In glomerular cells of puromycin aminonucleoside nephrosis rats both phosphorylated and total STAT3 levels increased during proteinuria

Halil İbrahim Saygıa,⁎, Zeynep Banu Güngörb, Fatma Kalayb, İsmail Seçkina
a İstanbul University, Cerrahpaşa Medical Faculty, Histology and Embryology Department, Turkey
b İstanbul University, Cerrahpaşa Medical Faculty, Biochemistry Department, Turkey

Abstract

Recent studies showed that JAK/STAT pathway plays role in glomerular damages. The fact that STAT3 could be activated also by oXidative stress make Puromycin Aminonucleoside (PAN) Nephrosis model very appropriate for examination of STAT3 expression changes in glomerular pathology. Along with a control group, three PAN groups sacrificed on different days were formed by the i.p. injection of PAN for 5 consecutive days. Throughout the experiment, 24-hour-urines were collected on specific days and proteinuria levels were monitored. At the end of the experiments, tissue specimens were stained immunohistochemically for both total and phosphorylated STAT3 and evaluated subjectively. They were also examined ultrastructurally in transmission electron micro- scope. The proteinuria levels did not increase significantly on 5th day but showed a dramatic increase on 10th and 15th days. On 20th and 25th days, urinary protein levels gradually decreased. Ultrastructural examinations showed glomerular damages such as significant decrease in slit pore number, a significant gradual increase in glomerular basement membrane thickness and podocyte hypertrophy on 5th and 15th days; besides significant increase in mesangial matriX. The first significant increases in phosphorylated and total STAT3 levels occurred in 5th day and 15th day groups respectively. These increases diminished in 25th day group. Regarding all the findings, it was deduced that STAT3 is one of the active factors in glomerular pathologies.

1. Introduction

Kidney diseases which can convert to end-stage renal disease (ESRD) if untreated, keeps having a high incidence in the world (Levey et al., 2007). Recent studies showed that the key point lies within the glomerulus where the filtration takes place (Haraldsson et al., 2008). Glomerulus is composed of three cell types all of which are indis- pensable for proper filtration process: endothelial cells that form the capillaries, mesangial cells that lie within the capillary tuft and podo- cytes that wrap around the capillaries (Haraldsson et al., 2008). Po- docytes which are terminally differentiated cells with a complex ul- trastructure thought to be more vulnerable compared to the other two due to its sensitive spot (TharauX and Huber, 2012). Podocytes have long processes called as foot processes and between them slit pores exist. In these pores slit diaphragm structures are present and they are central to glomerular filtration barrier.

One of the models used for glomerular kidney diseases is Puromycin Aminonucleoside (PAN) Nephrosis model. This model, depending on the application, could mimic minimal change disease (MCD) and focal segmental glomerulosclerosis (Pippin et al., 2009; Shen et al., 2016). PAN leads to foot process disorganization and slit diaphragm break- down that’s why it ends up in deterioration of glomerular filtration barrier and proteinuria (Burford et al., 2017). PAN is thought to be causing oXidative stress in podocytes (Diamond et al., 1986; Wang et al., 2009). However, the exact mechanism of PAN nephrosis could not be clarified.

JAK/STAT pathway was first described in inflammation signalling but later on it was realized that this pathway plays role also in many other cellular activities (Matikainen et al., 1999; Simon et al., 1998). In cancer and metastasis this pathway was found to be involved in in- flammation, cell proliferation, apoptosis and epithelial mesenchymal transitions (Teng et al., 2014; Wendt et al., 2014). In addition to many other diseases, recent studies showed that this pathway plays role widely also in kidney damages (Brosius and He, 2015; Chuang and He, 2010). Especially STAT3 was found to be of great importance for glo- merular pathology (Lu et al., 2009).

STAT3 firstly studied in mesangial cells of the glomerulus and found to be affected in Thy1 glomerulonephritis (Yanagita et al., 2001). Later on it was showed that STAT3 is the key player in HIV-Associated Ne- phropathy (He et al., 2004). STAT3 also have been studied in Diabetic Nephropathy and Adriamycin Nephrosis models and it was showed that the STAT3 levels had been significantly changed in these pathologies (Lu et al., 2009). However, the specific role of STAT3 in nephrosis is not clear enough and our literature search did not reveal any STAT3 study in PAN. Regarding the fact that the STAT3 can be activated via in- tracellular oXidative stress (Simon et al., 1998), it would be very pur- posive to study STAT3 levels in PAN nephrosis model. In this regard we aimed to form a PAN nephrosis model and examine the changes in both total and phosphorylated STAT3 levels.

