Diabetic condition induces hypertrophy and vacuolization in glomerular parietal epithelial cells

Diabetic nephropathy (DN) is accompanied by characteristic changes in the glomerulus, but little is known about the effect of diabetes on parietal epithelial cells (PECs). In this study, a descriptive analysis of PECs was undertaken in diabetic db/db mice and in diabetic patients. PEC hypertrophy was significantly more prominent in diabetic mice than in nondiabetic mice, and this was evident even at the early stage. Additionally, the number of vacuoles in PECs was markedly increased in diabetic mice, suggesting the presence of cellular injury in PECs in DN. Although rare, binuclear cells were observed in mice with early diabetes. In cultured PECs, a high glucose condition, compared with normal glucose condition, induced cellular hypertrophy and apoptosis. Flow cytometry showed that some PECs in the G0 phase reentered the cell cycle but got arrested in the S phase. Finally, in human diabetic subjects, hypertrophy and vacuolization were observed in the PECs. Our data showed that PECs undergo substantial changes in DN and may participate in rearrangement for differentiation into podocytes.


High glucose exposure induced PEC hypertrophy in vitro.
To elucidate the mechanisms for the hypertrophic changes in PECs, we used an established immortalized PEC cell line 23 . The expressions of PECspecific mRNAs, claudin-1, pax-2, pax-8, and CD10 were high, whereas the expressions of the podocyte-specific mRNAs, WT-1, nephrin, podocin, synaptopodin, nestin and desmin were low or barely detected (Fig. 2a). To further verify the morphological features of PECs in culture, we stained the cells with the cytoskeletal protein F-actin. There was a thin linear staining with F-actin in the cytoplasm and along the cell borders, reflecting the characteristic simple cytoskeletal structures of PECs, as previously described 23 (Fig. 2b). We examined the effect of high glucose exposure. On flow cytometry, cell size was significantly greater in the PECs cultured under high glucose (HG, 30 mM) conditions than in those under normal glucose (NG, 5 mM) plus 25 mM mannitol (Man) condition (Fig. 2c). The ratio of the total cellular protein to cell number, which is another indicator of cellular hypertrophy, revealed HG-induced hypertrophic changes in the PECs (Fig. 2d). The cellular size of PECs was not increased by simulation with other hypertrophic factors, including TGF-β1 24 , insulin, or aldosterone, with or without sodium chloride 25 (Fig. 2e). Because hypertrophy is often accompanied by cell cycle arrest 26 , we analyzed the cell cycle distribution in PECs using flow cytometry. In an HG condition, the percentage of cells decreased in the G0/G1 phase and increased in the S phase; these results indicated that some PECs in the G0/G1 phase entered the S phase after HG stimulation. However, these cells did not further progress into the G2 or M phase, indicating that the cell cycle was arrested in these cells (Fig. 2f).
HG exposure induced cellular apoptosis in PECs in vitro. Treatment of PECs with increasing concentrations of glucose for 24 h caused dose-dependent decreases in cell viability (Fig. 3a). Hoechst staining (Fig. 3b) and flow cytometry (Fig. 3c) revealed that treatment with increased concentration of glucose for 24 h increased apoptosis. Moreover, the live cell analysis system showed concentration-dependent increase in cell death (Fig. 3d) and apoptosis (Fig. 3e).

Hypertrophy and vacuolization in human PECs in DN. Hypertrophied and vacuolated PECs under
diabetic conditions were further examined using kidney tissue samples from patients with and without diabetes ( Table 1). Quantitative analysis using plastic-embedded semi-thin sections stained with toluidine blue showed that the cellular volume of PECs was significantly increased in the diabetes group compared with the control group (p < 0.05) (Fig. 4a-e). With the help of electron microscopy, we examined the morphological changes in PECs on human renal biopsy specimens of control patients and patients with diabetes. We observed flat cell Representative images of PAS staining (upper) and LTA staining (lower) in the 12-week-old db/m and db/ db mice. The dot plot graph shows the average of semiquantitative cuboidal PEC score in the db/m and db/db mice. (c) Immunostaining for CD44 in kidneys from the experimental mice groups (i.e., db/m, db/db, salinetreated, and adriamycin-treated). (d) Representative PECs (arrows) on transmission electron microscopy at 12 weeks of age in each mouse group. The dot plot graphs show the temporal changes in the cellular volume of PECs. (e,f) Temporal changes in the GBM thickness (e) and BBM thickness (f) in the db/m and db/db mice. (g) Representative PECs (arrows) on transmission electron microscopy at 24 weeks of age in the db/m and db/ db mice. Arrowheads indicate the vacuole in PECs. The right inserts are the views on higher magnification. Quantitative analysis of the number of vacuoles per cell is shown in the lower panel. (h) A binucleated PEC is identified in db/db mice at 12 weeks of age but not in db/m mice. Arrows indicate PECs. PECs, parietal epithelial cells. *P < 0.05 vs. db/m mice. Scale bars: (a up) 20 μm, (a low) 5    www.nature.com/scientificreports/ bodies and a little vacuolar changes in the control patients (Fig. 4f), whereas cellular enlargement and vacuolar changes in the patients with diabetes (Fig. 4g,h). Quantitative analysis showed that the ratio of vacuolated PEC per total PEC number in DN was high compared with that in control subjects but did not reach significance ( Fig. 4i).

