Fig 2: Promoting HIF-1 function reduces renal injury and ameliorates renal dysfunction in mouse models of diabetes.Kidneys were harvested from wild-type (WT) and Leprdb/db diabetic mice (db/db) that were treated with placebo (vehicle) or DMOG (A–C, G), and from non-diabetic control (Ctrl) or diabetic (Db) wild-type (WT) and Egln1+/- (+/-) mice (D–F, H). (A and D) Representative images of KIM-1 (red or green) and DAPI (blue) in kidney that were analysed using fluorescent immunohistochemistry. Quantifications of KIM-1 fluoresent signal are shown in corresponding histogram (A, n = 3–4; D, n = 3–6). (B and E) Representative images of KIM-1 and a-tubulin analyzed by western blotting. (C and F) Apoptotic cells were detected using TUNEL staining, and the percentage of TUNEL-positive cells were quantified (C, n = 4; F: n = 3–5). (G and H) Albuminuria is presented as the ratio of albumin (Alb) to creatinine in mouse urine (G, n = 7–13; H, n = 4–6). Data are shown as mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001 analysed using one-way ANOVA (A, C), Brown-Forsythe and Welch ANOVA (G) and two-way ANOVA (D, F, H) followed by multi-comparison test. Source data are shown in Figure 5—source data 1. Scale bar: 100 µm. Figure 5—source data 1.Evaluation of renal KIM-1 and TUNEL staining and albuminuria of mouse models.

Fig 3: Rho kinase inhibition reduces kidney injury induced by ischaemia and reperfusion.(a–c) Images of the rat renal cortex containing proximal tubules, showing isolectin B4 labelling kidney tubules (red), DAPI labelling nuclei (blue), and kidney injury molecule-1 (Kim-1) labelling as an injury marker (white lines indicate examples of injured tubules labelled in green), for control conditions (CONT) (a), after ischaemia and reperfusion (I/R) (b), and after ischaemia with hydroxyfasudil present during reperfusion (I/R + HF) (c).(d) Kim-1 levels for the three experimental conditions (six stacks, 3 animals for each group). Data are mean ± s.e.m. P values are corrected for multiple comparisons. Statistical tests used the number of animals as the N value (not the stack number). Figure 7—source data 1.Rho kinase inhibition reduces kidney injury induced by ischaemia and reperfusion.

Fig 4: Effects of the humanized anti-TLR4 mAb on the expression of KIM-1, Bax and Bcl-2 in renal tissues. The renal tissues were collected at 24 h after LPS treatment. (A) Immunohistochemical staining (magnification, ×200). Quantification of the immunohistochemical staining of (B) KIM-1, (C) Bax and (D) Bcl-2. #P<0.01 vs. control; *P<0.05 and **P<0.01 vs. LPS. TLR4, Toll-like receptor 4; mAb, monoclonal antibody; KIM-1, kidney injury molecule-1; LPS, lipopolysaccharide.

Fig 5: Functional characterization of uptake and efflux transporter activity in mouse kidney spheroids. (a) Apoptosis profile by MUSE analyzer in mouse kidney spheroids (mKidS) incubated with 20 µM cisplatin (CPT) with and without preincubation with cimetidine (CMT), an OCT-2 transporter inhibitor. Cell viability assay was performed by the Muse Count & Viability reagent (Millipore) following the manufacturer’s protocols. The results were obtained with Muse Count & Viability software module and the statistics are shown as the percentage of viable cells and annexin V-positive apoptotic cells. (b) Quantification of live/dead-stained mKidS incubated with 20 µM CPT with and without preincubation with CMT, an OCT-2 transporter inhibitor. (c) Quantitative RT-PCR analysis of kidney injury markers Havcr1, Lcn2, clusterin, and caspase-3 in mKidS compared with untreated controls. n = 12 for CTL, CPT, and CMT, respectively. (d) Apoptosis profile as analyzed by MUSE analyzer in mouse kidney spheroids (mKidS) incubated with 10 µM digoxin (DGX) with and without preincubation with verapamil (VRP), a p-Gp efflux transporter inhibitor. (e) Quantification of live/dead stained mKidS incubated with 10 µM DGX with and without preincubation with VRP. (f) Quantitative RT-PCR analysis of kidney injury markers Havcr1, Lcn2, clusterin, and caspase-3 in mKidS compared with untreated controls. n = 12 for CTL, DGX, and VRP, respectively. All data are shown as the means ± s.e.m. *p < 0.05 compared to control by unpaired Student’s t test.