2. Materials and methods

2.1. Animals and model formation

Twenty-four male Wistar albino rats weighing approXimately 120 g were used in the study. They were 6-week-old and obtained from EXperimental Animals Reproduction and Research Laboratory (Istanbul University, Turkey). They were housed three to a cage and were fed ad libitum with powdered standard rat chow and tap water under standard conditions (20-24oC temperature, 50–60% humidity, 12 h light/dark cycle). The cages were cleaned regularly. The study was conducted in compliance with ethical standards (Istanbul University, Animal Ethics Committee, Nu: 2015/37, 03/03/2015).

The rats were classified as 4 groups i.e. siX rats per group. The ne- phrosis model was induced as described by Sasaki et al. (1997). In this model the regimen followed causes a slow but sustained cytotoXicity to glomerular cells. The rats in the control group were injected 1 ml physiological salt solution whereas the rest of the rats were given puromycin aminonucleoside (PA) (sc-200112 A, Santa Cruz; Dallas, Texas, United States) as 1.5 mg/100 g i.p. in 1 ml physiological salt solution for five consecutive days. Control and 5th day group was sa- crificed on 5th day. 15th and 25th day groups were sacrificed on 15th and 25th days respectively.

2.2. Biochemical analysis

The rats were transferred to metabolic cages on −1, 5, 10, 15, 20 and 25th day and 24-h urines were collected. Protein amount in urine was measured quantitatively using the modified trichloroacetyl acid (T9159, Sigma-Aldrich; St. Louis, Missouri, United States) method (Choi et al., 1993).

2.3. Light and electron microscopic examinations

Animals were anesthetized and left kidneys were excised through abdomen. The kidneys were transferred to 10% neutral buffered for- malin and let them be fiXed before embedding in paraffin. Paraffin sections were stained with periodic acid-Schiff (PAS) stain. Mesangial matriX amounts in glomeruli were semi-quantitatively scored regarding PAS-positive material as performed by Raij et al. (1984). Briefly, at least fifty representative glomeruli scattered in the preparation assessed re- garding its mesangial matriX expansion. They were all assigned grades from 0 to +4 in proportion to glomerular involvement of mesangial matriX i.e. + 1 and +4 grades mean 25% and 100% glomerular in- volvement respectively. This grading was done by two observers in- dependently and blindly. The grades were summed and divided by the number of glomeruli examined. The final scores of the observers were averaged.

The cortical renal tissue divided into 1 mm3 pieces were transferred to 2.5% glutaraldehyde (Sigma-Aldrich) in a 0.1 M phosphate buffer solution for first fiXation. Post fiXation was achieved by 1% OsO4 pre- pared in the same buffer. Later the tissue samples were dehydrated with graded ethanol series (Merck, Kenilworth, New Jersey) and embedded in Araldite medium (10951, Sigma-Aldrich). Semi-thin sections,approXimately 1 μm, were cut by glass knives in the ultramicrotome and stained with 1% toluidine blue (prepared with 1% borax in bidis- tilled water) for observation under binocular light microscope (Olympus BX61). Ultra-thin sections were obtained in 50 nm thickness onto copper grids (300 mesh) with the same microtome, stained with uranyl acetate and lead citrate and they were investigated by trans- mission electron microscope (Jeol, JEM 1011).

Fig. 1. Proteinuria levels by days. Before the injections protein amounts in the urine was in normal range and did not display a statistically significant increase 5 days after the injections. However, protein excretion in the urine showed a significant increase on 10th and 15th days; and a significant decrease on 20th and 25th days. (*p < 0.05 compared to -1th, 5th, 20th and 25th days.) (N = 24 for -1th and 5th days. N = 12 for 10th and 15th days. N = 6 for 20th and 25th days).

Two morphometric analyses were carried out i.e. glomerular base- ment membrane (GBM) thickness measurement and slit pore (SP) number count in unit length by locating five different peripheral sites from three glomeruli at 20,000X magnification. SP count was held in 7 um length of GBM.

2.4. Immunohistochemical analysis

Immunohistochemical stainings were performed with mouse monoclonal anti-total-STAT3 (sc-8019, Santa Cruz) (tSTAT) and mouse monoclonal anti-phosphorylated-STAT3 (Tyr705) (sc-8059, Santa Cruz) (pSTAT) antibodies. The sections of 5 μm thickness were placed onto slides coated with poly-L-Lysine (P8920, Sigma-Aldrich) then deparaffinized and rehydrated in graded alcohol series. ImmunoCruzTM mouse LSAB Staining System (sc-2050, Santa Cruz) was used and the protocol suggested by the manufacturer was followed. Sections were counter-stained with toluidine blue. The specificity of the stainings were proved via omitting primer antibody step.

