severe glomerular injury, casts and protein reabsorption droplets in proximal tubule cells (depicted by black arrows).

Figure 4. Accumulation of proteins in proximal tubule cells of megalin-KO/NEP25 mouse given LMB2

In (a)–(h), black arrows depict megalin (+) proximal tubule cells (PTCs), and yellow arrows depict megalin (−) PTCs. (a) and (b) are from adjacent sections stained for megalin and albumin, respectively. (c) and (d) are from adjacent sections stained for megalin and immunoglobulin light chain (IgL), respectively. (e) and (f) are from adjacent sections stained for megalin and IgA, respectively. (g) and (h) are from adjacent sections stained for megalin and IgG, respectively. (b), (d), (f) and (h) show immunostained droplets of respective protein in megalin (+) PTCs. (i)–(l) are from serial sections of megalin-KO/NEP25 mouse kidney stained for megalin (i), IgL (j), IgA (k) and IgG (l), respectively. Note that megalin (+) PTCs (black arrows) contain IgL, IgA and IgG, but megalin (−) PTCs (yellow arrows) do not.

Figure 5. Percentage of protein-accumulating cells in megalin (+) vs. (−) proximal tubule cells of megalin-KO/NEP25 mice given LMB2

In these figures, each pair of dots connected with a line represents average values for megalin (+) vs. (−) proximal tubule cells (PTCs) from the same mosaic mouse. (a), percentage of albumin accumulating cells. (b), percentage of immunoglobulin light chain (IgL) accumulating cells. (c), percentage of IgA accumulating cells. (d), percentage of IgG accumulating cells. All four species of protein assessed were accumulated preferentially in megalin (+) PTCs. Relatively low percentage of IgG accumulation likely reflects low affinity of the antibody used for staining of the antigen.

Figure 6. Expression of early tubule injury markers in proximal tubule cells of megalin-KO/NEP25 mouse given LMB2

In each picture, black arrows depict megalin (+) proximal tubule cells (PTCs), and yellow arrows depict megalin (−) PTCs. (a) and (b) are from adjacent sections stained for megalin and heme-oxygenase-1 (HO-1), respectively. HO-1 is selectively expressed in megalin (+) PTCs. (c) and (d) are from adjacent sections stained for megalin and monocyte chemoattractant protein-1 (MCP-1), respectively. MCP-1 is selectively expressed in megalin (+) PTCs. (e) is a section double-stained for megalin and TUNEL. Only megalin (+) PTCs are undergoing apoptosis. (f) and (g) are from adjacent sections stained for megalin and aquaporin-1 (AQP-1), respectively. Expression of AQP-1 is diminished in megalin (+) and (−) PTCs similarly.

Figure 7. Percentage of early injury marker-expressing cells in megalin (+) vs. (−) proximal tubule cells of megalin-KO/NEP25 mice given LMB2

In (a), (b) and (c) , each pair of dots connected with a line represents total values for megalin (+) vs. (−) cells from each mosaic mouse. (a), percentage of heme-oxygenase-1 (HO-1) expressing cells. (b), percentage of monocyte chemoattractant protein-1 (MCP-1) expressing cells. (c), percentage of apoptotic cells. HO-1 expression, MCP-1 expression and apoptosis were observed preferentially in megalin (+) proximal tubule cells (PTCs). (d), percentage of aquaporin-1 (AQP-1) diminishing cells. Each pair of dots connected with a line represents average values for megalin (+) vs. (−) cells from each mosaic mouse. Diminished AQP-1 was observed similarly in megalin (+) and (−) PTCs.