Diffuse Glomerular Nodular Lesions in Diabetic Pigs Carrying a Dominant-Negative Mutant Hepatocyte Nuclear Factor 1-Alpha, an Inheritant Diabetic Gene in Humans

<div><p>Glomerular nodular lesions, known as Kimmelstiel-Wilson nodules, are a pathological hallmark of progressive human diabetic nephropathy. We have induced severe diabetes in pigs carrying a dominant-negative mutant hepatocyte nuclear factor 1-alpha (HNF1α) P291fsinsC, a maturity-onset diabetes of the young type-3 (MODY3) gene in humans. In this model, glomerular pathology revealed that formation of diffuse glomerular nodules commenced as young as 1 month of age and increased in size and incidence until the age of 10 months, the end of the study period. Immunohistochemistry showed that the nodules consisted of various collagen types (I, III, IV, V and VI) with advanced glycation end-product (AGE) and <i>N</i>^ε-carboxymethyl-lysine (CML) deposition, similar to those in human diabetic nodules, except for collagen type I. Transforming growth factor-beta (TGF-β) was also expressed exclusively in the nodules. The ultrastructure of the nodules comprised predominant interstitial-type collagen deposition arising from the mesangial matrices. Curiously, these nodules were found predominantly in the deep cortex. However, diabetic pigs failed to show any of the features characteristic of human diabetic nephropathy; e.g., proteinuria, glomerular basement membrane thickening, exudative lesions, mesangiolysis, tubular atrophy, interstitial fibrosis, and vascular hyalinosis. The pigs showed only Armanni-Ebstein lesions, a characteristic tubular manifestation in human diabetes. RT-PCR analysis showed that glomeruli in wild-type pigs did not express endogenous HNF1α and HNF1β, indicating that mutant HNF1α did not directly contribute to glomerular nodular formation in diabetic pigs. In conclusion, pigs harboring the dominant-negative mutant human MODY3 gene showed reproducible and distinct glomerular nodules, possibly due to AGE- and CML-based collagen accumulation. Although the pathology differed in several respects from that of human glomerular nodular lesions, the somewhat acute and constitutive formation of nodules in this mammalian model might provide information facilitating identification of the principal mechanism underlying diabetic nodular sclerosis.</p></div

Reverse transcription-PCR for endogenous HNF1α and HNF1β in wild-type pigs at 4 weeks of age.

<p>Both HNF1α (<b>A</b>) and HNF1β (<b>B</b>) were negative in isolated glomeruli. Liver was used as a positive control, and heart as a negative control. G  =  isolated glomeruli; L  =  liver; H  =  heart.</p

Transmission electron microscopy at age 4 weeks (A,B,E) and 5 months (C,D) in transgenic pigs.

<p><b>A</b>) In 4-week-old transgenic pigs, mesangial widening is associated with fiber deposition in the mesangial matrices. Magnification: 2,000×. <b>B</b>) Fibers accumulated at mesangial areas, forming early lesion. Magnification: 500×. <b>C</b>) At 5 months, established glomerular nodules showed that mesangial areas and capillary lumens are filled with bright fibers (arrows). Vacuolations of proximal tubules were also seen (arrowheads). Magnification: 300×. <b>D</b>) Subendothelial widening with loss of endothelial fenestration and mesangial interposition are shown. Note that collagen is also found in the subendothelial spaces (arrows). Magnification: 1,500×. <b>E</b>) The nodules consist of fibril collagens with cross striation, indicating interstitial-type forms of collagen fibrils. Magnification: 10,000×. <b>F</b>) Thickness of glomerular basement membranes in transgenic pigs was no different from those in wild-type pigs at 4 weeks and 5 months old. Transgenic pigs; n = 1, wild-type pigs; n = 3. Tg  =  transgenic pigs; WT  =  wild-type pigs; GBM  =  glomerular basement membrane.</p

Analysis of biochemical parameters in transgenic (Tg) and wild-type (WT) pigs at age 1, 5 and 10 months.

