Ten-fourteen-week-old male wild-type (WT) C57BL/6J (stock no. 000664) and type-1 diabetic (Akita) C57BL/6-Ins2 ^Akita /J (stock no. 003548) mice were bought from the Jackson Laboratory (Bar Harbor, ME). The mice were maintained in the University of Louisville’s animal facility and fed ad libitum with standard chow and water. The animal experiments were performed according to the approved protocols (Approval No. 20683, dated 2 December 2020) by the institutional animal care and use committee (IACUC) of the University of Louisville School of Medicine and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication, 2011), U.S.A. WT and Akita mice were either treated with saline or Nimbidiol (0.40 mg kg⁻¹ d⁻¹) for 8 weeks with a micro-osmotic pump. The dose of Nimbidiol was determined based on the optimized dose in our previous study (Juin et al., 2023). The four experimental groups were termed as ‘WT + Saline’, ‘WT + Nimbidiol’, ‘Akita + Saline’ and ‘Akita + Nimbidiol’. At the end of the experiment, 2X tribromoethanol (TBE) was used to euthanize the mice, and the samples were collected.

Dihydroethidium (cat. no.: D11347) was purchased from Thermo Fisher Scientific (Carlsbad, CA). The detection kit for GSH: GSSG (cat. no. ab138881) was procured from Abcam (Cambridge, MA). Assay kits for SOD (cat. no. 706002) and catalase (cat. no. 707002) activity were purchased from Cayman Chemicals (Ann Arbor, MI). Tween 20 (cat. no. M147) was from VWR Chemicals, LLC (Solon, OH), and polyvinylidene fluoride (cat. no. 1620177) membrane was from Bio-Rad (Hercules, CA). Non-fat dry milk powder (cat. no. M17200) and Bovine Serum Albumin (cat. no. A30075) were procured from Research Products International Corp. (Mt. Prospect, IL, USA). Agarose (cat. no. BP-160) was bought from Fisher Scientific (Fair Lawn, NJ). Optimal Cutting Temperature compound (cat. no. 23-730–571) was purchased from Fisher Healthcare, (Houston, TX). Nimbidiol (cat. no. SMB00209) was purchased from Sigma-Aldrich (St. Louis, MO).

Blood pressure (BP) of the mice was measured every fortnight by the tail-cuff method using the ‘CODA™ Non-Invasive Blood Pressure System’ (Kent Scientific Corporation, Torrington, CT) as described previously (Majumder et al., 2022). Mice were trained with BP holder in a few 15 min sessions a week prior BP measurement. Mice were placed on an animal warming platform putting in the BP holder and acclimatised for 10 min before measuring the BP.

The mice were anesthetized with isoflurane and then placed on a warm (37.5°C) platform supinely. An ultrasound transmission gel (Other-Sonic; Pharmaceutial Innovations, Inc., Newark, NJ, USA) was applied on the depilated skin and ultrasound imaging was performed using a Vevo 2,100 system (VisualSonics, Toronto, ON, Canada, USA). The left kidney was scanned in the short axis by the transducer, MS550D (22–55 MHz). Peak systolic velocity (PSV) and end-diastolic velocity (EDV) (mm/sec) of the renal arterial blood flow was recorded in the Pulsed-Wave Doppler mode. Renal arterial resistive index (RI) was obtained by analysing the cine loops.

Barium angiography was performed to evaluate renal vascular density as described previously (Pushpakumar et al., 2020). Barium sulfate (0.1 g/mL) was introduced into the kidney through the infrarenal aorta using a PE10 catheter (ID - 0.28 mm; Franklin Lake, NJ, USA). Renal angiogram was obtained using Kodak In-Vivo Imaging Systems FX Pro (Molecular Imaging System, Carestream Health Inc., Rochester, NY, USA). Density of the vessels was quantified using ‘VesSeg software tool’.

Dihydroethidium (DHE) staining was used to measure a major ROS, i.e., superoxide (O2^•−) in the kidney as described elsewhere (John et al., 2017). In brief, ice-cold acetone fixed kidney cryosections were incubated with freshly prepared 5 µM DHE solution for 15 min at room temperature in a humidified chamber avoiding exposure to light. The stained sections were imaged by an Olympus FluoView1000 confocal microscope (B&B Microscope, Pittsburgh, PA, USA) and the fluorescence intensity was determined using ‘ImageJ’ software.

