Insights into the renal pathogenesis in Schimke immuno-osseous dysplasia: A renal histological characterization and expression analysis

Abstract

Schimke immuno-osseous dysplasia (SIOD) is a pleiotropic disorder caused by mutations in the SWI/SNF2-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like-1 (SMARCAL1) gene, with multiple clinical features, notably end-stage renal disease. Here we characterize the renal pathology in SIOD patients. Our analysis of SIOD patient renal biopsies demonstrates the tip and collapsing variants of focal segmental glomerulosclerosis (FSGS). Additionally, electron microscopy revealed numerous glomerular abnormalities most notably in the podocyte and Bowman's capsule. To better understand the role of SMARCAL1 in the pathogenesis of FSGS, we defined SMARCAL1 expression in the developing and mature kidney. In the developing fetal kidney, SMARCAL1 is expressed in the ureteric epithelium, stroma, metanephric mesenchyme, and in all stages of the developing nephron, including the maturing glomerulus. In postnatal kidneys, SMARCAL1 expression is localized to epithelial tubules of the nephron, collecting ducts, and glomerulus (podocytes and endothelial cells). Interestingly, not all cells within the same lineage expressed SMARCAL1. In renal biopsies from SIOD patients, TUNEL analysis detected marked increases in DNA fragmentation. Our results highlight the cells that may contribute to the renal pathogenesis in SIOD. Further, we suggest that disruptions in genomic integrity during fetal kidney development contribute to the pathogenesis of FSGS in SIOD patients.

Keywords: DNA fragmentation; FSGS; SIOD; SMARCAL1; Schimke immuno-osseous dysplasia; focal segmental glomerulosclerosis; kidney; kidney pathology.

Figure 1.

Histological characterization of the renal biopsies from SIOD patients. (A–D) Analysis of the renal biopsy from SIOD Patient 1. (A) Hematoxylin and eosin (H&E) stain, (B) Masson’s trichrome (MASS) stain, (C) Periodic acid–Schiff (PAS) stain, (D) Jones’ methenamine silver (JMS) stain. These analyses demonstrate the FSGS tip variant (arrow points to the tip region) in Patient 1. (E–H) Analysis of the renal biopsy from SIOD Patient 2. (E) H&E, (F) MASS, (G) PAS, and (H) JMS stains demonstrate the FSGS collapsing variant in Patient 2. (I–L) Analysis of the renal biopsy from SIOD Patient 3. (I) H&E, (J) MASS, (K) PAS, and (L) JMS stains reveal the FSGS tip variant in Patient 3 (arrow points to the tip lesion). Scale, 20 μm.

Figure 2.

Characterization of the tubule-interstitial changes in SIOD patients. (A–C) Analysis of the renal biopsy from SIOD Patient 1. (A) Hematoxylin and eosin (H&E) stain reveals no overt tubule-interstitial changes in Patient 1. (B) Masson’s trichrome (MASS) stain demonstrates minimal interstitial fibrosis (arrow). (C) Periodic acid–Schiff (PAS) stain reveals minimal tubule basement membrane thickening (arrow). (D–F) Analysis of the renal biopsy from SIOD Patient 2. (D) Interstitial infiltrates (lymphocytes; arrow) and protein casts (arrowhead). (E) Moderate interstitial fibrosis (arrow). (F) Basement membrane thickening in tubules (arrow), arteriole thickening (arrowhead). (G–I) Analysis of the renal biopsy from SIOD Patient 3. (G) Interstitial infiltrates (lymphocytes; arrow) and tubular necrosis (arrowhead). (H) Tubular protein casts (arrowhead) and Interstitial fibrosis (arrow). (I) Tubular basement membrane thickening. (J) Morphometric analysis of the percentage of interstitial fibrosis in renal biopsies from SIOD patients. Patient 1 demonstrates 3.42% (± 0.246%) interstitial fibrosis, Patient 2 demonstrates 11.6 % (± 0.923%) interstitial fibrosis, and Patient 3 demonstrates 25.8% (± 1.43%) interstitial fibrosis. (K) Analysis of the percentage of atrophic tubules in renal biopsies from SIOD patients. Patient 1 demonstrates 1.06% (± 0.342%) atrophic tubules, Patient 2 demonstrates 11.4% (± 1.15%) atrophic tubules, Patient 3 demonstrates 15.3% (± 1.43%) atrophic tubules. Scale, 100 µm.

Figure 3.

