On the basis of previous data ^[1][2], it was hypothesized that exogenous administration of AZGP1 may inhibit fibrotic processes in the kidney. To test this, it was induced experimental renal fibrosis by UUO in male C57Bl/6J mice treated with recombinant AZPG1 (rAZGP1) or with vehicle. After surgery, mice were injected intravenously with rAZGP1 (150 µg per injection) or vehicle every other day until day 11 (Figure 1A). On day 14, kidneys were harvested, and blood was taken to determine AZGP1 serum levels. Three days after the last injection, serum AZGP1 levels were still significantly higher in rAZGP1 treated mice (Figure 1B). This was accompanied by better preservation of proximal tubular integrity as shown by Lotus tetragonolobus lectin (LTL) binding (Figure 1C,D) and by reduced collagen deposition as shown by Picrosirius Red staining (Figure 1C,E). In parallel, it was observed significantly lower expression of Havcr1 (coding for kidney injury marker KIM-1), Acta2 (coding for the fibroblast activation marker α-smooth muscle actin; αSMA), and Col1a1 (coding for collagen type I) in kidneys of AZGP1 treated mice (Figure 1F–H). Together, these data indicated anti-fibrotic effects of rAZGP1 administration in kidneys challenged by UUO stress.

To consolidate the observed effects by an independent method, it was generated a mouse model with transgenic AZGP1 expression driven by a proximal tubule-specific promoter. Transgenic mice carrying murine Azgp1 cDNA with an upstream loxP-Stop-loxP sequence were crossed with Slc34a1-CreERT2 mice to achieve tamoxifen-inducible AZGP1 overexpression in proximal tubular cells (Figure 2A). Successful Cre recombination in proximal tubules was confirmed by additional tdTomato reporter gene co-expression (Figure 2B). Tamoxifen-induced overexpression of AZGP1 resulted in a mean 4,6-fold increase of Azgp1 transcript levels in control kidney homogenates. (Figure 2C). This increase in healthy control mice was smaller than anticipated and was only associated with a small but non-significant increase in serum AZGP1 (Figure 2D). Tamoxifen-induced transgenic mice and control mice underwent UUO surgery, and kidneys were examined after two weeks. AZGP1 transcript, as well as serum levels, showed a significant decrease after UUO (Figure 2C,D). While it was found no significant differences in tubular LTL binding or Picrosirius Red staining in the AZGP1 overexpressing mice (Figure 2E–G), it observed significantly reduced up-regulation of Havcr1, Acta2, and Col1a1 transcripts in the tamoxifen-treated group (Figure 2H–J). These data are consistent with a protective effect of transgenic tubular AZGP1 expression, although the observed differences were smaller as compared to the effects obtained with systemic rAZGP1 treatment.

It previously linked anti-fibrotic activities of AZGP1 to TGF-beta antagonizing mechanisms and reduced fibroblast activation ^[1][3][4]. Additional mechanisms, in particular a potential involvement of AZGP1 in altered lipid metabolism of the kidney, have not been explored. Given its properties as a lipid degradation factor, it evaluated changes in lipid accumulation in UUO kidneys. Quantification of Oil Red O staining revealed a significant reduction of tubular lipid droplet deposition in rAZGP1 treated kidneys (Figure 3A,B). Concomitantly, the expression of key players of tubular fatty acid oxidation (FAO) was significantly different between the groups. Quantitative PCR revealed elevated transcripts for carnitine palmitoyl-transferase 1A (Cpt1), peroxisome-proliferator-activated receptor α (Ppara), and PPARgamma coactivator-1a (Ppargc1a) in contralateral (con) as well as ligated (UUO) rAZGP1 treated kidneys (Figure 3C–E). These data suggested that AZGP1 plays a role in stabilizing renal tubular lipid balance and FAO during stress conditions. Accordingly, it was observed an inverse pattern in kidneys from AZGP1 knockout mice, which showed a significantly decreased expression of Cpt1, Ppara, and Ppargc1a (Figure 3F–H).

To further differentiate primary effects of AZGP1 from secondary changes due to UUO, it analyzed isolated primary tubular epithelial cells (PTEC) from kidneys of AZGP1 knockout and wildtype mice. In AZGP1 deficient PTEC, it was observed increased lipid droplets by Oil Red O staining (Figure 4A,B). The increased lipid accumulation was accentuated when PTEC were challenged with 0.4 mM palmitic acid (PA) for 24 h (Figure 4A,B). On the transcriptional level, knockout PTEC showed significantly lower levels of Cpt1 and Ppargc1a, as well as a trend for lower Ppara expression (Figure 4C–E). Together, these results provide evidence for the involvement of AZGP1 in tubular epithelial lipid metabolism.