Total protein was extracted from the kidney by RIPA buffer (Boston BioProducts, Worcester, MA, USA), containing phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Sigma, St. Louis, MO, USA). The protein was quantified by Bradford assay. An equal amount protein was resolved by SDS-PAGE and Western blotting was performed following the standard protocol as described previously (Juin et al., 2021). GAPDH was used as a loading control for normalization of the relative expression of protein. A list of antibodies along with the sources, catalog numbers, and dilutions was provided in Table 1. ‘ImageJ’ software was used to quantify the protein bands by densitometric analysis.

An assay kit (refer to the ‘Chemicals and Antibodies’ section) was used to evaluate the ratio for the reduced to oxidized glutathione (GSH: GSSG) following the manufacturer’s instructions as mentioned elsewhere (Pushpakumar et al., 2020).

Total SOD and catalase activity was determined using Assay kits (refer to the ‘Chemicals’ section) according to the manufacturer’s protocol as described previously (Pushpakumar et al., 2020).

Total RNA was extracted from the kidney using Trizol reagent (cat. no. 15596-026, Invitrogen, Carlsbad, CA, USA). cDNA was synthesized from 1 µg of isolated total RNA by reverse-transcription using an EasyScript cDNA Synthesis kit (cat. no. G234, MidSci, St. Louis, MO, USA) according to the manufacturer’s protocol. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to amplify cDNA using the GoTaq Hot Start Green Master Mix (cat. no. M5122, Promega, Madison, WI, USA) as per manufacturer’s instructions. After PCR amplification, the product was subjected to electrophoresis on 1.5% agarose gel and a ChemiDoc XRS system (Bo-Rad, Hercules, CA) was used to visualize the bands. The band intensity was analysed by densitometry with ‘ImageJ’. A list of the primer sequences (Invitrogen, Carlsbad, CA, USA) are shown in Table 2.

The experimental data are represented as mean ± standard deviation (SD) from 6 mice/group. Statistical significance was determined by the one-way or two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test using GraphPad Prism 9 software. p < 0.05 was considered to be statistically significant.

At the beginning of the experiment (0 weeks), saline-treated diabetic Akita mice showed higher systolic, diastolic, and mean blood pressure (BP) compared the age-matched WT control (Figures 1A–C). BP in Akita mice continued to increase and reached to the peak at 4 week and thereafter remained constant (Figures 1A–C). Interestingly, Nimbidiol-treatment to Akita mice significantly reduced systolic, diastolic, and mean BP at 4 weeks onwards compared to that of saline-treated Akita mice (Figures 1A–C). The reduction of BP was maximum at 6 week of the Nimbidiol-treatment and thereafter it remained constant till the end of the experiment, i.e., 8 week (Figures 1A–C). BP was unchanged in the Nimbidiol-treated WT mice compared to the saline-treated WT control throughout the experiment (Figures 1A–C).

Renal arterial resistive index (RI) is the measure of vascular resistance that serves as a prognostic marker of renal outcome associated with diabetes and hypertension (Weber et al., 2017; Li et al., 2021). Therefore, we assessed renal arterial RI of the mice by ultrasonography and tested whether Nimbidiol influences vascular resistance. There was no significant change in RI between WT mice-treated with saline and Nimbidiol (Figures 2A, B). Akita mice exhibited significantly elevated renal arterial RI compared to that of WT control (Figures 2A, B). Nimbidiol-treatment to Akita mice significantly decreased renal arterial RI compared to the saline-treated Akita mice (Figures 2A, B).

In order to evaluate renal vasculature, barium angiography was performed. Compared to the WT control, total renal vascular density of the Akita mice was significantly reduced (Figures 3A, B). Nimbidiol-treatment to Akita mice significantly increased vascular density compared to the Akita mice that received saline (Figures 3A, B). No significant change in total vasculature was observed between saline- and Nimbidiol-treated WT mice (Figures 3A, B).

