A new mouse model for renal lesions produced by intravenous injection of diphtheria toxin A-chain expression plasmid

B6C3F1 (CLEA Japan, Inc., Tokyo, Japan; a hybrid between C57BL/6N and C3H/HeN) male mice, aged 8 to 15 weeks, were used for intravenous injection of plasmid DNA. Adult MNCE-36 transgenic mice (aged 8–15 weeks) [25] were also used as a control for monitoring CAG promoter specificity in renal tissue. They expressed enhanced green fluorescent protein (EGFP) ubiquitously under control of the CAG promoter [25].

Mice were kept on a 12 h light/12 h dark schedule (lights on from 0700 h to 1900 h) and allowed food and water ad libitum. Experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals at Tokai University. All efforts were made to minimize the number of animals used and their suffering.

For expression of DT-A, pCAG/DT-A plasmid DNA (Figure 1A) was used. A 0.6-kb Hin dIII and Bam HI fragment containing a DT-A gene was isolated from pBI106 [26]. This fragment was Klenow-filled and then inserted into the Eco RI site (the site had been destroyed by T4 DNA polymerase treatment) in the 3rd exon of rabbit β-globin gene in pBsCAG-2, [27] a derivative of pCAGGS [8]. pBsCAG-2 [hereafter referred to as pCAG lacking the DT-A gene (Figure 1A)] was used as a negative control. For expression of EGFP, pCE-29 plasmid [28] (Figure 1A) was used. In these three plasmids, the CAG promoter was employed for systemic expression of a downstream cDNA or gene.

All solutions used for intravenous DNA injection were prepared at a volume of 200 μl/mouse. Forty μl of FuGENE™6 (Boehringer Mannheim GmbH, Mannheim, Germany), a transfection reagent (lipid), was diluted with 60 μl of PBS(-), and then added to 20 μg of circular plasmid DNA dissolved in 100 μl of PBS(-) according to the manufacturer's protocol. For control injection (mock injection), a solution containing 40 μl of FuGENE™6 dissolved in PBS(-) (without DNA) or PBS(-) only was prepared.

Solution containing plasmid DNA/FuGENE™6 complex, FuGENE™6 only or PBS(-) only was injected into the tail vein of unanesthetized B6C3F1 males with a 1-ml plastic disposable syringe (Terumo, Tokyo, Japan) fitted with a 27-gauge needle (Nipro, Osaka, Japan). Injections were performed at a speed of 200 μl/8–10 sec. This injection was repeated every day for up to 6 days (Figure 1B).

Genomic DNA of organs was isolated as previously described [29] with several modifications [30]. PCR amplification reactions were performed, as previously described [31]. One set of primers (DTA-S and DTA-2RV) for detection of the introduced pCAG/DT-A is listed in Figure 1A. This primer set yields 267-bp fragments. DTA-S (5'-ATG TTG TTG ATT CTT CTA AAT-3') and DTA-2RV (5'-GCG AGA ACC TTC GTC AGT CCT-3') correspond to nucleotides 87 to 107 and 333 to 353 in the DT-A gene sequence, [26] respectively.

Total RNA was isolated from organs by the method of Chomczynski and Sacchi [32]. RT-PCR was performed using sense primer βA-1 (Figure 1A) and reverse primer DTA-RV (Figure 1A), as previously described [33]. βA-1 (5'-TCT GAC TGA CCG CGT TAC TCC CAC A-3') corresponds to nucleotides -1,011 to -987 of the chicken β-actin gene sequence, [34] and DTA-RV (5'-CAG AGT ATC CCG CAG CGT CGT-3') to nucleotides 254 to 274 in the 5' region of the DT-A gene sequence [26]. The primer set (βA-1/DTA-RV) was designed to produce PCR fragments of 300 bp. The PCR primer set (mβA-S/mβA-RV) [33] for detection of mouse β-actin mRNA was also used.

