Selective depletion of mouse kidney proximal straight tubule cells causes acute kidney injury

Abstract

The proximal straight tubule (S3 segment) of the kidney is highly susceptible to ischemia and toxic insults but has a remarkable capacity to repair its structure and function. In response to such injuries, complex processes take place to regenerate the epithelial cells of the S3 segment; however, the precise molecular mechanisms of this regeneration are still being investigated. By applying the "toxin receptor mediated cell knockout" method under the control of the S3 segment-specific promoter/enhancer, Gsl5, which drives core 2 β-1,6-N-acetylglucosaminyltransferase gene expression, we established a transgenic mouse line expressing the human diphtheria toxin (DT) receptor only in the S3 segment. The administration of DT to these transgenic mice caused the selective ablation of S3 segment cells in a dose-dependent manner, and transgenic mice exhibited polyuria containing serum albumin and subsequently developed oliguria. An increase in the concentration of blood urea nitrogen was also observed, and the peak BUN levels occurred 3-7 days after DT administration. Histological analysis revealed that the most severe injury occurred in the S3 segments of the proximal tubule, in which tubular cells were exfoliated into the tubular lumen. In addition, aquaporin 7, which is localized exclusively to the S3 segment, was diminished. These results indicate that this transgenic mouse can suffer acute kidney injury (AKI) caused by S3 segment-specific damage after DT administration. This transgenic line offers an excellent model to uncover the mechanisms of AKI and its rapid recovery.

Fig. 1

Generation and analyses of the B6-Gsl5 TRECK transgenic (Tg) mice. A The Gsl5-hDTR construct contains kidney proximal tubule cell-specific promoter/enhancer regions amplified by PCR with the BAC367O1 clone as a template and the coding sequence of hDTR obtained from the pMS7 plasmid (Saito et al. 2001). E1 and E1′ are exons of the c2GnT gene in the BAC. Arrowheads show the PCR primers. B RT–PCR analysis of various organs in a Tg mouse. To detect the expression of hDTR mRNA, DTR-F1 and DTR-R2 primers were used. RT, reverse transcription; Sub. Gl., submaxillary gland. C Immunoprecipitation-western blotting. Mouse kidney membrane fractions were immunoprecipitated with anti-hDTR antibody. Western blotting analysis with anti-hDTR antibody showed a specific band in the fractions of the Tg mice bound to the IP complex, but not for the wild-type (WT) mouse or for the unbound fraction of the Tg mouse. rhDTR, recombinant human DTR. D Immunohistochemical study of the kidneys of Tg and WT mice. Human DTR protein was stained dark brown with metal-enhanced DAB reagent and the sections were counterstained with toluidine blue. The bar indicates 50 μm. E Immunohistochemical staining of a Tg mouse kidney. The sections were double-labeled with hDTR (green) and kidney segment specific antibodies (red; megalin, AQP2, THP and podocin). Scale bars = 50 μm

Fig. 2

Urinary sediments excreted 2 days after 50 μg/kg DT administration. A A urine sample was spread onto a glass slide, fixed with methanol and stained with Giemsa solution. B Another urine sample on the slide was fixed with paraformaldehyde and incubated with rabbit anti-AQP7 antibody followed by an AlexaFluor594-conjugated anti-rabbit IgG antibody (red in b and c). DAPI was used to detect nuclei (blue in a and c). We can find cell debris expressing AQP7 along with the intact cells in urine

Fig. 3

Urinary excretion of proteins after DT administration. DT was injected at doses of 1 (n = 5), 10 (n = 4), 50 (n = 5), 75 (n = 6) and 100 (n = 5) μg/kg body weight for Tg mice and 100 (n = 5) μg/kg for WT littermates. Values were normalized with the concentration of urinary creatinine. Vertical bars are SDs

Fig. 4

Identification of mouse serum albumin excreted into the urine of a Tg mouse after DT administration. A SDS–PAGE analysis of urine samples of a Tg mouse before (0) and 1–11 days after DT administration of 50 μg/kg. The CBB-stained protein bands (a to f) were excised. The proteins in gel pieces were digested with trypsin, and the digested peptides were extracted and analyzed by a tandem mass spectrometry. a, b and c were identified as mouse serum albumin; d a mouse major urinary protein, MUP3; e another major urinary protein, MUP1; and f a mouse α_2U globulin V. St, Protein standards; BSA, bovine serum albumin. B Tandem mass spectrometric identification of the 65 kDa protein (band b). Upper, the amino acid sequence of mouse serum albumin is shown. Red characters indicate the amino acid sequences determined by tandem mass spectrometric analysis. Lower, the product ion spectrum of the precursor fragment with 1149.65 of mass value obtained from band b. This spectrum identified the amino acid sequence LVQEVTDFAX, corresponding to amino acids 66–75 in mouse serum albumin. Amino acids Q, V and D, indicated by the product ion fragments, b7, y4, y6 and y8, are characteristic for mouse serum albumin but not for bovine or human albumin

Fig. 5

Concentration of blood urea nitrogen (BUN) after DT administration Vertical bars are SDs

Fig. 6

Histochemical studies after DT administration. A The boundary region between the cortex and medulla on day 3 after 50 μg/kg DT administration in Tg (a) and WT (b) mice. Dotted line shows the border between the cortex and the medulla. Stained with HE. B PAS stained sections of the kidney of a Tg mouse died on day 6 after 100 μg/kg DT administration. (a) In proximal tubules, not only the S3 segment but also the more proximal segments S1 and S2 were injured. Cast formation (arrows) was observed in the distal tubules. (b) White arrowheads indicate acute tubular injury observed in the S3 segment. (c) In the S3 segment, tubular cells were exfoliated into lumen, and some tubules were denuded (white arrow). Some distal tubules were stuck with casts (black arrow). (d) The glomerulus (arrow) seemed to be intact. C PAS stained sections of Tg mouse kidney on day 14 after 75 μg/kg DT administration. Both damaged (a) and regenerated (b) S3 segments were observed (arrowheads)

Fig. 7

Immunohistochemistry of the kidney after DT administration. AQP7 localized in the S3 segment cells vanished from the kidney of a Tg mouse on day 3 after 75 μg/kg of DT administration (DT(+)), whereas a control mouse without DT administration (DT(−)) showed no abnormality. In the Tg mouse, megalin expressed in proximal tubule cells also partially disappeared. AQP2 in the collecting duct cells, THP in TAL cells, podocin in a glomerulus were not affected. Scale bars = 50 μm

Fig. 8

Western blotting analysis of the kidney after DT administration. A Western blot analysis of the membrane fractions prepared from kidney of DT (+) and DT (−) mouse using anti-hDTR (top) and anti-megalin (bottom) antibodies. For hDTR, the equivalent amount of one-eighth of the kidney was applied on the gel. For megalin, 5 μg of the kidney membrane protein was applied. B Densitometric quantification of hDTR and megalin expression levels in A. DT (+), a Tg mouse on day 3 after 75 μg/kg of DT administration; DT (−), a control mouse without DT administration