Discussion
In this study, we focused on the morphological alterations in PEC, which is one of the glomerular cells, in DN. We found hypertrophy of PECs and decrease in cuboidal PECs in diabetic mice. We analyzed the morphological changes on HG stimulation causing cell cycle arrest and cellular hypertrophy. Moreover, in mouse models with diabetes, HG stimulation caused PEC injury, which was represented by vacuolization. Although the pathologic implications remain to be clarified, our study identified several histologic characteristics of PECs in DN and may provide some information for further investigation on PECs.
In human DN, PEC hypertrophy was described as enlargement of the nuclear diameter and the presence of euchromatic nuclei on transmission electron microscopy, although the timing of occurrence was not clarified and the detailed mechanism was not elucidated 12 . On electron microscopy, PEC hypertrophy was observed in the early stage in the mice model of DN. PEC hypertrophy was apparent as early as the occurrence of BBM thickening; this suggested that PEC hypertrophy plays an important role in the hyperproduction of the BBM by PECs. In renal cells, HG has been reported to induce cellular hypertrophy in podocytes 17,18,[27][28][29] , mesangial cells 30-32 and renal tubular cells 33,34 . We tested various stimuli, including HG, insulin, TGF-β1 and aldosterone and demonstrated that only HG caused hypertrophy of the PECs. The hypertrophic changes in podocytes have been reported to be involved with the cell cycle 35 . Consistently, our cell cycle analysis suggested reentry into the cell cycle and the S phase arrest of hypertrophied PECs.
In the process of searching for the hypertrophied PECs in diabetic mice, we carefully distinguished them from activated PECs and cuboidal PECs. In activated PECs, various morphological changes were reported, including cell enlargement, nuclear swelling, structural changes from flat to cuboidal shape, and extracellular matrix production 10,36 . CD44 is a marker of activated PEC 4,9 , and the expression is higher in crescentic glomerulonephritis and in FSGS 16,37,38 . The relationship between CD44 expression and hypertrophy was suggested in DN 12,39 . Indeed, the expression of CD44 on PEC was found in patients with DN, especially in the late phase 12 . However, our findings showed hypertrophy of PECs without CD44 expression in the early stage of diabetic model mice, suggesting that PEC hypertrophy in the early stage of DN was independent of CD44 activation. The CD 44 expression in human samples with DN in the early stage needs to be further investigated. Cuboidal PECs have been defined by their shape, localization on the Bowman's capsule and the presence of brush borders and abundant mitochondria, similar to the shape of proximal tubular cells. These cells were recently reported to be more easily activated compared with classical flat PECs in disease conditions 11 . We found that the area lined with cuboidal PECs was decreased in diabetic mice, implying that the hypertrophied PECs may extrude cuboidal PECs away into the proximal tubular area. Sex hormones [40][41][42] , age 43,44 and differences in mouse strain 40 have been described to affect the distribution of cuboidal PECs, although the factor that reduces the cuboidal PECs in diabetic mice remain to be clarified.