Urine samples were collected by grasping mice and immediately transferred to urine testing paper (#1333; Sankyo Medicals Inc., Tokyo, Japan) to evaluate the levels of protein and occult blood semi-quantitatively. Serum was prepared by separation of the coagulated whole blood by cava puncture after urine collection under pentobarbital-induced anesthesia. Serum concentrations of blood urea nitrogen (BUN), creatinine (CRT), Na⁺, Cl^-, and K⁺ were measured by an automated analyzer in SRL Lab. Ltd. (Tokyo, Japan).

After mice introduced with plasmid DNA/FuGENE™6 complex, FuGENE™6 only or PBS(-) only were perfused with 4% paraformaldehyde (PFA) in PBS(-), major organs (including brain, heart, lung, pancreas, liver, kidney and intestine) were dissected, post-fixed with 4% PFA in PBS(-) at 4°C for over 10 days except for the kidney, and then subjected to standard histological processing. The kidney was cut into several slices approximately 3–5 mm thickness. Some specimens were subjected to immunohistochemical, immunoelectron microscopic and electron microscopic analyses, as described below. Major organs including the kidney were paraffin-embedded and then stained with hematoxylin-eosin (H-E). Some of these paraffin-embedded specimens were stained with periodic acid-Schiff (PAS), and periodic acid-Schiff-methenamine silver (PAM).

For inspection of EGFP fluorescence, B6C3F1 mice intravenously injected with pCE-29/FuGENE™6 complex, MNCE-36 transgenic mice or their non-transgenic littermates were perfused with 4% PFA in PBS(-). Organs were dissected from these mice and cut into several pieces approximately 3–5 mm in thickness. Some of these specimens were immediately subjected to observation for EGFP fluorescence under a fluorescence stereomicroscope (SZX12, Olympus, Tokyo, Japan) with DM505 filters (BP460–490 and BA515IF, Olympus). This allowed us to examine gene expression directly in the intact organ, thereby avoiding potential artifacts that may be introduced during sample preparation for histochemical examination. The other specimens were further fixed with 4% PFA in PBS(-) at 4°C overnight, dehydrated in a sucrose series in PBS(-) and embedded in O.C.T. compound (Tissue-Tek, No. 4583, Miles Scientific, Naperville, IL) for cryostat sectioning. The sections were observed for EGFP fluorescence using a Olympus BX60 microscope under UV illumination. Microphotographs of the specimens were taken using a digital camera (FUJIX HC-300/OL; Fuji Film, Tokyo, Japan) attached to an Olympus BX60 or SZX12 microscope and printed out using a digital color printer (CP700DSA; Mitsubishi, Tokyo, Japan).

For immunohistochemistry and immunoelectron microscopic analyses, mice were first perfused with 4% PFA in PBS(-). The kidneys were isolated, sliced and further fixed with 4% PFA in PBS(-) at 4°C for 3 days, treated with 10% sucrose in PBS(-) at 4°C for 3 days, embedded in O.C.T. compound, and frozen at -80°C. Sections (4 μm thick) were cut with a cryostat and subjected to immunohistochemistry using anti-GFP antibodies (#8367-2; Living Colors A.v. Peptide Polyclonal Antibody, Clontech, MA) and an avidin-biotin coupling (ABC) peroxidase technique performed with the aid of a kit (Vectstain Elite; Vector, Burlingame, CA). The sections were washed in PBS(-) and incubated with 0.02% diaminobenzidine (DAB) in 0.05 M Tris buffer (pH7.6) containing 0.01% H₂O₂ for 3 min. After washing in PBS(-), the sections were fixed with 2.5% glutaraldehyde for 3 min at room temperature. The sections were washed in PBS(-), post-fixed with 1% osmium tetroxide in 0.1 M sodium phosphate buffer (pH 7.4) for 3 min, washed in distilled water, dehydrated in a graded ethanol series, and flat-embedded in Epon 812 (TAAB Laboratories Equipment Ltd., Berkshire, United Kingdom). After polymerization of the Epon 812, we examined the sections by light microscopy, clipped selected fields, and cut them with an ultramicrotome into ultrathin sections, which were then examined for DAB-stained sites with an electron microscope (JEM-1200EX; JEOL Ltd., Tokyo, Japan).

