Severity and Frequency of Proximal Tubule Injury Determines Renal Prognosis

Abstract

AKI increases the risk of developing CKD, but the mechanisms linking AKI to CKD remain unclear. Because proximal tubule injury is the mainstay of AKI, we postulated that proximal tubule injury triggers features of CKD. We generated a novel mouse model to induce proximal tubule-specific adjustable injury by inducing the expression of diphtheria toxin (DT) receptor with variable prevalence in proximal tubules. Administration of high-dose DT in mice expressing the DT receptor consistently caused severe proximal tubule-specific injury associated with interstitial fibrosis and reduction of erythropoietin production. Mild proximal tubule injury from a single injection of low-dose DT triggered reversible fibrosis, whereas repeated mild injuries caused sustained interstitial fibrosis, inflammation, glomerulosclerosis, and atubular glomeruli. DT-induced proximal tubule-specific injury also triggered distal tubule injury. Furthermore, injured tubular cells cocultured with fibroblasts stimulated induction of extracellular matrix and inflammatory genes. These results support the existence of proximal-distal tubule crosstalk and crosstalk between tubular cells and fibroblasts. Overall, our data provide evidence that proximal tubule injury triggers several features of CKD and that the severity and frequency of proximal tubule injury determines the progression to CKD.

Keywords: acute renal failure; chronic kidney disease; proximal tubule.

Figure 1.

DTR (hHBegf) is selectively expressed in proximal tubules of Ndrg1^CreERT2/+:iDTR mice after the administration of tamoxifen. (A) Schema of Ndrg1^CreERT2/+ mice and iDTR mice. (B) Real–time RT-PCR analysis confirmed the high expression of hHBegf in the kidney. The expression levels were normalized to those of GAPDH and expressed relative to the expression in the brain. (C–K) Immunostaining of hHBegf in the kidney. (D and E) hHBegf was expressed in aquaporin 1 (AQP1) -positive proximal tubules. hHBegf was expressed in all epithelial cells in (D) the S1 and S2 segments but not in (E) the S3 segment of proximal tubules. (F and G) hHBegf did not colocalize with Tamm–Horsfall protein (THP) or NaCl cotransporter (NCC), markers of distal tubules. (H and I) There are a very few hHBegf-positive cells in collecting ducts colocalizing with (H) AQP2 (a marker for principal cells) or (I) V-ATPase (a marker for intercalated cells). (J) hHBegf did not colocalize with nestin (a marker for podocytes). (K) The expression of hHBegf was not detected in the kidneys of Ndrg1^CreERT2/+:iDTR mice before the administration of tamoxifen. (L) Recombination efficiencies of hHBegf in proximal tubules (PTs), distal tubules (DTs), collecting ducts (CDs), and glomeruli (G) (n=3). Scale bars, 10 μm. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 2.

Administration of DT induces proximal tubule injuries in Ndrg1^CreERT2/+:iDTR mice. (A) We injected high-dose DT (25 ng/g) to Ndrg1^CreERT2/+:iDTR mice after the administration of tamoxifen and euthanized them at various time points. (B) Serum creatinine and BUN were elevated after DT administration (n=3–5). (C–H) Histologic analysis of the kidney of Ndrg1^CreERT2/+:iDTR mice 3 days after the administration of DT. Scale bars, 10 μm. (C) PAS-stained section revealed diffuse acute tubular injury after the administration of DT. (D) Electron microscopy revealed proximal tubule injuries with loss of brush border, vacuolization, and degeneration of mitochondria and lysosome. (E and F) DT induced the expression of (E) Kim-1 and (F) cleaved caspase-3 in (G) 14.9%±3.0% of proximal tubules. (H) Some of the proximal tubule epithelial cells were positive for Ki-67. (I) Scoring of tubular injury (n=3–4). (J–M) Real–time RT-PCR analysis revealed (J) the induction of Kim-1 mRNA and (K–M) the reduction of megalin, organic anion transporter 1 (OAT1), and OAT3 mRNA, respectively, after the administration of DT (n=3–4). (N) Glucosuria was detected after DT administration (n=3–4). Statistical analysis by (G and I) t test and (B and J–N) ANOVA followed by Bonferroni post hoc analysis. AQP1, aquaporin 1; Ctrl, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *P<0.05; **P<0.01.

Figure 3.

