**3. Results**

#### *3.1. Recruitment of MSCs and HMSCs into Nephritic Not Normal Kidneys*

Detection of fluorescence in the kidneys after intrarenal arterial administration of fluorescent MSCs is illustrated in Figure 1C. High levels of GFP were visualized under UV light and the fluorescence was found in the nephritic kidneys (Figure 1C3,4) but not in the normal kidney (Figure 1C1,2). Fluorescence cells were found at glomeruli in the MSC treated groups. CD44 positive cells were found in glomeruli of MSC or HMSC treated kidneys, but not in the control kidney (Figure 2A). These results confirm the recruitment of MSCs and HMSCs in injured kidneys. There was no significant di fference in tra fficking CD44 positive cell numbers between the MSC and HMSC groups.

**Figure 2.** Representative pictures of immunohistochemical stained sections for CD44, a marker for MSC, in ten groups of rats show CD44 positive stains in the glomeruli indicated by arrows in MSCs or HMSCs treated kidneys compared to control kidney (**A1–10**). Anti-Thy1.1 evoked mesangial lysis (indicated by +) and the mesangial matrix accumulation (indicated with \*) in the nephritic rats. Intrarenal MSCs transplantation ameliorated anti-Thy1.1-induced nephritis in the rat model. Normoxic (MSC) and hypoxic MSCs (HMSC) markedly reduced the inflammatory cell infiltration in the glomeruli in H&E stains (**B**). The severity of glomerulosclerosis was explored in both normoxic MSC and HMSC treated rats in PAS stains (**C**) and in Masson stains (**D**). Thy1-induced nephritis significantly elevated urinary protein level and the elevated urinary protein level was significantly reduced by intrarenal MSC or HMSC treatment (**E**). Glomerular sclerotic index calculated by the PAS sections among the experimental groups are presented in (**F**). Thy1-induced nephritis significantly increased glomerular sclerosis in all nephritis treated groups as compared to Con group. MSC at cell number (2–5) × 10<sup>5</sup> level and HMSC at cell number (1–5) × 10<sup>5</sup> level significantly reduced sclerosis degree. Glomerular fibrosis determined by the hydroxyproline contents among all groups of animals are presented in (**G**). Thy1-induced nephritis significantly increased renal hydroxyproline content in all nephritis treated groups as compared to Con group. MSC and HMSC significantly reduced renal fibrotic degree. Each graph is amplified at 400×. The scale bar (20 μm) is indicated in each graph. \* *p* < 0.05 vs. Con group. # *p* < 0.05 vs. Thy1 group. a *p* < 0.05 vs. Thy1 + 1MSC group. b *p* < 0.05 vs. Thy1 + 2MSC group. c *p* < 0.05 vs. Thy1 + 5MSC group.

#### *3.2. MSC or HMSC Ameliorates Nephritic Severity in the Rat GN Model*

The success of glomerulonephritis induction in our model was confirmed by the elevated urine protein concentrations and typical characteristics in the histopathologic examination, including mesangial cell proliferation, mesangialysis, and sclerosis appearance 5 days after injury. In H&E stains of both MSC and HMSC treated kidneys, the severity of glomerulosclerosis was attenuated and decreased numbers of inflammatory cells in glomeruli were found (Figure 2B). The glomerulosclerosis index by PAS stains sections (Figure 2C) are 0.01 for the control, 0.5 for anti-Thy1 with placebo, 0.19 for 1 × 10<sup>5</sup> MSC treated group, 0.16 for 2 × 10<sup>5</sup> MSC treated group, 0.03 for 5 × 10<sup>5</sup> MSC treated group, 0.03 for 1 × 10<sup>5</sup> HMSC treated group, and 0 for both 2 and 5 × 10<sup>5</sup> MSC treated groups (Figure 2F). Both normoxic and hypoxic MSC reduced the severity of proteinuria in GN rats. The level of proteinuria reduction did not di ffer among 1, 2, 5 × 10<sup>5</sup> MSC infusion, but there exists a trend in HMSCs with a higher cell number having a lower proteinuria (Figure 2E). The severity of glomeruli fibrosis was also ameliorated by MSC and HMSC administration, which is shown in the Masson stains (Figure 2D) and in the hydroxyproline content (Figure 2H).