For electron microscopic analysis, after perfusion with 4% PFA in PBS(-), the kidneys were isolated, sliced, immersed for 2 h in 0.1 M sodium phosphate buffer (pH 7.4) containing 2% glutaraldehyde, post-fixed in 2% osmium tetroxide in 0.1 M cacodylate buffer for 1 h, dehydrated in acetone, and embedded in Epon 812. Semithin sections were stained with toluidine blue and examined by light microscopy to identify regions of the blocks containing several glomeruli. Ultrathin sections were stained with uranyl acetate and lead acetone and were examined with a JEM-1200EX electron microscope.

To evaluate the extent of glomerular nephritic lesions, at least 100 glomeruli in H-E-stained sections were randomly chosen per kidney, and lesions were quantified and graded from 0 to 2: score 0, normal; score 1, weak to mild change (exhibiting mild mesangial cell proliferation and mesangial matrix expansion); score 2, severe change (exhibiting lobular structure of glomeruli and adhesion of glomeruli to the inner surface of Bowman's capsule). The extent of glomerular lesions was expressed as a score (%) using the following formula: The number of glomeruli exhibiting each score/total numbers of glomeruli examined.

All data are means ± SD (n = 4 to 6 for each treatment group). For comparison of pairs, statistical analysis the unpaired t-test was used with the level of significance set at P < 0.05.

In this study, we used CAG promoter for expression of DT-A in mouse kidney, since it has been suggested to be active in murine kidney, particularly glomeruli [9]. Cryostat sections of MNCE-36 kidney revealed bright fluorescence in glomeruli [indicated by arrows in (a) of Figure 1C] and relatively weak fluorescence in some portions of tubules [indicated by arrowheads in (a) of Figure 1C]. Non-transgenic control kidney was completely negative for fluorescence [(b) of Figure 1C]. Other lines, MNCE-39 and -53, [25] exhibited similar distributions of EGFP in kidney (data not shown), eliminating the possibility that the above findings were due to the transgene integration site used. Immunohistochemical staining of cryostat sections revealed that immunoreactive deposits were present on the surface of glomerulus facing Bowman's space [indicated by arrows in (c) of Figure 1C]. Non-transgenic kidney was completely negative for immunoreaction [(d) of Figure 1C]. Immunoelectron microscopic analysis further revealed that immunoreactive deposits were present mainly in glomerular epithelium [indicated by arrows in (e) of Figure 1C] and slightly in glomerular endothelium [indicated by arrows in (f) of Figure 1C]. A portion of mesangial cells was also found to be slightly positive for staining by the antibody (data not shown). Thus, the CAG promoter was concluded to be predominantly active in glomerular epithelial cells.

We used FuGENE™6 as reagent for tail-vein-mediated gene delivery to mouse kidney, since it has been proven useful for gene delivery to mid-gestational fetuses after intravenous injection [35]. We performed 6 repeated injections of pCE-29/FuGENE™6 complex for up to 6 days, and examined fluorescence on day 4. Each treatment yielded deposition of EGFP fluorescence in some organs including the lung, heart, kidney and pancreas. The inner surface of heart (indicated by arrows in Figure 2a), and a portion of inner area (indicated by arrows in Figure 2b) and outer surface (indicated by arrows in Figure 2c), probably endothelial cells, of lung were successfully transfected. This is probably because DNA/lipid complexes have maximal accessibility to these cells upon intravenous injection. In the kidney, EGFP expression was observed in almost all (more than 80%) of the glomeruli (indicated by arrows in Figure 2d), although the intensity of staining varied among glomeruli. Expression in kidney declined when inspected on day 7 (data not shown). Other organs (including brain, liver, intestine and spleen) did not fluoresce at a detectable level. Cryostat sectioning of EGFP-expressing kidney confirmed the previous finding that EGFP fluorescence was localized to glomeruli (indicated by arrows in Figures 2e,2f) and a portion of renal tubules (indicated by arrowheads in Figure 2e), although the distribution and intensity of EGFP fluorescence were more patchy and weaker than in the MNCE kidneys [Figures 2e,2f vs. (a) of Figure 1C]. These findings indicated that the overall pattern of EGFP expression in kidney after tail-vein-mediated gene transfer was almost the same as that in MNCE transgenic kidney.