Ultra-structural analysis of the renal biopsies from SIOD patients. (A) Schematic model of the cells of the glomerulus. (B–E) Transmission electron microscopy of the renal biopsy from Patient 1. (B) Adhesions to the Bowman’s capsule (white arrow). (C) High magnification reveals podocyte hypertrophy (black arrow) and adhesions to the parietal epithelial with irregular thickening of the Bowman’s capsule (white arrow). (D) Red blood cells in the urinary space (left black arrow) and podocyte foot process effacement (right black arrow). (E) Magnification at 30,000× reveals fused and flattened podocyte foot processes, which are effaced (black arrow). (F–I) Transmission electron microscopy of the renal biopsy from Patient 2. (F) Podocyte hypertrophy and hyperplasia (black arrow). (G) Glomerular capillary collapse with increased mesangial matrix. (H) Podocyte foot process effacement (black arrow). (I) Magnification at 30,000× reveals fused and flattened podocyte foot processes and clear foot process effacement (black arrow). (J–M) Transmission electron microscopy of the renal biopsy from Patient 3. (J) Interstitial edema. (K) Increased mesangial matrix (black arrows). (L) Podocyte foot process effacement (black arrow). (M) Magnification at 30,000× reveals fused and flattened podocyte foot processes with marked effacement (black arrow). (N–P) Measurements of glomerular basement membrane (GBM) thickness at 30,000× magnification in SIOD Patients 1, 2 and 3. (Q) Mean GBM thicknesses of 189 ± 7.70 nm for Patient 1, 296 ±16.6 nm for Patient 2 and, 267 ± 9.47 nm for Patient 3. Data are expressed as the mean ± SEM. Scale (B, D, J) 10 µm; (C, F, G, H, K, L) 2 µm; (E, I, M) 500 nm.

Figure 4.

SMARCAL1 protein expression in developing and mature human kidney. (A) Schematic model of nephron segments. (B–D) SMARCAL1 expression in the postnatal adult kidney. (B, C) Low-power image demonstrates SMARCAL1 expression in the kidney cortex and medulla showing expression in most but not all kidney cells. (D) High magnification of a representative glomerulus demonstrating SMARCAL1 expression in tubules adjacent to the glomerulus, parietal epithelial cells (top black arrow), and cells in the glomerular tuft in a pattern consistent with the podocyte (bottom black arrow). (E) Schematic representation of a developing kidney showing mesenchyme (blue), ureteric epithelium (red), stroma (pink), and morphological stages of developing nephrons (navy blue). (F) SMARCAL1 is expressed in the stroma and ureteric epithelium. Low levels of expression are also observed in the mesenchyme and renal vesicle in a developing 7-week fetal kidney. (G, H) SMARCAL1 expression in a developing 11-week fetal kidney. (G) SMARCAL1 is observed in the nephrogenic zone, specifically in the mesenchyme, stroma, collecting duct, renal vesicle and S-shaped body. (H) SMARCAL1 is expressed in the parietal epithelial cells and in some but not all cells of the glomerular tuft in a pattern consistent with the podocyte (black arrow). (I, J) SMARCAL1 expression in 16-week developing kidney. (I) SMARCAL1 is expressed in the developing podocyte cell layer in the S-shape body (black arrow). (J) SMARCAL1 is observed in the glomerulus in a pattern consistent with the podocyte (black arrow). CB-comma shaped body, CD-collecting duct, G-glomerulus, M-mesenchyme, PE-parietal epithelial cell, P-podocyte, RV-renal vesicle S-stroma, SB-S-shaped body, T-tubules, UE- ureteric epithelium. Scale (B, C), 200 µm; (D, F, G, H, I, J), 20 µm.

Figure 5.

Localization of Smarcal1 with glomerular markers in adult mouse kidney. (A, B) Serial sections of an adult mouse kidney incubated with Smarcal1 or Wilm’s Tumor 1 (Wt1; a nuclear marker of podocytes) and counterstained with hematoxylin. (A) Smarcal1 is detected in a number of cells within the glomerulus in a pattern identical to that seen in human tissue. (B) Wt1 is expressed in selective cells of the glomerulus. Some Wt1-positive cells are also Smarcal1-positive (circled nuclei #1–4). Not all Smarcal1 nuclei correspond to Wt1-positive cells (dotted lines a, b). (C, D) Serial sections of an adult mouse kidney incubated with Smarcal1 or Erg (a nuclear marker of endothelial cells) and counterstained with hematoxylin. (C) Smarcal1 is detected in a number of cells within the glomerulus (D) Erg is expressed in selective cells of the glomerulus. Some Erg-positive cells are also Smarcal1-positive (circled nuclei #1–4). Not all Smarcal1 nuclei correspond to Erg-positive cells (dotted lines a, b). Scale, 20 µm.

Figure 6.

Kidneys from SIOD patients exhibit increased DNA fragmentation. (A) TUNEL assay on normal human adult kidney demonstrating no DNA fragmentation as measured by the absence of TUNEL-positive brown nuclei. (B) Kidney biopsy from a postnatal non-SIOD patient with FSGS demonstrating few TUNEL-positive cells with a mild degree of DNA fragmentation. (C, D) SIOD Patients 1 and -2 exhibit high levels of DNA fragmentation in the majority of cells. (E) SIOD Patient 3 demonstrates low levels of DNA fragmentation in the majority of cells. Scale, 20 μm.