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A Nuphar Lutea Plant Active Ingredient, 6,6′-dihydroxythiobinupharidine, Ameliorates Kidney Damage And Inflammation In A Mouse Model Of Chronic Kidney Disease – Scientific Reports – Renal.PlatoHealth.ai

Animals

This study was approved by the Ben-Gurion University of the Negev Animal Use and Care Committee, protocol number IL-39-07-2018(D). All protocols comply with the NIH Guidelines and were reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). Animals were housed in standard laboratory cages. Food and water were given ad libitum. After preliminary safety and dosing experiments: we used 8 weeks old male C57BL/6J mice, and provided them with an Adenine diet (0.3% for 9 days, then 0.2% for the rest of time) to induce CKD31. Mice (Harlan Laboratories Inc. Rehovot, Israel) were treated with IP injections of a non-toxic dose of DTBN (25 ug QOD) or saline, forming 4 experimental groups: control (C), CKD, C-DTBN and CKD-DTBN. CKD was induced by adenine diet for 8 weeks as follows: first, a 0.3% adenine, 0.9% phosphorus and 75 ppm iron diet was given for 9 days, and then switched to a 0.2% adenine diet and unchanged phosphorus and iron until the end of the experiment. Control groups were fed with a control diet (0.3% phosphorus, ~ 75 ppm iron). All diets were purchased from Envigo Teklad, (Huntingdon, UK).

6,6′-Dihydroxythiobinupharidine (DTBN, 30343-70-5) (Fig. 1) was purchased from Sigma/Merck (SMB00609), was diluted in DMSO to a 1 mg/ml stock solution and further diluted in saline. Intra peritoneal (i.p.) injections of a working solution containing 25 µl DTBN stock, 7.5 µl DMSO and 67.5 µl saline (25 μg/0.1 ml) were given three times a week for 8 weeks. Mice were sacrificed at experiment end, using lethal anesthesia with ketamine and xylazine, collecting: blood, and liver and kidney tissues.

Blood analyses

Tail blood samples for complete blood count (CBC) and urea were taken two times during experiment and prior to sacrifice. Blood counts were determined using a veterinary heamatology analyzer (Exigo H400, Boule, FL, USA). Tail blood urea was analyzed using an enzyme linked immunosorbent (ELISA) commercial kit (Abcam, Cambridge UK). Serum creatinine, was analyzed using the AU2700 analyzer (Beckman-Coulter, CA, USA), based on the Jaffe method for determination of mouse serum creatinine. Urine albumin was tested by the Coomassie blue reaction on urine samples that were loaded on an SDS-PAGE. Bovine serum albumin (fraction V, MP biomedicals, OH, USA) served as control.

RNA extraction and real time PCR

RNA was extracted using a commercial kit (Macherey–Nagel, Dueren, Germany). cDNA was transcribed using qScript cDNA synthesis kit (Quanta biosciences, MD, USA) and qPCR assays were performed with power SYBR green PCR master mix (Applied Biosystems, Foster City, CA, USA) as previously described32, using the ABI Prism 7300 sequence detection system (Applied Biosystems, CA, USA). Primers for quantification (Sigma-Aldrich, Rehovot, Israel) are summarized in Table 1.

Table 1 Primers used for RT-PCR.

Western Immunoblot analysis

The following antibodies were used for evaluation of the kidney extracts: β-actin (MP Biomedicals, OH, USA), STAT3 and pSTAT3 (Cell Signaling Technology, MA, USA), as previously described33.

Kidney histology

Kidney segments were fixed in 4% formalin for 48 h, then embedded in paraffin and cut. Kidney sections were deparaffinized, rehydrated and stained with Masson’s trichrome (Bio-Optica, Milano, Italy). Histological analyses were done by two Pathologists (AFT and DB) blinded to study groups. Damaged area quantification of Masson’s trichrome staining was performed as described by Chen et al. using the ImageJ software34.

Immunohistochemistry staining was performed as previously described5. Primary antibodies against F4/80 (BM-8) (diluted 1:30; Santa Cruz biotechnology Inc., Dallas Texas, USA) and Myeloperoxidase (MPO) (diluted 1:50; Abcam, Cambridge, UK), NF-κB p65 (Bio-Rad, CA, USA), IL-1 and IL-6 (GeneTex, CA, USA) were used for the immunohistochemistry staining. For image processing, Topika Analysis software (Topika, Tel Aviv, Israel) was used. Area quantification of immunohistochemical staining was performed using an ImageJ software as described.

Data analysis

Since our data do not comply with parametric test like ANOVA, we used the comparable non-parametric test Kruskal–Wallis to compare multiple groups, and the non-parametric test Mann–Whitney for comparison of each two groups. The null hypotheses were rejected at the 5% level. Values along the manuscript are presented as medians ± interquartile range (IQR). Asterisks indicate a significant difference between the indicated and the control group, where hash marks indicate a significant difference between the indicated and the CKD group. ****/#### indicates p value < 0.0001, ***/### indicates p value < 0.001, **/## indicates p value < 0.005, */# indicates p value < 0.05.