B6C3F1 male mice were injected 6 times with pCAG/DT-A/FuGENE™6 complex, pCAG/FuGENE™6 complex, FuGENE™6 only (mock injection) or PBS(-) only (mock injection). All mice in both experimental and control mock-injected groups appeared healthy during the 6-day gene delivery periods, with the exception that one mouse injected with pCAG/DT-A/lipid complex died suddenly immediately after the final injection. Major organs were immediately dissected from the dead mouse and subjected to molecular biological analysis for detection and expression of pCAG/DT-A. The remaining mice were first subjected to collection of serum and urine, and then perfused with 4% PFA in PBS(-) prior to isolation of organs and subsequent histological analyses on day 4 and weeks 3 and 5.

H-E staining of the kidneys sampled on day 4 after injection of pCAG/DT-A/FuGENE™6 complex revealed remarkable alterations of glomerular components. All specimens (8 tested) exhibited similar phenotypic alterations. These alterations included formation of segmental glomeruli (glomerular lobulation) (Figures 3d,3e,3f), mesangial cell proliferation (as evaluated by the presence of mitotic figures in the mesangial region; Figures 3a,3d,3e,3f), loss of Bowman's space probably due to mesangial cell proliferation (indicated by arrows in Figures 3a,3b), appearance of cellular degeneration (indicated by arrows in Figures 3c,3e), and attenuation of glomerular capillaries (indicated by arrowheads in Figures 3c,3f). Notably, inflammatory response (infiltration of mononuclear lymphocytes) in the tubulointerstitium was also observed in some specimens (indicated by arrowheads in Figure 3g). However, none of the mice receiving pCAG/FuGENE™6 complex exhibited such glomerular abnormalities (6 kidneys examined; Figure 3j). All mice receiving FuGENE™6 only or PBS(-) exhibited only normal glomeruli (each of 8 kidneys examined; Figure 3k). The kidneys of intact mice were also normal (2 kidneys examined; Figure 3l). Inspection of H-E-stained specimens revealed no clear abnormality in the other major organs (including brain, heart, lung, pancreas, liver and intestine) tested, although heart, lung and pancreas have previously been identified as susceptible to transfection by exogenous DNA (see Figures 2a,2b,2c).

Electron microscopic analysis of the kidneys (2 kidneys from different mice) treated with pCAG/DT-A/FuGENE™6 complex confirmed the above findings. Hypercellularity in the mesangial area of the DT-A expression vector-treated mice was due to increase in number of mesangial cells, accompanied by expansion of matrix (Figure 4a). The most prominent abnormalities observed were formation of focal deposits in glomerular basement membrane (GBM) (indicated by arrows in Figures 4c,4d,4e,4f). Initially, small deposits were also noted in GBM (indicated by arrowheads in Figures 4d,4e). However, other glomerular components including endothelial and epithelial cells appeared almost normal. These cells thus survived without suffering transfection by the DT-A expression plasmid/lipid complex. Degeneration of mesangial cells was sometimes observed (indicated by the arrow in Figure 4a). The control kidney mock-injected with PBS(-) exhibited only normal glomerular structure (2 kidneys from different mice; Figure 4b). These phenotypic alterations were thus thought to have initiated from cellular ablation of glomerular epithelium, glomerular endothelium and some mesangial cells, followed by successive formation of focal deposits in GBM and mesangial cell proliferation. Electron microscopic analysis revealed that the tubules sometimes had electron-dense droplets, probably due to readsorbtion of DNA/lipid complex (indicated by arrows in Figure 4g).