ImmunoperoXidase staining of the rats’ glomeruli was evaluated as performed by Chen et al.(2006). This evaluation has been done by two observers blindedly in all rats of each group in at least fifty re- presentative glomeruli scattered in the preparation. Immunopositivity was graded between 0 to 3; from no staining to strong staining. Then the grades were summed and divided by the number of glomeruli ex- amined.

2.5. Statistical analysis

Values were expressed as mean ± SD. Comparison of means was performed by One Way ANOVA Dunnett T3 test using SPSS21.0 sta- tistical software. p< 0.05 was considered statistically significant.

3. Results

3.1. Proteinuria

Proteinuria levels were followed by days and plotted in Fig. 1. Be- fore the injections, proteinuria of the rats was averagely 3.95 ± 0.84 mg/day. On 5th day, there was a proportionally high in- crease in average proteinuria level (6.57 ± 0.87 mg/day) but this in- crease was not statistically significant. However, on 10th day a sig- nificant increase was observed and the proteinuria became
138.56 ± 2.83 mg/day. This high levels persisted on 15th day (131.17 ± 6.71 mg/day). On 20th and 25th days, protein amount in the urine decreased significantly (31.10 ± 25.90 and 11.25 ± 5.10 mg/day respectively). The urine volumes did not display any significant changes among the groups (data now shown).

3.2. Light microscopic findings

Mesangial matriX amounts were semi-quantitatively evaluated and presented in Fig. 2. Mesangial matriX score of control rats turned out to be 0.498 ± 0.173. In 5th day group, the score increased significantly and became 1.330 ± 0.178. The high amount of mesangial matriX within the glomeruli found to be persisting in 15th day group (1.450 ± 0.155). However, in 25th day group it showed a significant decrease compared to 15th day group and became 1.140 ± 0.139. An example glomerulus for each scoring was shown in Fig. 2.The semi thin sections stained with toluidine blue were evaluated based on the morphological criteria and with this evaluation many sites within the mesangium were thought to be sites of angiogenesis in 5th and 15th day groups (Fig. 3).

3.3. Electron microscopic findings

In ultrastructural examination many pathological changes were detected in podocytes (Fig. 4). Firstly, podocytes found to have un- dergone hypertrophy gradually in 5th and 15th day groups. These hy- pertrophic podocytes showed poor electron-density and poor ultra- structure in addition to many protein absorption granules. Foot processes were effaced and slit diaphragms were displaced apically. Effaced foot processes displayed many vesicles, formed or forming.

Two morphometric evaluations, GBM thickness (Fig. 5) and SP number in unit length (Fig. 6), were done by electron microscopy. Average GBM thickness was 118.79 ± 5.44 nm in control group. Al- though an increase was seen in 5th day group (121.79 ± 2.12 nm) it was not statistically significant. However, in 15th day group GBM thickness showed a significant increase and became 142.93 ± 11.68 nm. In 25th day group, with a significant decrease, it came close to normal levels by becoming 121.39 ± 2.52 nm.

Fig. 3. Toluidine blue staining of semi-thin sections from 5th and 15th day groups. (A) Sites of angiogenesis. Endothelial cells were thought to be forming new capillaries within the glomerular tuft. (B) Endothelial cells on lumen formation stage. Cells that had been migrated to new spots within the glomerular tuft were assumed to begin forming lumen amongst themselves. (Insets are showing the circles) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Slit pore number in unit length was averagely 18 ± 0.6 in control group. A dramatic decrease was seen in 5th day group and it became 5 ± 0.4. In 15th day group a small increase was seen (6 ± 1.6) but it was not statistically significant. However, in 25th day group with a significant decrease it came close to control levels and became 14 ± 1.1.

3.4. The changes in total and phosphorylated STAT3 levels

Subjective evaluation of the tissue sections immunohistochemically with STAT3 antibodies showed that both tSTAT3 and pSTAT3 levels were effected in PAN nephrosis (Fig. 7). The sections used as negative control showed no staining. The glomeruli of control rats displayed mostly 0 and +1 im- munoreactivity for tSTAT3 and the overall score of the group was 1.130 ± 0.038. The score of 5th day group was similar to control group (1.190 ± 0.084). In 15th day group, the grades of glomeruli were mostly +2 and +3. So the overall score displayed a dramatic increase (2.010 ± 0.111). In 25th day group (1.581 ± 0.185), al- though the score retracted, it was still high compared to control and 5th day groups.However, in pSTAT3 levels first dramatic increase was detected in 5th day group (1.749 ± 0.092) compared to control group (0.337 ± 0.071). The 5th day group mostly displayed glomeruli with grades +2 and +3 whereas control group displayed just 0 and +1 grades. The highness persisted in 15th group (1.740 ± 0.075). In 25th
day group (1.030 ± 0.083) pSTAT3 levels fell but still had a significant highness compared to control group.Although double-staining is necessary to be sure, regarding the staining pattern it could be said that all three glomerular cells, podo- cytes, mesangial and endothelial cells had immunoreactivity for pSTAT3 (Fig. 8).

