Ramipril therapy in integrin α1-null, autosomal recessive Alport mice triples lifespan: mechanistic clues from RNA-seq analysis

Abstract

The standard of care for patients with Alport syndrome (AS) is angiotensin-converting enzyme (ACE) inhibitors. In autosomal recessive Alport (ARAS) mice, ACE inhibitors double lifespan. We previously showed that deletion of Itga1 in Alport mice [double-knockout (DKO) mice] increased lifespan by 50%. This effect seemed dependent on the prevention of laminin 211-mediated podocyte injury. Here, we treated DKO mice with vehicle or ramipril starting at 4 weeks of age. Proteinuria and glomerular filtration rates were measured at 5-week intervals. Glomeruli were analyzed for laminin 211 deposition in the glomerular basement membrane (GBM) and GBM ultrastructure was analyzed using transmission electron microscopy (TEM). RNA sequencing (RNA-seq) was performed on isolated glomeruli at all time points and the results were compared with cultured podocytes overlaid (or not) with recombinant laminin 211. Glomerular filtration rate declined in ramipril-treated DKO mice between 30 and 35 weeks. Proteinuria followed these same patterns with normalization of foot process architecture in ramipril-treated DKO mice. RNA-seq revealed a decline in the expression of Foxc2, nephrin (Nphs1), and podocin (Nphs2) mRNAs, which was delayed in the ramipril-treated DKO mice. GBM accumulation of laminin 211 was delayed in ramipril-treated DKO mice, likely due to a role for α1β1 integrin in CDC42 activation in Alport mesangial cells, which is required for mesangial filopodial invasion of the subendothelial spaces of the glomerular capillary loops. Ramipril synergized with Itga1 knockout, tripling lifespan compared with untreated ARAS mice. © 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Keywords: Alport syndrome; glomerular basement membrane; glomerulonephritis; integrin α1β1; laminin; podocyte injury; ramipril; slit diaphragm.

Figure 1.. Comparative analysis of proteinuria, fibrosis, and lifespan in Alport, integrin α1-null Alport, and ramipril-treated integrin α1-null Alport mice.

(A) Proteinuria is markedly reduced in Itga1 null Alport mice treated with ramipril compared with untreated α1-null Alport mice. Urine was collected from the indicated treatment groups at the indicated ages. Total protein was measured by Bradford assay and normalized to urinary creatinine. Line represents Loess regression with a span of 1.5. (B) progression of fibrosis in ARAS mice, DKO mice, and DKO mice treated with ramipril as a function of disease progression (C) Itga1-null Alport (DKO) mice treated with ramipril live twice as long as untreated DKO mice and 3 times as long as untreated Alport mice, even when ramipril therapy is started after fibrosis is evident (10 wks). Animals from the indicated treatment groups were allowed to live until they lost >10% of their peak body weight and then euthanized (a humane endpoint, considered age of death). The age of death is indicated in weeks. Shaded regions indicate the 95% confidence interval.

Figure 2.. Kinetics of laminin α2 accumulation in the GBM of DKO compared to ramipril-treated DKO mice.

(A) Laminin 211 is abundant in the GBM in ramipril-treated DKO mice at 25 weeks of age relative to 20 and 15 weeks of age compared to untreated DKO mice at 15 weeks of age. Dual immunofluorescence analysis was performed on cryosections from wild type, 20, and 25-week-old DKO/ramipril mice. Sections were immunostained using antibodies for laminin-211 and laminin α5 (a GBM marker). Scale bar, 10 μm. (B) The progression of GBM laminin-211 accumulation as a function of disease development was quantified using ImageJ as described in Materials and methods. An example of this process is illustrated in Supplementary material, Figure S2A. * = P < 0.05, *** = P < 0.001 using one way ANOVA with Tukey’s Honest Significant Difference test.

Figure 3.. Kinetics of GFR reduction in DKO versus ramipril-treated DKO mice.

(A) A significant reduction in GFR was observed between 9 and 15 weeks in untreated DKO mice. Serial GFR measures were made at the indicated ages. (B) A marked reduction in GFR is observed at 30 and 32 weeks relative to 25 weeks in ramipril-treated DKO mice versus Wildtype. Serial GFR measures were made using ramipril-treated DKO and Wildtype mice. GFR measures were at the indicated ages. NS = Not Significant; * = P < .05, using one way ANOVA with Tukey’s Honest Significant Difference test for A and Dunnett’s test for B.

Figure 4.. Progressive reduction of nephrin and podocin transcripts and protein in Alport mouse models.

(A) Foxc2, encoding a transcriptional regulator of Nphs1 and Nphs2 was down-regulated in ARAS mice, DKO mice, and ramipril-treated DKO mice with the same kinetics as the Nphs1 and Nphs2 transcripts, which encode the slit diaphragm proteins nephrin and podocin, respectively, suggesting a functional relationship. SV5WKO, 129 Sv Alport 5-wks; SV7WKO, 129 Sv Alport 7-wks DKO10U, DKO 10-wk untreated; DKO15U, DKO 15-wk untreated; DKO15T, DKO 15-wk ramipril-treated; DKO25T, DKO 25-wk ramipril-treated. (B) Total Protein Normalized Densitometry analysis of Nephrin and Podocin Western Blots of 5 and 7-wk old Alport mice. (C) Nephrin and podocin proteins were decreased in the glomerular extracts of Alport mice as a function of glomerular disease progression. Total protein is shown as a loading control. (D) Podocyte counts as a function of disease progression in Alport, DKO, and ramipril-treated DKO mice. DKO20T, DKO 20-wk ramipril-treated. Other labels same as in A. NS = Not Significant.

Figure 5.. Ramipril-treated DKO mice show slowed progression of podocyte foot process effacement.

(A–D). TEM analysis of glomerular capillary loops from Ramipril-treated DKO mice show a progressive loss of intact foot processes. (A) Sv Alport 7-wk, (B) Ramipril-treated DKO 15-wk, (C) Ramipril-treated DKO 20-wk, (D) Ramipril-treated DKO 25-wk. Scale bar, 2 μm. (E) The number of morphologically normal podocyte foot processes progressively decrease with time in ramipril-treated DKO mice. “Morphologically normal” podocyte foot process pedicles were quantified for multiple capillaries in multiple mice as described in the methods. ** = P < 0.01; *** = P < 0.001 using one way ANOVA with Tukey’s Honest Significant Difference Test.

Figure 6.. Reduced migration and CDC42 activation in integrin α1-null mesangial cells compared to wild type mesangial cells.

(A) α1-null mesangial cells show a failure to activate CDC42 in response to Lipopolysaccharide (LPS) Stimulation. Wild type and α1-null mesangial cells were stimulated with lipopolysaccharide and activated CDC42 measured by G-LISA. Experiments were performed three independent times. LPS = Lipopolysaccharide; NT = non treated. (B) Wild type and Itga1-null mesangial cells were migrated with or without fetal calf serum in Boyden chamber migration assays. These were done in the presence or absence of the indicated cell signaling inhibitors. Cell migration is shown relative to FCS alone. In all cases experiments were performed three independent times. Asterisks indicate significant differences (** = P < 0.01, *** = P < 0.001 using one way ANOVA with Tukey’s Honest Significant Difference Test). ILK inh., Integrin linked kinase inhibitor; RAC1 inh., Rac Family Small GTPase 1 inhibitor; Inta1 null MES, integrin α-null mesangial cells; TAG 374 focal adhesion kinase inhibitor.