#### *3.3. Hypoxic Preconditioning Upregulated HIF-1*α *and VEGF Expression*

Hypoxic preconditioning significantly upregulated HIF-1 α concentration (Figure 3A) and VEGF (Figure 3D) expression in the MSCs but downregulated several growth factors with growth factors array assay (Figure 3B) in the conditioned medium (Figure 3C).

**Figure 3.** The concentration of HIF-1α (**A**) and multiple growth factors array (**B–D**) from conditioned medium and MSC were determined. The upregulation and downregulation were denoted with the arrows.

#### *3.4. MSCs or HMSCs Reduce ED1, ER Stress, Autophagy, and Apoptosis with Western Blot*

The oxidative stress index of renal ED-1 (Figure 4A), GRP78 (Figure 4B), Beclin-1 (Figure 4C), LC3-II (Figure 4D), Bax/Bcl-2 ratio (Figure 4E), Caspase-3 (Figure 4F), PARP (Figure 4G) was significantly elevated in anti-Thy1.1 treated glomeruli. MSCs with three dosages did not significantly reduce these oxidative parameters in the anti-thy1.1 treated kidneys. However, HMSCs treated anti-thy1.1 kidneys with the number of 5 × 10<sup>5</sup> significantly reduced these parameters as compared to anti-thy1.1 treated group.

**Figure 4.** The oxidative stress index of renal ED-1 (**A**), GRP78 (**B**), Beclin-1 (**C**), LC3-II (**D**), Bax/Bcl-2 ratio (**E**), Caspase-3 (**F**), PARP (**G**) and β-actin (**H**) is determined with western blot among eight groups of rats. The quantitative data showed that anti-Thy1.1 significantly enhanced ED-1, GRP78, Beclin-1, LC3-II, Bax/Bcl-2 ratio, Caspase 3, and PARP in the damaged kidneys. MSCs with three amounts did not significantly reduce these oxidative parameters in the anti-thy1.1 treated kidneys. HMSCs treated anti-thy1.1 kidneys with the number of 5 × 10<sup>5</sup> significantly reduced these parameters as compared to anti-thy1.1 treated group. \* *p* < 0.05 vs. Con group. # *p* < 0.05 vs. Thy1.1 group.

#### *3.5. MSCs or HMSCs Reduce ED-1 Infiltration, ER Stress, Autophagy, and Apoptosis by IHC*

The representative pictures of immunohistochemical stained sections and semi-quantitative analyses of ED-1, GRP78, LC3-II, caspase 3, TUNEL and collagen IV in the study groups are presented in Figure 5. With the semi-quantitative analysis of the histochemical stained kidney sections for ED-1, anti-Thy1.1 administration markedly increased the numbers of ED-1positive cells in the glomeruli. Infusion of MSCs reduced the number of ED-1 positive cells that infiltrated in the kidneys, and infusion of HMSCs had a better reduction of the ED1 positive cell number (Figure 5A1–5) than MSCs. Similar results were found for GRP78, LC3-II, caspase 3, TUNEL, and collagen IV. These sections show that anti-Thy1.1 administration increased stress index protein (GRP78) accumulation (Figure 5B1–5), autophagy index protein (LC3-II) detection (Figure 5C1–5), caspase 3 positive cells (Figure 5D1–5), apoptotic (TUNEL+) cells (Figure 5E1–5), and collagen IV accumulation (Figure 5F1–5). MSC infusion ameliorated the increase in GRP78, LC3-II, caspase 3, TUNEL, and collagen IV in kidneys after anti-Thy1.1 infusion. HMSC infusion had a greater effect on ameliorating inflammatory cell infiltration, stress protein accumulation, apoptotic cells and autophagy in glomeuli vs. MSC treatment.