4. Discussion

Although the studies trying to clarify the pathogenesis of kidney diseases have increased up, underlying mechanisms of glomerular pa- thology could not have been revealed yet. One of the models used in these researches is PAN nephrosis model which is thought to be based on oXidative stress (Diamond et al., 1986; Wang et al., 2009). Some of the recent studies has put forward JAK/STAT pathway, a principal cellular pathway, as a significant player in kidney diseases (Lu et al., 2009; Marrero et al., 2006). This pathway, especially STAT3 member, has gathered many attention with the recent findings showing its ac- tivity in many critical intracellular functions related to growth factors and intracellular factors such as oXidative stress (Simon et al., 1998; Zhuang, 2013). Accordingly, in this study, we aimed to study PAN nephrosis model and to detect changes in phosphorylated and total STAT3 protein levels immunohistochemically along with the morpho- metric and light microscopic analysis of glomerular changes.PAN nephrosis models are monitored via measuring the amount of protein excretion in the urine (Lowenborg et al., 2000). So the proteinuria level was monitored for the confirmation of model forma- tion and the course of the nephrosis. While the protein levels in the urine did not display a significant increase on the 5th day, a dramatic increase was detected on the 10th and 15th days and then proteinuria levels retracted to control levels gradually on the 20th and 25th days. Together with the morphological findings, these results proved the formation of the model. The presence of protein absorption granules in podocytes (Messina et al., 1987), the decrease in the SP number and the gradual thickening of the GBM (Lowenborg et al., 2000) are known to be concomitant indicators of nephrosis along with proteinuria.

Fig. 4. Ultrastructural findings in 15th day group. (A) Healthy podocytes (*) from the control group. (B) The foot processes of the podocytes appears normal in control group. (C) Hypertrophy of podocytes. (D) Protein absorption granules in podocytes. (E) Apical displacement of slit diaphragm (black arrows). (F) Foot process effacement and distinct steps of vesicle formation in foot processes (white arrows).

Although STAT3 is basically known for its role in inflammation signalling (Matikainen et al., 1999), it is also associated with oXidative stress, angiogenesis, apoptosis and proliferation in many tissues (Yu et al., 2009). STAT3 has a great influence on cellular functions. Nu- merous genes such as p53, p21, Bcl2, Bcl-XL, Survivin, Akt and TGF-B were attributed to be STAT3 targets and this clearly makes it very important for the cellular events (Carpenter and Lo, 2014).

The correlation we found between the STAT3 levels and proteinuria implied that STAT3 is an active factor in the pathogenesis of PAN ne- phrosis. pSTAT3 levels had increased on 5th day and this increase continued on 15th day. However, on 25th day the increase disappeared and the levels returned back to control levels. tSTAT3 had showed its first statistically significant increase on 15th day and although retracted on 25th day it was still statistically significant compared to control group. Regarding tSTAT3 levels on 5th day, it could be said that the increase in pSTAT3 levels on 5th day was not due to the increase in tSTAT3 levels.

Fig. 5. GBM thickness measurements. (A–D) An example measurement from each group; control, 5th, 15th and 25th day groups respectively. (E) Group averages of GBM thickness. The first dramatic increase in GBM thickness which was in normal range in control and 5th day group was detected in 15th day group. This increase disappeared in 25th day group. (*p < 0.05 compared to control; 5th and 25th day groups.) (N = 6 for each group).

Many recent studies disclosed STAT3 as an active regulator of mesangial matriX. In a study held with Thy1 glomerulonephritis model, it was shown that the increase in intraglomerular mesangial matriX had been caused by Gas6 autocrine growth factor via STAT3 phosphoryla- tion (Yanagita et al., 2001). Moreover, many other studies held with diabetic nephropathy models showed increased STAT3 protein levels and these increases were found to be correlated with mesangial matriX expansion caused by TGF-B, collagen IV and fibronectin expressions (Lu et al., 2009; Marrero et al., 2006). So our findings declaring a corre- lation between STAT3 levels and mesangial matriX increase are con- sistent with the literature.