We demonstrated that HG exposure led to PEC apoptosis in a dose-dependent manner in vitro. PEC apoptosis has been reported in high fat feeding mice 45 , albumin overload model mice and rats 46 , transgenic mouse model of human immunodeficiency virus-associated nephropathy 47 and mice with electrocoagulation of the renal cortex 48 in vivo. Moreover, apoptosis has been reported in cultured PECs exposed to albumin 46 and IFN-β exposure 49 . On transmission electron microscopy, our results did not show any apoptotic PECs, but there was vacuolization of PECs in early diabetes mice model and in diabetic human subjects. Because vacuolization is one of the characteristics of cell injury in podocytes 21,22 , this result suggested the presence of PEC injury in DN. In addition, the presence of binuclear PECs in diabetic mice suggested that PECs in DN undergo mitotic catastrophe instead of apoptosis. Mitotic catastrophe is cell death due to aberrant mitosis, and this implies PEC detachment from the BBM and death, as previously described in podocytes 50-52 . Pathologically, this PEC injury was speculated to reduce glomerular regeneration in DN, because, recently, the function of PECs as progenitors of podocytes has been noticed 53 . PEC injury may also lead to periglomerular inflammation through cytokine secretion 54 , which can result in periglomerular fibrosis, as observed previously in diabetic model mice 55 and human diabetic patients 56 .
We acknowledge some limitations in this study. First, the number of human subjects for the assessments of PEC morphology was small. Further studies including larger populations of patients with DM are necessary. Second, our examination on human diabetic sample was performed only at the late stage of DN, which is associated with profound proteinuria and elevated creatinine. Generally, there are few chances of performing renal biopsy of patients with early stage of DN. However, hypertrophy and vacuolization of PECs was observed even at late stage of DN. Further studies on human subjects with only microalbuminuria and early histologic changes are needed.
In conclusion, diabetic condition induced hypertrophy and injury in PECs. Because PECs are considered progenitors of podocytes, this injury to PECs might impair glomerular regeneration or periglomerular inflammation in a later stage. Further studies are warranted to elucidate the potential pathologic role of these morphological changes in PECs in DN.      . (g,h) The PECs in patients with DM were enlarged (arrow) and exhibited cytoplasmic vacuolization (arrowhead). Each figure was obtained from corresponding patients in Table 1 www.nature.com/scientificreports/ (Applied Biosystems) 59 . The relative mRNA level for each gene was normalized to the mRNA express level of β-actin. The primer sequences for the forward and reverse primers are listed in Table 2.

Methods
Immunohistochemical analysis. Immunohistochemistry was performed, as previously described 59 .
Briefly, paraffin-embedded kidney pieces were stained using rat monoclonal antibody against CD44 (1:500; BD bioscience, San Diego, California; catalog number 550538). Before incubation, the tissue sections underwent antigen retrieval in 0.01 mol/l of sodium citrate buffer (pH 6.0) at 120 °C for 20 min. Then, the sections were incubated with biotin-labeled goat anti-rat IgG (1:200; Vector Laboratories, Burlingame, CA) antibody then treated with the Vectastain Elite ABC Kit (Vector Laboratories). Sections were examined using a biological microscope (Olympus BX53; Olympus Corporation, Tokyo, Japan). Quantitative computer-assisted image analysis was performed by a blinded observer using the Image-Pro Plus software (version 5.1; Media Cybernetics, Bethesda, MD), which is an image analysis software program.
Immunofluorescence staining. Immunofluorescence staining for PAX8 was performed on paraffinembedded sections using rabbit polyclonal anti-PAX8 antibody (1:100; Proteintech Group, Rosemont, IL; catalog number 10336-1-AP). Alexa Fluor 594 donkey anti-rabbit IgG (Invitrogen) was used as the secondary antibody. Immunofluorescence staining for Fluorescein-labeled LTA (Vector Laboratories) was performed in a similar manner. The slides were premounted with VECTASHIELD mounting medium with DAPI (Vector Laboratories) and kept in darkness to dry. For fluorescence microscopy, all sections were stained and analyzed simultaneously to exclude artifacts due to variable decay of the fluorochrome. The sections were examined using a confocal fluorescence microscope (Carl Zeiss LSM-710) at a magnification of 400 × .

Identification of flat PECs and cuboidal PECs.
