Feasibility of repairing glomerular basement membrane defects in Alport syndrome

Abstract

Alport syndrome is a hereditary glomerular disease that leads to kidney failure. It is caused by mutations affecting one of three chains of the collagen α3α4α5(IV) heterotrimer, which forms the major collagen IV network of the glomerular basement membrane (GBM). In the absence of the α3α4α5(IV) network, the α1α1α2(IV) network substitutes, but it is insufficient to maintain normal kidney function. Inhibition of angiotensin-converting enzyme slows progression to kidney failure in patients with Alport syndrome but is not a cure. Restoration of the normal collagen α3α4α5(IV) network in the GBM, by either cell- or gene-based therapy, is an attractive and logical approach toward a cure, but whether or not the abnormal GBM can be repaired once it has formed and is functioning is unknown. Using a mouse model of Alport syndrome and an inducible transgene system, we found that secretion of α3α4α5(IV) heterotrimers by podocytes into a preformed, abnormal, filtering Alport GBM is effective at restoring the missing collagen IV network, slowing kidney disease progression, and extending life span. This proof-of-principle study demonstrates the plasticity of the mature GBM and validates the pursuit of therapeutic approaches aimed at normalizing the GBM to prolong kidney function.

Figure 1.

NEFTA and Rosa26-rtTA drive expression of T-Col4a3 and deposition of transgene-derived protein into the GBM. (A) The (TetO)₇/CMV-Col4a3 (T-Col4a3) transgene is induced by a doxycycline (Dox)-bound rtTA. (B) To achieve rtTA expression in podocytes, the nephrin (Nphs1) promoter was placed upstream of the rtTA-3G cDNA and the SV40 large T-antigen poly A signal (SV40pA) to generate the NEFTA transgene. (C–E) Kidney sections from a 10-day-old NEFTA; T-HISGFP; T-hLAMA5 mouse induced with doxycycline prenatally shows green fluorescent protein (GFP) fluorescence in podocyte nuclei and continuous staining for human laminin α5 in the GBM (C). Immunostaining for Wilms tumor 1 (D) identifies podocyte nuclei, all of which are GFP-positive (green/yellow in the merge in E). (F–H) Kidney sections of 23-week-old Col4a3^+/− (F), Col4a3^−/−; NEFTA; T-Col4a3 (G), and Col4a3^−/−; Rosa26-rtTA; T-Col4a3 (H) mice fed doxycycline from birth were stained for COL4A3. Original magnifications: C, ×200; D and E, ×600; F–H, ×400. g, glomerulus.

Figure 2.

Podocyte-specific expression of T-Col4a3 from postnatal day (P) 0 and P21 attenuates Alport glomerulosclerosis. Genotypes: Col4a3^+/−, control; Col4a3^−/−, nonrescued Alport; NEFTA-P0-R23W, rescued Alport induced at P0 for 23 weeks; NEFTA-P21-R20W, rescued Alport induced at P21 for 20 weeks. (A) Immunostaining for assembled collagen α3α4α5(IV) NC1 hexamers. Basement membranes of glomeruli (G) and tubules (T) are positive in the control (a) and negative in the mutant (b). Only GBM is positive in the podocyte-specific rescue mice (c and d). (B) Double staining for COL4A2 (green) and COL4A4 (red), as indicated. COL4A4 is only detected in control (e) and rescued (g and h) GBMs. COL4A2 is normally present primarily in the mesangium (a and e), but is increased in the mutant GBM (b and f). More COL4A2 is present in the P21 rescue GBM (d and h) versus P0 rescue (c and g). Arrows indicate GBM. (C) Periodic acid-Schiff staining of paraffin sections shows some glomerulosclerosis (gs), a crescentic glomerulus (cg), and protein casts (P) in the mutant (b) versus control (a), but more normal kidney architecture in the rescues (c and d). (D) Ultrastructural analysis of the glomerular capillary wall shows the normal ribbon-like GBM (arrowheads) in most segments of control (a) and rescues (c and d); areas of GBM thickening (arrows) are present diffusely in the mutant (b) but only segmentally in the rescues (c and d). rbc, red blood cells within the glomerular capillaries. Original magnifications: A and B, ×400; C, ×200; D, ×7500.

Figure 3.

Survival of doxycycline-fed Col4a3^−/−; NEFTA; T-Col4a3 mice versus nonrescued Col4a3^−/− mice correlates with albuminuria and GBM contour. (A and B) SDS-PAGE analysis of 1 µl of urine from control (C), mutant (M), and rescued (R) mice induced at embryonic day 14 (E14), birth (P0), or 21 days (P21). Ages at urine collection are indicated in weeks (W). Littermates are indicated by brackets below the gels. BSA standards at 1 and 5 µg are shown for each. (C) Survival curve showing age at ESRD for 19 Col4a3^−/− and 14 Col4a3^−/−; NEFTA; T-Col4a3 rescued (NEFTA-R) mice induced with doxycycline at various ages, E11-P21. All mice expressing T-Col4a3 survived longer than the longest-surviving nonexpressor. (D–F) Scanning electron microscopic analysis of decellularized GBM shows it to be rough and blebbed in the Col4a3^−/− at 12 weeks (D), smooth in the control at 40 weeks (E), and slightly blebbed yet smooth in the 40-week-old rescued mouse fed doxycycline from P21 (F). Original magnifications: main images, ×6000; insets, ×40,000.