Background Renal intratubular casts are frequently observed in the distal nephron segments of the kidney and have long been regarded as a sign of renal disease. However, the composition and pathological significance of intratubular casts have remained understudied.

Methods We leveraged Hematoxylin and Eosin (H&E) staining to identify intratubular casts along with concurrent Co-detection by indexing (CODEX) multiplexed spatial protein imaging on human kidney biopsy sections from the Kidney Precision Medicine Project (KPMP). We also conducted immunoblotting of Prominin-1 (PROM1) in urine and assessed its levels from publicly available urinary proteomics datasets of the KPMP consortium.

Results We analyzed 424 intratubular casts across 33 individuals with kidney disease or healthy controls. We identified PROM1 and IGFBP7 as major constituents of casts (positive staining in 90.1% and 35.6%, respectively). Staining for UMOD, an established cast component, was present in 86.1%. These components exhibited distinct alterations depending on the disease state. Intratubular casts were predominantly detected in the distal nephron segments, and their presence was associated with a marked loss of NCC and AQP2 expression in the cast-containing tubular epithelium, suggesting underlying injury. The loss of these membrane transporters correlated with protein components within casts, and the presence of intra-cast PROM1 showed the strongest association, with an odds ratio of 30.8 (95% confidence interval: 13.4-71.0). Urinary PROM1 secretion was confirmed by immunoblotting and was increased in patients with acute kidney injury (AKI) compared to healthy controls (p = 0.01).

Conclusions We identified PROM1, a dedifferentiation and injury marker expressed in epithelial cells, as a novel major constituent of intratubular casts. Our studies suggest that protein composition signature within casts varies with disease state and is associated with tubular injury in distal nephron segments. Our study also suggests that urinary PROM1 may serve as a biomarker for AKI.

Key Points

✓ Utilized CODEX multiplex protein imaging to elucidate the intratubular cast components and the associated tubular alterations.

✓ Identified PROM1, a dedifferentiation marker, as a major constituent of intratubular casts.

✓ Protein components within casts were altered by disease state and were associated with the injury of the surrounding tubular epithelium.

The authors have declared no competing interest.

This work was supported by National Institute of Health, National Institute for Diabetes and Digestive and Kidney Diseases R01DK111651 for TME, VA Merit Award 5I01BX003935 for TME, Dialysis Clinic Inc for TME, and Takeda Science Foundation for AN. The Kidney Precision Medicine Project (KPMP) is supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) through the following grants: U01DK133081, U01DK133091, U01DK133092, U01DK133093, U01DK133095, U01DK133097, U01DK114866, U01DK114908, U01DK133090, U01DK133113, U01DK133766, U01DK133768, U01DK114907, U01DK114920, U01DK114923, U01DK114933, U24DK114886, UH3DK114926, UH3DK114861, UH3DK114915, and UH3DK114937.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

Institutional Review Board of University of Washington gave ethical approval for this work (Approval no. 20190213). Institutional Review Board of Biopsy Biobank Cohort of Indiana gave ethical approval for this work (Approval no. 1906572234).

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