Administration of DT induces interstitial fibrosis in Ndrg1^CreERT2/+:iDTR mice. (A–F) Immunostaining of the kidney of Ndrg1^CreERT2/+:iDTR mice 3 days after the administration of high-dose DT. (A) Immunostaining revealed the increased expression of collagen 1 after the administration of DT. (B) GFP–positive collagen–producing fibroblasts were increased in the interstitium of Ndrg1^CreERT2/+:iDTR:Coll1a1-GFP mice. (C) DT administration increased the expression of α-SMA in the interstitium. (D and E) α-SMA–positive myofibroblasts emerged around (D) Kim-1 or (E) cleaved caspase-3–positive proximal tubules. (F) DT administration increased the number of F4/80-positive cells. Scale bars, 10 μm. (G, H, J, and K) Real–time RT-PCR analysis of the kidney after the administration of DT. (G) Col1a1 and (H) Acta2 mRNA were elevated after DT administration, whereas (J) Epo mRNA was significantly decreased. (K) Expression of Emr1 mRNA was increased after DT administration (n=3–4). (I) Quantification of α-SMA–positive area in the interstitium (n=3–4). Statistical analysis by (I) t test and (G, H, J, and K) ANOVA followed by Bonferroni post hoc analysis. Ctrl, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPF, high power field. *P<0.05; **P<0.01.

Figure 4.

Mild proximal tubule injury triggers reversible fibrosis, whereas repeated mild injuries cause sustained interstitial fibrosis. (A) Ndrg1^CreERT2/+:iDTR mice were injected with low-dose DT (0.25 ng/g) after tamoxifen treatment and euthanized 3 and 35 days after the administration. (B) Histologic analysis of Ndrg1^CreERT2/+:iDTR mice 3 days after the administration of low-dose DT. Low-dose DT induced acute proximal tubular injury and the emergence of Kim-1–positive proximal tubules and α-SMA–positive myofibroblasts. (C) Histologic analysis 35 days after low–dose DT administration revealed the complete recovery of pathologic findings. Neither a Kim-1–positive proximal tubule nor a α-SMA–positive myofibroblast was detected. (D and E) Scoring of tubular injury and quantification of α-SMA–positive area in the interstitium indicated the regeneration of proximal tubules and the reversibility of fibrosis (n=3–4). (F) Kim-1, Acta2, and Fn mRNA were elevated at day 3 and returned to the baseline at day 35 after low–dose DT administration (n=3–8). Statistical analysis by ANOVA followed by Bonferroni post hoc analysis. (G) Ndrg1^CreERT2/+:iDTR mice were repeatedly injected with low-dose DT (0.25 ng/g three times) after tamoxifen treatment and euthanized 3–4 weeks after DT administration. (H) Histologic analysis after the repeated DT injections revealed severe interstitial fibrosis. Immunostaining showed the presence of Kim-1–positive proximal tubules and α-SMA–positive myofibroblasts. Massive infiltration of F4/80-positive cells was also observed after the repeated DT injections. (I) Acta2 and Fn mRNA were elevated after the repeated DT injections, whereas the elevation of Kim-1 mRNA was not significant (n=3–8). (J) Scoring of tubular injury. Some of proximal tubules had not completely recovered (n=3–4). (K) Quantification of α-SMA–positive area in the interstitium. The α-SMA–positive area was increased after the repeated DT injections (n=3–4). Statistical analysis by t test. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPF, high power field; NT, non treat. Scale bars, 10 μm. *P<0.05; **P<0.01.

Figure 5.