To assess reactive oxygen species (ROS) levels, dihydroethidium (DHE) staining was performed. The ROS production remained unchanged in the kidney of WT mice-treated saline and Nimbidiol (Figures 4A,B). Compared to the WT control, saline-treated Akita mice demonstrated an elevation in the ROS production as evidenced a significant increase in the fluorescence intensity in the glomerular and tubular regions of the Akita mice receiving saline (Figures 4A,B). It was interesting to note that Nimbidiol treatment to Akita mice significantly reduced the ROS production that is comparable to that of WT control (Figures 4A, B).

The lipid peroxidation product, 4-hydroxynonenal (4-HNE) is widely considered as a crucial oxidative stress marker that contributes to the hypertension and diabetic complications including DN (Calabrese et al., 2007; Yang et al., 2014; Csala et al., 2015; Yu et al., 2018; Dham et al., 2021). Therefore, we measured the expression of 4-HNE in the kidney. Our results showed that there was no significant difference in 4-HNE expression between WT mice treated with saline and Nimbidiol (Figure 4C). We observed a significant upregulation of 4-HNE in the kidney of saline-treated Akita mice, which was mitigated by Nimbidiol treatment (Figure 4C).

The ratio of reduced to oxidized glutathione (GSH: GSSG) serves as an important indicator of oxidative stress in the diabetic complications including DN (Zitka et al., 2012). To investigate whether elevated ROS production was associated with the imbalance in the ratio of the reduced to oxidized glutathione, we evaluated reduced and oxidized glutathione in the kidney of the experimental mice groups. Our results showed that there was no significant difference in the ratio of the reduced to oxidized glutathione in the kidney of saline- and Nimbidiol-treated WT mice (Figure 4D). However, the ratio was drastically decreased in the diabetic kidney compared to that of WT control (Figure 4D). Interestingly, Nimbidiol treatment ameliorated the adverse ratio in diabetic mice (Figure 4D).

NADPH oxidase (Nox) system serves as the important source of ROS production (Pushpakumar et al., 2017). Nox4, the most abundant isoform of Nox present in the kidney, and another important Nox subunit p22phox have been reported to be involved in diabetes and hypertension (Etoh et al., 2003; Watanabe et al., 2013; Munoz et al., 2020). Therefore, the expression levels of Nox4 and p22phox were measured by Western blot analyses. Akita mice showed elevated expression of Nox4 and p22phox in the kidney compared to the WT control (Figure 5A). Nimbidiol treatment to Akita mice significantly reduced their expression levels, that were comparable to the WT mice (Figure 5A). Nox4 and p22phox expression in Nimbidiol-treated WT mice remained statistically unaltered compared to the WT mice receiving saline (Figure 5A). Further, we evaluated the expression of reactive oxygen species modulator 1 (ROMO1), an important regulator of the ROS production. Results revealed that ROMO1 was upregulated in the kidney of saline-treated Akita mice compared to that of WT control (Figure 5A). Interestingly, compared to the saline-treated Akita mice, ROMO1 expression was significantly reduced in Akita mice treated with Nimbidiol (Figure 5A). No significant difference in ROMO1 expression was observed between WT mice treated with saline and Nimbidiol (Figure 5A).

To understand whether elevated ROS generation was related to the imbalance of crucial antioxidative enzymes, we evaluated the expression of SOD-1, SOD-2, and catalase in the kidney. Western blot analyses showed a significant decrease in the renal expression of SOD-1, SOD-2, and catalase in diabetic mice compared to the WT control (Figure 5B). Nimbidiol-treatment to diabetic mice normalized their expression in the kidney (Figure 5B). However, Nimbidiol did not change the expression of these enzymes in the WT mice (Figure 5B). Then, we measured the activity of the total SOD and catalase in the kidney. Results revealed that there was no significant difference in the SOD and catalase activity in the kidney of the WT mice-treated with saline and Nimbidiol (Figures 5C, D). Compared to the WT control, on the other hand, diabetic mice showed a sharp decline in the SOD and catalase activity in the kidney (Figures 5C, D). Interestingly, Nimbidiol-treatment to diabetic mice normalized their activity, comparable to the WT control (Figures 5C, D).