Fig. 6. Slit Pore (SP) number counts. (A–D) An example counting from each group; control, 5th, 15th and 25th day groups respectively. (E) Group averages of SP count. Foot processes which appeared normal in control group displayed normal amount of slit pore number. However, SP numbers decreased significantly in 5th and 15th day groups. In 25th day group, foot processes re-established normal structure as a result SP number returned to near normal levels with a significant increase.(*p < 0.05 compared to control group; **p < 0.05 compared to 5th and 15th day groups.) (N = 6 for each group).

Highest STAT3 activation in our study was detected in endothelial cells and we thought that this finding would shed light on how en- dothelial cells are affected in glomerular kidney damages. JAK/STAT pathway of endothelial cells was thought to be active in the course of angiogenesis and vasculogenesis as shown in many studies focused on different tissues (Valdembri et al., 2002). Additionally, glomerular an- giogenesis was found to be activated in PAN nephrosis (Seckin et al., 2012). Along with the suggested VEGFR2–JAK2/STAT3 relation in angiogenesis (Dong et al., 2010) the critical role of VEGF not only in endothelial cells but also in podocytes (Wang et al., 2015) make VEGF signalling important for glomerular functioning. So it is not a surprise that JAK2/STAT3 pathway is operative in glomerular damages. Besides many studies showed that in endothelial cells this pathway was acti- vated during oXidative stress (Arany et al., 2006). Cellular movement protein Rac1 was found to be activated in endothelial cells via STAT3 activation due to oXidative stress (Mattagajasingh et al., 2012). As a result, in relation to all this literature, we thought that JAK2/STAT3 pathway is active also in podocytes and endothelial cells of glomerulus TRPC6 could be STAT3 as suggested by Kunichika et al. (2004) More- over Angiotensin II receptor blockage was found to ameliorate proteinuria in PAN (Takahashi et al., 2014). As a result, it could be deduced that Angiotensin II related STAT3 activation in podocytes and mesangial cells is an important activity for glomerular diseases. This eventuality probably leads to hypertrophy in podocytes which are fully differentiated and post-mitotic. All these data put STAT3 forward as an important factor regarding the stability of podocyte and mesangial cell activities.

Fig. 7. (A) tSTAT3 immunoreactivity scoring. An example image for each grade was represented. Control and 5th day groups were including mostly 0 and +1 grades. However, in 15th day group +2 grade was dominant. In 25th day group +1 grade was common along with +2 grade. (B) pSTAT3 immunoreactivity scoring. An example image for each grade was represented. Control group was including only 0 and +1 grades. In 5th and 15th day groups +2 grade was dominant with extensive +1 and +3 grades. In 25th day group only little +2 and +3 grades were present but mostly +1 grade was detected. (C) The overall immunoreactivity scores for tSTAT3 and pSTAT3. (***p < 0.05 compared to control and 5th day groups; **p < 0.05 compared to control, 5th and 15th day groups; *p < 0.05 compared to control and 25th day groups.) (N = 6 for each group) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 8. Close inspection of pSTAT3 im- munoreactivity localizations. All the glo- merular cells, podocytes (arrowheads), en- dothelial cells (white arrows) and mesangial cells (black arrows) showed immunoreactivity (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

In summary, we patterned the levels of STAT3 proteins during the different periods of PAN nephrosis. Subjective evaluation of immunoreactivities showed that before the occurrence of proteinuria, STAT3 phosphorylation increased and this increase thought to be cor- related with the first pathological changes within the glomeruli such as angiogenesis, cell hypertrophy and mesangial matriX increase. This shows that STAT3 is effected and activated before the onset of protei- nuria. In the group that showed a dramatic increase in proteinuria, pSTAT levels kept increasing and tSTAT levels also showed a statisti- cally significant increase correlating with the evident glomerular changes such as GBM thickening, decrease in slit pore number. In re- covery period, pSTAT levels retracted back to control group levels whereas tSTAT levels retained a high level.

All these findings support the idea that STAT3 is an active cellular factor during nephrosis development so it could be a pharmacological target to develop specific therapies for nephrosis. However, more stu- dies focusing on the cellular factors related to STAT3 are needed and in this respect, utilization of STAT3 inhibitors in animal models could be informative about nephrosis pathogenesis.

Declaration of interest

The authors declare that there are no conflicts of interest. The au- thors alone are responsible for the content and writing of the paper.

Funding

This study was funded by Academic Training Program Institutional Coordination Unit of Istanbul University.

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