Scattered proximal tubule injury does not induce interstitial fibrosis. (A) Ndrg1^CreERT2/+:iDTR mice were pretreated with various doses of tamoxifen (0.025 mg/g ×1 day, 0.05 mg/g ×1 day, 0.15 mg/g ×1 day, and 0.15 mg/g ×5 days [the protocol of previous experiments]) and injected with high-dose DT (25 ng/g). These mice were euthanized 3 days after the administration of DT. (B) Immunostaining revealed that the prevalence of hHBegf–positive proximal tubules increases along with dose and frequency of tamoxifen administration. (C) Real–time RT-PCR also revealed that hHBegf expression increases along with dose and frequency of tamoxifen administration (n=3). (D) Serum creatinine levels after the administration of high-dose DT increase along with dose and frequency of tamoxifen administration (n=3–4). (E and H) PAS–stained kidney sections of Ndrg1^CreERT2/+:iDTR mice treated with DT and scoring of tubular injury revealed that the severity of proximal tubule injury increases along with dose and frequency of tamoxifen administration. (F) Immunostaining revealed that Kim-1–positive proximal tubules emerge at lower doses of tamoxifen (0.025×1 and 0.05×1) than do α-SMA–positive myofibroblasts. (G) Although Kim-1 mRNA increased in the kidney treated with low-dose tamoxifen (0.05×1), Acta2 and Fn mRNA did not (n=3–4). (I) Quantification of α-SMA–positive area in the interstitium. α-SMA–positive myofibroblasts increased at higher doses of tamoxifen (0.15×1 and 0.15×5). Statistical analysis by ANOVA followed by Bonferroni post hoc analysis. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPF, high power field. Scale bars, 20 μm. *P<0.05; **P<0.01.

Figure 6.

Fibroblasts cocultured with injured tubular epithelial cells express higher levels of collagen, α-SMA, and inflammatory genes. (A) Schema of coculture experiment. Renal fibroblasts (NRK49F) were cocultured with tubular epithelial cells (NRK52E) pretreated with vehicle or TGFβ1 (10 ng/ml). NRK52E cells were vigorously rinsed three times before coculture. (B) Col1a1 and Acta2 mRNA of NRK49F was increased when cocultured with NRK52E cells pretreated with TGFβ1. (C) Ccl2 and IL-6 mRNA of NRK49F was increased when cocultured with NRK52E cells pretreated with TGFβ1 (n=4). Statistical analysis by t test. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *P<0.05; **P<0.01.

Figure 7.

Proximal tubule injury triggers distal tubule injury. (A) Ngal mRNA was increased after the administration of high-dose DT (25 ng/g). (B) Ngal emerged in the distal tubule stained by Tamm–Horsfall protein (THP) and NaCl cotransporter (NCC) after DT administration. Ngal staining in proximal tubules indicates the reabsorption from the urine. (C) Analysis of LacZ-positive cells in Ndrg1^CreERT2/+:iDTR:USAG-1^+/LacZ mice. The expression of USAG-1 (Sostdc1) and uromodulin mRNA, distal tubule markers, was high, whereas the expression of megalin mRNA, a proximal tubule marker, was low in LacZ-positive cells (P3). (D) Analysis of LacZ-positive cells in Ndrg1^CreERT2/+:iDTR:USAG-1^+/LacZ mice after the administration of DT. Ngal and osteopontin mRNA, markers of tubule injury, was increased in LacZ–positive distal tubule cells after DT administration. (E) Expression of Ngal mRNA after the administration of various doses and frequencies of tamoxifen and high-dose DT (25 ng/g). Ngal mRNA was increased only when treated with high-dose tamoxifen (n=3–4). Samples used in Figure 5G were analyzed in this experiment. (F) Immunostaining revealed that Ngal–positive distal tubules emerged when treated with high-dose tamoxifen (arrows). Statistical analysis by ANOVA followed by Bonferroni post hoc analysis. Ctrl, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Scale bars, 10 μm. *P<0.05; **P<0.01.

Figure 8.

Repeated mild tubular injuries cause glomerulosclerosis and atubular glomeruli. (A) Ndrg1^CreERT2/+:iDTR mice were injected with high-dose DT (25 ng/g) after the administration of tamoxifen and euthanized 3 days after DT administration. (B) PAS–stained kidney sections of Ndrg1^CreERT2/+:iDTR mice revealed that the glomeruli were structurally maintained. (C and D) Analysis by (C) scanning electron microscopy and (D) transmission electron microscopy showed that podocytes maintained their structure. (E) Almost all glomeruli were negative for α-SMA. (F) Next, Ndrg1^CreERT2/+:iDTR mice were repeatedly injected with low-dose DT (0.25 ng/g) after the administration of tamoxifen and euthanized 3–4 weeks after the last DT administration. (G) Sclerotic glomeruli were observed after repeated DT administration. (H) About 14% of glomeruli were positive for α-SMA, and (I) approximately 42% of glomeruli were atubular (n=3). Ctrl, control. Scale bars, 10 μm in B, E, G, and H; 2 μm in C and D.