The two important isoforms of nitric oxide synthases (NOSs) viz., Endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) play crucial role in the regulation of oxidative stress, vascular function, blood pressure and DN (Trachtman et al., 2002; Kumar et al., 2005; Forstermann and Sessa, 2012; Dellamea et al., 2014; Pushpakumar et al., 2017; Juin et al., 2023). Therefore, we assessed the expression levels of eNOS and iNOS in the kidney. Akita mice showed a downregulation of eNOS and an upregulation of iNOS expression compared to the WT mice (Figure 6). Interestingly, Nimbidiol treatment to Akita mice normalized their expression levels that were comparable to the WT mice (Figure 6). Nimbidiol did not statistically alter the expression levels of eNOS and iNOS in WT mice compared to the saline-treated WT mice (Figure 6).

Oxidative stress induces progression and development of hypertension and nephropathy in diabetes (Shi et al., 2013). SIRT1 plays a pivotal role in the regulation of oxidative stress in hypertension and DN by regulating ACE2 expression (Yacoub et al., 2014). The results exhibited that there were no significant differences in the expression levels of SIRT1 and ACE2 between WT mice receiving saline and Nimbidiol (Figure 7). A distinct downregulation of the expression levels of SIRT1 and ACE2 was observed in Akita mice compared to the Winduce oxidative stress leading to the renal injury. Nimbidiol mitigatesT control (Figure 7). It was noteworthy that Nimbidiol treatment to Akita mice significantly increased the levels of SIRT1 and ACE2 expression, comparable to that of WT control (Figure 7).

A plethora of evidence suggest the pathogenic role of proinflammatory cytokine IL17 and protective role of anti-inflammatory cytokine IL10 in diabetes and hypertension (Gunnett et al., 2002; Didion et al., 2009; Madhur et al., 2010; Weber et al., 2017; Qiu et al., 2021). To evaluate the mRNA and protein expression levels we performed semi-quantitative RT-PCR and Western blot analyses. Our results exhibited a distinct upregulation of IL-17 and a downregulation of IL-10 both at mRNA and protein levels in the kidney of Akita mice compared to the WT control (Figures 8A, B). In Akita mice receiving Nimbidiol, the expression levels of IL-17 and IL-10 were normalized (Figures 8A, B). The mRNA and protein expression levels of IL-17 and IL-10 remained unaltered in the WT mice treated with saline and Nimbidiol (Figures 8A, B).

Kidney injury molecule-1 (KIM-1) present in the proximal tubules of the kidney is largely recognized as an important biomarker of the progressive renal damage in diabetes (Papu John et al., 2019; Majumder et al., 2022). Further, Lipocalin-2 (LCN-2) also serves as a crucial prognostic marker of renal injury in diabetes and hypertension (Johnson et al., 2022; Majumder et al., 2022). Therefore, we investigated whether diabetic conditions induced kidney damage and Nimbidiol treatment ameliorated renal injury in Akita mice. The Western blot analyses revealed that there was no significant change in the expression levels of KIM1-1 and LCN-2 between WT mice treated with saline and Nimbidiol (Figure 9). We observed that KIM1-1 and LCN-2 were substantially elevated in the kidney of the Akita mice compared to the WT control (Figure 9). Of note, Nimbidiol treatment to Akita mice significantly reduced the levels of KIM1-1 and LCN-2 that were comparable to the WT control (Figure 9).

NF-κB signaling plays a critical role in the development of different kindney diseases including DN by regulating macrophage polarization and gene expressions of various inflammatory cytokines and chemokines (Baker et al., 2011). Therefore, we measured the expressions of phosphorylated-NF-κB (p65) [p-NF-κB (p65)] and IkBα in the kidney by Western blot analysis. p-NF-κB was found to be significantly upregulated and IkBα was significantly downregulated in the diabetic kidney compared to the WT control (Figure 10). Nimbidiol treatment normalized the expression levels of p-NF-κB and IkBα in Akita mice (Figure 10). However, there were no significant differences in the protein levels of p-NF-κB and IkBα between saline- and Nimbidiol-treated WT mice (Figure 10).