PECs comprise mainly of flat and cuboidal PECs. We discriminated between flat and cuboidal PECs using PAX8 which stain flat PECs and cuboidal PECs in Bowman's capsule) 60 , and LTA which only stain cuboidal PECs in Bowman's capsule 11 . Flat PECs were identified as PAX8 positive LTA negative cells internally lining the Bowman's capsule. We measured flat PEC nuclear volume as a substitute for flat PEC cellular volume, because the ratio between nuclear and cytoplasmic size has long been observed to be maintained at a constant value 61 . The flat PECs' nuclear area was measured as PAX8 positive area per cell, and then the nuclear volume was calculated as described in morphological analysis. Cuboidal PECs were identified as LTA positive cells in the Bowman's capsule for immunofluorescent staining 11 . For morphometric analysis of cuboidal PECs, each glomerulus within the kidney section was ranked on a scale of zero to five, depending on the degree of cuboidal PECs. Each rank was represented as follows: 0 = no cuboidal PECs; 1 = cuboidal PECs up to one-fourth of the glomerular circumference; 2 = cuboidal PECs < 50% of the glomerular circumference; 3 = cuboidal PECs < 75% of the glomerular circumference; 4 = cuboidal PECs < 100% of the glomerular circumference and 5 = entirely cuboidal PECs without the classical flat PECs. Age 46,47 , gender [43][44][45] and genetic background 43 affect the distribution of cuboidal PECs and flat PECs; therefore, we matched age, gender, and genetic background to compare cuboidal PECs other than compared conditions. We compared cuboidal PEC score of male db/m mice with that of male db/db mice. The background of mice is shown in Supplementary  Table S1. Thirty glomeruli were obtained for each sample, and the total mean score was calculated.
Electron microscopy. The kidney tissues were fixed on 2% glutaraldehyde and embedded in epoxy resin.
The semi-thin sections stained with toluidine blue were inspected under the light microscope. The ultrathin sections, stained with uranyl acetate and lead citrate, were investigated and snapped using an electron microscope. To evaluate PEC morphometry, electron micrographs of at least five glomeruli per kidney were randomly obtained from each mouse and observed at a magnification of 2000 × . To evaluate GBM thickness and BBM thickness, a mean of 30 measurements were taken per kidney at random sites where the GBM and BBM were displayed in best cross-section as previously described 62 .  TGT GCA TGG CCT CTT GTT  AGG GCC TTT CCT ATG GAT GAG AGT   PAX-2  AGC TAC ATG CCC ACT AAA GCA CAG  CAC GGG ACC ATG TTC GTC A   PAX-8  TGC TAT CGC AGG CAT GGT G  ACT ACA GAT GGT CAA AGG CTG TGG   CD10  TTA TGT CCT GTC ATA GCA GCC AAA G CAG CAG GAA TCT AGG CAC CAGAG   WT-1  TGA AGA CCC ACA CCA GGA CTC  TGT GAT GGC GGA CCA ATT C   Nephrin  GTG ATG ACG TCA CAG AAG CAA GAA  TTT GTG TGG CAT ACA CTG  www.nature.com/scientificreports/ Human renal biopsy specimens. We obtained renal needle biopsy specimens from 15 patients. We retrospectively identified eight control cases without diabetes and seven cases with diabetes. Control cases were obtained from patients without glomerular abnormalities in light microscopy. Before study enrollment, written informed consent was obtained from all patients. The study was performed in accordance with the Declaration of Helsinki, and the study protocol was approved by the human ethics review committee of the Department of Internal Medicine, School of Medicine, Keio University. Patients for whom fewer than nine PECs were observed on biopsy were excluded. PECs with sclerotic glomeruli were excluded. All visible PECs from each human sample were assessed by morphology. All the biopsies were categorized based on the pathologic classification of the Renal Pathology Society (RPS) Diabetic Nephropathy Classification 3 . PEC area was measured in plasticembedded semi-thin sections stained with toluidine blue using light microscopy at a magnification of 1000 × . The area measurement was performed using Image-Pro Plus software. The volume was calculated as described in morphological analysis below. Vacuolated PECs were counted and assessed by the ratio to the total PEC number using electron microscopy. The patient clinical data at the time of renal biopsy and the pathological data are summarized in Table 1.
Morphological analysis. Podocyte volume has been estimated using various methods [63][64][65][66] . However, to the best of our knowledge, a method to quantitatively estimate PEC volume and PEC nuclear volume has not been reported to date. Therefore, we substituted the measurement methods of podocytes for those of PECs. First, for counting the PEC number, we used the Weibel and Gomez method. Briefly, the PEC number was calculated from the cell density per volume (Nc v ) and the volume density of PECs (Vc v ) according to the equation: Nc v = k/β × Nc A Statistical analysis. Data are expressed as the mean ± standard error of mean and were analyzed by oneway analysis of variance, followed by the Bonferroni post hoc test. The criterion for statistical significance was a P value < 0.05.