Cellular Origins of Type IV Collagen Networks in Developing Glomeruli

Laminin and type IV collagen composition of the glomerular basement membrane changes during glomerular development and maturation. Although it is known that both glomerular endothelial cells and podocytes produce different laminin isoforms at the appropriate stages of development, the cellular origins for the different type IV collagen heterotrimers that appear during development are unknown. Here, immunoelectron microscopy demonstrated that endothelial cells, mesangial cells, and podocytes of immature glomeruli synthesize collagen α1α2α1(IV). However, intracellular labeling revealed that podocytes, but not endothelial or mesangial cells, contain collagen α3α4α5(IV). To evaluate the origins of collagen IV further, we transplanted embryonic kidneys from Col4a3-null mutants (Alport mice) into kidneys of newborn, wildtype mice. Hybrid glomeruli within grafts containing numerous host-derived, wildtype endothelial cells never expressed collagen α3α4α5(IV). Finally, confocal microscopy of glomeruli from infant Alport mice that had been dually labeled with anti-collagen α5(IV) and the podocyte marker anti-GLEPP1 showed immunolabeling exclusively within podocytes. Together, these results indicate that collagen α3α4α5(IV) originates solely from podocytes; therefore, glomerular Alport disease is a genetic defect that manifests specifically within this cell type.Basement membranes are thin sheets of extracellular matrix that underlie epithelial cells, including the vascular endothelium, and surround all muscle cells, Schwann cells, and adipocytes. They are composed of polymers of laminin and type IV collagen, and also contain nidogen/entactin, and proteoglycans. During glomerulogenesis, a basement membrane beneath developing endothelial cells fuses with a separate basement membrane layer beneath differentiating podocytes, to produce the glomerular basement membrane (GBM) shared on opposing surfaces by both cell types.¹Unlike most basement membranes in the body, the laminin and collagen IV composition of the GBM changes temporally as the glomerulus develops.² The earliest GBMs of comma- and S-shaped nephrons contain laminin α1β1γ1 (laminin 111), whereas those at later developmental stages and in adulthood contain laminin α5β2γ1 (laminin 521).^2,3 Previously, we showed by postfixation immunoelectron microscopy that both endothelial cells and podocytes synthesize laminin α1 and β1 initially, and both cells then undergo a laminin isoform switch and synthesize laminin α5 and β2 as glomeruli mature.⁴ The mechanism and reason why laminin replacement occurs are unknown, but this may be necessary for achievement and maintenance of the highly differentiated states assumed by glomerular endothelial cells and podocytes. For example, mice with genetic deletions of laminin α5 fail to develop vascularized glomeruli and die before birth with incomplete neural tube closure and placental vascular defects.⁵ By transplanting embryonic day 12 (E12) metanephroi into kidney cortices of newborn wildtype mice, we partially rescued the kidney phenotype and observed well vascularized hybrid glomeruli within grafts containing wildtype endothelial cells and laminin α5 mutant podocytes.⁶ However, the GBMs that form within these glomerular hybrids are stratified and contain laminin α5 on their inner, subendothelial surfaces and laminin α1 on their outer, podocyte halves. Intriguingly, the podocytes fail to form foot processes, suggesting that an absence of the laminin α1-α5 isoform switch stunts podocyte differentiation.⁶ Unlike laminin α5, laminin β2 expression is not required for normal glomerular development. However, mice with genetic delection of the Lamb2 gene become proteinuric by 8 d after birth, display effaced foot processes, and suffer renal failure by 4 wk of age.⁷ Recently, a human mutation mapped to the LAMB2 locus has been linked to Pierson syndrome, a disease of variable phenotype that entails congenital nephrosis, diffuse mesangial sclerosis, perinatal or childhood renal failure, ocular abnormalities, and neuromuscular deficits.^8–10Six genetically distinct collagen type IV α chains form three different triple helical heterotrimers (protomers) in separate compartments of the mature glomerulus.^11,12 A network of collagen α1α2α1(IV) is found in the mesangial matrix, collagen α3α4α5(IV) is present in the GBM, and Bowman''s capsule contains networks of both α1α2α1(IV) and α5α6α5(IV).^3,13 The immature GBMs of comma, S-shaped, and early capillary loop stage glomeruli all contain collagen α1α2α1(IV).² Beginning in capillary loop stages, this network is replaced by α3α4α5(IV),² which is the only collagen IV network normally present in mature GBM. Like laminins, the mechanism for collagen IV isoform substitution in the GBM is unknown. Nevertheless, the mature α3α4α5(IV) isoform is more resistant to proteolytic degradation than α1α2α1(IV),¹⁴ which may be critical for establishment and maintenance of glomerular permselective barrier properties.Importantly, the collagen α3(IV) and α4(IV) chains associate exclusively with α5(IV), to form the α3α4α5(IV) protomer.^15,16 The carboxyl terminal noncollagenous 1 (NC1) domains of the α chain polypeptides interact specifically to select and register appropriate chains for triple helix assembly. Collagen networks then assemble through head to head interactions between NC1 domains of two protomers, to produce a NC1 hexamer structure.^11,12 Similarly, the amino termini of four protomers associate in an anti-parallel fashion, to form a three-dimensional network of polymerized collagen IV.^11,12 Mutations of the human COL4A3, COL4A4, and/or COL4A5 genes prevent the proper assembly of a stable α3α4α5(IV) network, resulting in Alport disease.^11,12,15,16 This disorder is characterized in kidney by an absence of collagen α3α4α5(IV) and persistence of collagen α1α2α1(IV) in the GBM, thickening and multilamination of the GBM, proteinuria, and in most cases eventually, renal failure. Mice homozygous for a Col4a3 mutation possess a strikingly similar phenotype, and therefore, represent an attractive experimental model of human Alport disease.^17–19As indicated earlier, we have previously shown that both glomerular endothelial cells and podocytes synthesize the different laminin isoforms at appropriate stages of glomerular development.⁴ Here, we sought to define unambiguously the cellular origins of GBM collagen α1α2α1(IV) and α3α4α5(IV) during glomerular development. Additionally, we attempted to rescue the murine Alport phenotype by grafting E12 kidneys from Col4a3 knockout mice into renal cortices of wildtype newborn hosts to generate glomerular hybrids containing wildtype endothelial cells and Alport podocytes. Our findings show that collagen α3α4α5(IV) originates only from podocytes, which indicates that Alport disease in the glomerulus is a genetic disorder of this cell type specifically.