<p>Aberrations: Tg  =  transgenic pigs; WT  =  wild-type pigs; *: n = 1.</p

Renal pathological findings at age 4 and 19 weeks in transgenic pigs.

<p><b>A</b>) In transgenic pigs, mesangial expansion commenced as early as 4 weeks. At 19 weeks, distinct glomerular nodules had formed. Magnification: 400×. <b>B</b>) The number of glomeruli with nodules as a fraction of the total number was compared between the superficial cortex and deep cortex. <b>C</b>) Glomerular tuft area in superficial and deep cortexes was compared between wild-type pigs and transgenic pigs. Transgenic pigs; n = 3, wild-type pigs; n = 3. *<i>P</i><0.05. WT  =  wild-type pigs; Tg  =  transgenic pigs.</p

<i>In vitro</i> development of the chimeric embryos produced by injection or aggregation method.

*<p>Blastomeres isolated from single embryos.</p>**<p>Blastomeres isolated from two embryos.</p

Production of chimeric blastocysts with donor ICM and parthenogenetic host embryos.

<p>(A, D) A donor ICM (stained with Dil) aggregated with host blastomeres isolated from parthenogenetic embryos at the morula (A) or 4–8 cell stage (D). (B, E) Bright field images of chimeric blastocysts developed from the aggregated embryos. (C, F) Confocal fluorescence images of chimeric blastocysts showing DiI fluorescence in ICMs. Single confocal sections of fluorescence were overlaid on the bright field images. (G-I) Parthenogenetic host morulae injected with DiI-stained donor ICM (G) and resultant chimeric blastocysts (H, I). Arrow heads, ICM. Scale bars = 50 µm.</p

Design of ZFNs targeting the pig <i>IL2RG</i> gene and isolation of nuclear donor cells.

<p>(A) Schematic representation of ZFNs binding to pig <i>IL2RG</i>. The coding and untranslated regions are indicated by gray and white boxes, respectively. A ZFN consists of a nuclease domain (Fok I) and a DNA-binding domain (zinc finger proteins), and the recognition sequences of the zinc finger proteins are underlined. (B) Flow chart for the isolation of nuclear donor cells (clone #98) for SCNT. (C) ZFN-induced mutation in cell clone #98. The upper and lower sequences represent the WT and clone #98 sequence of <i>IL2RG</i>, respectively. The deletion mutation and nucleotide substitution in clone #98 are indicated by a hyphen and black box, respectively. The initiation codon of <i>IL2RG</i> is shown in a dotted box. The ZFN-binding and ZFN-cleavage sites are double-underlined and boxed, respectively. The major transcription initiation site is indicated with a circle.</p

Generalized scheme for the production of chimeric porcine blastocysts and fetuses by the aggregation method.

<p>For <i>in vitro</i> analysis of the chimeric blastocyst formation, donor ICMs were isolated from parthenogenetic blastocysts derived from IVM oocytes. Isolated ICMs stained with DiI were aggregated with blastomeres isolated from parthenogenetic host embryos in a microwell made on the bottom of a culture dish. For <i>in vivo</i> analysis of chimeric fetus formation, the donor ICMs were isolated from blastocysts fertilized <i>in vitro</i> by transgenic boar sperm carrying the fluorescent huKO gene. ICMs of the IVF blastocysts were similarly aggregated with the parthenogenetic host embryos as the DiI-stained ICMs, and the resultant blastocysts were transferred to recipient pigs to obtain chimeric fetuses.</p

Phenotypes of <i>IL2RG</i> KO pigs.

<p>(A, B) The thymic phenotype in WT and <i>IL2RG</i> KO pigs. The white arrowheads indicate normal thymuses in WT pigs. (C, D) Histological analysis of the spleens of WT and <i>IL2RG</i> KO pigs. The white pulp of the spleen is indicated by a dotted white circle. Bar = 100 µm. (E) The proportion of lymphocytes in the peripheral blood (PB) of WT and <i>IL2RG</i> KO pigs. The data represent the means ± SD values for 4 pigs. The asterisk indicates a statistically significant difference (P<0.01) between the values for WT and <i>IL2RG</i> KO pigs (n = 4).</p