Expression of osteogenic proteins in kidneys of cats with nephrocalcinosis

Abstract

Background: Nephrocalcinosis is a common pathological finding in cats with chronic kidney disease and nephrolithiasis. Understanding its pathogenesis may identify future therapeutic targets.

Hypothesis: Nephrocalcinosis is associated with expression of an osteogenic phenotype.

Animals: Kidneys with medullary mineralization were obtained from 18 cats (10 with and 8 without nephroliths) undergoing necropsy.

Methods: Cross-sectional study. Microradiography and histopathology (modified von Kossa stain) were used to confirm parenchymal mineralization. Immunohistochemistry for 5 osteogenic markers was performed to determine their co-localization with nephrocalcinosis. The proportion of kidneys with stronger immunointensity in mineralized versus non-mineralized regions was analyzed using 1-tailed sign tests. The proportion of kidneys with co-localization of nephrocalcinosis and each marker was compared between kidneys with and without nephroliths using Fisher's exact tests.

Results: Nephrocalcinosis co-localized with osteopontin immunoreactivity in all 18 cats (100%) and with osteocalcin in 12 cats (67%). Both osteogenic markers had stronger immunointensity in mineralized regions compared with non-mineralized regions. Limited co-localization was observed with other markers: bone morphogenic protein-2 in 2 kidneys (both with nephroliths) and tissue non-specific alkaline phosphatase in 1 kidney (without nephroliths); runt-related transcription factor-2 was undetected. No statistically significant differences were found in the co-localization of nephrocalcinosis with osteogenic proteins between kidneys with and without nephroliths.

Conclusions and clinical importance: Expression of osteogenic proteins in areas of nephrocalcinosis indicates that nephrocalcinosis is associated with the development of an osteogenic phenotype. Targeting these processes could offer a novel approach to prevent nephrolithiasis at its origin.

Keywords: chronic kidney disease; immunohistochemistry; mineralization; nephroliths; radiography.

FIGURE 1

Faxitron microradiographs of kidneys before and after colorization (red) to identify parenchymal mineralization. Mineralization appeared as radiating striations extending from the cortex inward toward the renal papilla in the kidney with nephroliths (A and B) and without nephroliths (C and D). Punctate mineralization is represented in blue box (B). The radiopaque nephroliths were colored yellow.

FIGURE 2

Photomicrographs of renal medulla stained with von Kossa for detecting minerals and mouse anti‐Runx2 for RUNX2 immunoreactivity. At the renal papilla, minerals were abundant (A; ×10 magnification). Panel (B) is the higher magnification (×40 magnification) of the insert in (A) showing mineralization primarily located at the basement membrane of renal tubules (arrow). At the outer medulla, fewer von Kossa‐positive regions were observed (C; ×10 magnification). Panel (D) is the higher magnification (×40 magnification) of the insert in (C), showing von Kossa‐stained minerals within tubules (arrow). The immunoreactivity of RUNX2 is negative.

FIGURE 3

Photomicrographs of renal medulla immunoreactivity for individual osteogenic proteins (left panel) and double stained for the corresponding osteogenic protein and von Kossa (right panel). Osteopontin immunoreactivity co‐localized with von Kossa (A and B; ×20 magnification), osteocalcin immunoreactivity co‐localized with von Kossa (C and D; ×20 magnification). Bone morphogenic protein‐2 immunoreactivity co‐localized with von Kossa (E and F; ×20 magnification). Tissue non‐specific alkaline phosphatase immunoreactivity co‐localized with von Kossa (G and H; ×10 magnification). Runt‐related transcription factor 2 immunoreactivity not co‐localized with von Kossa (I and J; ×40 magnification).