**Figure 5.** Immunohistochemical stains for ED1, GRP78, LC3-II, Caspase1, TUNEL, and Collagen IV in the study groups. Detection of ED1+ cell in the study groups are demonstrated in (**A1–4**). Semiquantitative assessment revealed infusion of MSCs and showed a reduction in numbers of macrophage/monocyte infiltration (ED1+ cell). HMSCs possessed a better reduction effect (**A5**). Representative pictures of sections for stress index protein acculumation (**B**1–4), autophagy by LC3-II (**C1–4**), apoptotic cells by caspase 1(**D1–4**) and TUNEL (**E1–4**) and collagen deposition (**F1–4**) in glomeruli. The semiquantitative assessment of these sections revealed anti-Thy1.1 administration increased stress index protein (GRP78) accumulation, autophagy index protein (LC3II) detection, apoptotic (TUNEL, caspase 1) cells, and collagen IV accumulation. MSC infusion ameliorated the increase in GRP78, LC3 II, caspase 1, TUNEL, and collagen IV in kidneys after anti-Thy1.1 infusion. HMSC infusion had greater therapeutic reduction effects. Each graph is amplified at 400×. The scale bar (20 μm) is indicated in the Figure. \* *p* < 0.05 vs. Con group. # *p* < 0.05 vs. Thy1 group. a *p* < 0.05 vs. Thy1 + 5MSC group.

#### *3.6. HMSCs Promote Nuclear Nrf2 Expression, Reduce NF-kB Expression, Rescue ROS Enzymatic Scavengers and Elevate Anti-Oxidative Response Element Proteins*

Figure 6 demonstrates the reactive oxygen species (ROS) injury index and the intrinsic anti-oxidative mechanisms expression among the experimental groups. The accumulation of ROS by 4 HNE histochemical stain among the groups are demonstrated in Figure 6A1–4, the semi-quantitative analyses of these sections revealed that anti-Thy1.1 infusion greatly increased ROS. In the treatment groups, only a higher HMSC cell number ameliorated the ROS accumulation (Figure 6A5). ROS enzymatic scavenger expressions including MnSOD, Cu/ZnSOD, and catalase were significantly reduced by antiThy-1.1 infusion. These enzymes were rescued by HMSC transplantation, but not by MSCs (Figure 6B1–3). As for Nrf2 signaling, the master regulator of ROS injury, the results showed that nuclear Nrf2 expression was not changed, while NF-kB expression was elevated by anti-Thy1.1 infusion. Infusion of HMSCs significantly increased nuclear Nrf2 and reduced NF-kB expressions in anti-Thy1.1 treated rat kidneys. Infusion of MSCs had a lesser e ffect on nuclear Nrf2 elevation and NF-kB expression. However, the e ffect did not reach statistical significance (Figure 6C1–3). Semi-quantitative detection of anti-oxidative response element (ARE) protein expression (GCLC, GCLM and GPX) by Western blotting in the kidneys are shown (Figure 6D1–3). These proteins had a consistent expression trend in the experimental groups. Briefly, the anti-Thy1.1 antibody infusion reduced ARE protein expressions, and hypoxic preconditioning MSCs rescued these protein expressions. Administration of MSCs did not significantly increase protein expression, though it tended to increase their levels. Figure 6E demonstrated a summary diagram of this study.

*Antioxidants* **2020**, *9*, 2

**Figure 6.** The index of ROS, ROS enzymatic scavenger expression, master anti-oxidative injury regulator Nrf2 expression, and anti-oxidative response protein expressions among the experimental groups. The accumulation of ROS by 4 HNE stain among the groups are demonstrated from the amplified graph at 400× (scale bar = 20 μm, **A1–4**). The semi-quantitative analyses of these sections revealed that anti-Thy1.1 infusion greatly increased ROS, and HMSCs ameliorated the ROS accumulation (**A5**). ROS enzymatic scavengers including MnSOD, Cu/ZnSOD and catalase were significantly reduced by antiThy-1 infusion. These enzymes were rescued by HMSCs (**B1–3**). The nuclear Nrf2 expression is not triggered by anti-Thy 1.1 infusion and is enhanced in the HMSC treated group. Significantly elevated nuclear NFkB expression is noted in anti-Thy1.1 group, which is efficiently suppressed in HMSCs treated group (**C1–3**). Downstream antioxidative response element protein expression including GCLC, GCLM, GPX are suppressed by anti-Thy 1.1 infusion. These proteins are rescued by HMSC transplantation (**D1–3**). \* *p* < 0.05 vs. Con group. # *p* < 0.05 vs. Thy1.1 group.
