Chondroitin sulfate N-acetylgalactosaminyltransferase 1 promotes the progression of renal fibrosis mediated by versican 1 in mouse remnant kidney

Aim: Renal fibrosis is a final common pathway for progressive chronic kidney diseases. Immune cell infiltration and production of tumor growth factor-ß (TGF-ß) are essential factors for fibrosis development. We examined the role of chondroitin sulfate (CS) proteoglycan, which is one of the main extracellular matrix components induced by TGF-ß signaling. We also examined CS N-acetylgalactosaminyltransferase 1 (T1), an enzyme that catalyzes the first step of CS-specific synthesis. Methods: T1-/- mice, genetically lacking T1, and T1+/+ mice underwent 5/6 nephrectomy (Nx) or sham operation. Kidney function, urine marker, mRNA expression, and TGF-ß signaling were evaluated 1 month after Nx or sham operation. Renal fibrotic area was quantified 3 months later. Results: Both T1+/+ and T1-/- mice with Nx showed equivalent loss of kidney function, however, a tubular damage marker, upregulation of TGF-ß and collagen expression, and renal fibrosis were suppressed in T1-/- mice with Nx. Versican, one of the core proteins of CS proteoglycan, was exclusively upregulated in T1+/+ mice with Nx. Among the versican splicing variants, versican 1 (V1) was expressed in the medullary interstitium of the remnant kidney in T1+/+ mice. V1 was produced in the interstitial macrophages, fibroblasts/myofibroblasts, and endothelial cells, whereas TGF-ß was expressed in endothelial cells. Phosphorylation of the TGF-ß signaling molecules Smad2/3 was not induced in T1-/- mice with Nx. In vivo administration of TGF-ß inhibitor into Nx mice reduced V1 and Tgfb expression. Conclusion: T1 was essential for the initial induction of V1 upregulation, effective TGF-ß signaling, and subsequent renal fibrosis. Overall design: Eleven-week-old male T1+/+ mice underwent five-sixth nephrectomy. Briefly, the right kidney was exposed through a flank incision and removed. After a week, the left kidney was exposed and the upper and lower poles were ligated with a 3-0 non-absorbable suture until the coil diameter was half of the kidney. For control animals (sham), each flank of the kidney underwent sham surgery by incising flanks, exposing the kidney, and suturing each flank. Four weeks after surgery, the mice were sacrificed under anesthesia to collect kidney samples. Each single nucleus was captured using the Chromium System (10x Genomics) from frozen kidney samples for single-nucleus RNA sequencing.