On morphometric analysis of the H-E-stained kidney specimens derived from injection of pCAG/DT-A/FuGENE™6 complex, the percentage of glomeruli scored 2 was approximately 80 % [(4-d) column in Figure 5]. In the kidneys with injection of pCAG/FuGENE™6 complex and those from mock injections, the percentages of glomeruli scored 2 were all below 3% [(4-d) column in Figure 5]. When the paraffin-embedded specimens derived from the kidney treated with pCAG/DT-A/FuGENE™6 complex were stained for PAS and PAM, there was extensive deposition of PAM-positive materials in the mesangial area (2 kidneys from different mice examined; Figure 6f), indicating expansion of mesangial matrix. The PAS-positive materials did not differ among specimens tested (Figure 6b vs. Figure 6e).

The next step was to evaluate whether the above-mentioned renal lesions would persist over 3 weeks after final administration of a DT-A expression vector.

At week 3, gradual recovery of injured glomeruli was observed in all mice (8 kidneys) tested that had been injected with pCAG/DT-A/FuGENE™6. Almost all glomeruli (more than 80%) at this stage were scored 1 [(3-w) column in Figure 5]. Notably, a portion of glomeruli was still enriched with proliferated mesangial cells, and cellular debris was often observed in the Bowman's space (indicated by the arrow in Figure 3h). PAS and PAM staining also confirmed the above finding that mesangial deposition was much reduced compared with that in the samples collected on day 4 (2 kidneys from different mice; Figures 6e,6f vs. Figures 6h,6i, respectively).

At week 5, several abnormal findings found in glomeruli sampled at week 3 had nearly been lost: These included expansion of glomerular capillaries and reduction of mesangial cell proliferation and mesangial matrix, although proteinaceous substances were often observed in the Bowman's space (Figure 3i). Electron microscopic observation revealed almost complete recovery of the glomeruli (Figure 4h). This was confirmed by morphometric analysis of H-E-stained specimens: Almost all glomeruli (more than 70%) at this stage were scored 0 [(5-w) column in Figure 5]. Staining with PAS and PAM revealed glomerular structures indistinguishable from those of normal kidney specimens (Figures 6b,6c vs. Figures 6k,6l, respectively).

To obtain evidence that the abnormalities in kidneys mentioned above are in fact caused by introduction and expression of a DT-A expression vector, we performed PCR and RT-PCR analyses. As shown in Figure 7A, the pCAG/DT-A introduced was detected in all major organs tested (including brain, where it is believed to be difficult for exogenous substances in blood to be transferred due to the blood-brain barrier). RT-PCR analysis revealed that several organs (including lung, heart, kidney and intestine) exhibited a band of the expected size (Figure 7B). Organs derived from mock injection [PBS(-) only] were negative for expression of DT-A mRNA (Figure 7B). These findings suggest that the glomerular injuries reported here were actually caused by expression of the DT-A gene.

Serum levels of CRT, an endogenous marker of renal function, were transiently elevated in the pCAG/DT-A/FuGENE™6 complex-administered mice, with a peak at day 4, while the other biochemical markers including BUN, Na⁺, Cl^-, and K⁺ were unaltered through day 4, and at weeks 3 and 5 (Figure 8). Mice treated with pCAG/FuGENE™6 complex, FuGENE™6 only or PBS(-) only exhibited no change in serum parameters at any time-point tested (Figure 8).

Urine levels of proteins were significantly elevated in the mice treated with pCAG/DT-A/FuGENE™6 complex through day 4 and at weeks 3 and 5 (Table 1). Furthermore, urine levels of occult blood were transiently elevated in the pCAG/DT-A/FuGENE™6 complex-administered mice, with a peak at day 4 (Table 1). These findings suggest that the renal damage caused by DT-A expression is correlated well with alterations in biochemical parameters in serum and urine.