SS rats were rendered diabetic with streptozotocin as previously reported (Miller et al., 2018). These rats, when maintained on low salt diet, do not develop hypertension and do not have metabolic syndrome making them suitable to study type 1 diabetes after streptozotocin injection. However, Dahl SS rats have some vascular dysfunction at baseline (Ren et al., 2014) and have an impaired response to increased perfused pressure even on low salt diet (Takenaka et al., 1992). As we reported previously, microvascular renal responses in SS rats with induced DN are lost due to p66Shc-mediated signaling (Miller et al., 2018). Afferent arterioles of hypertensive SS rats exhibit impaired myogenic response to increased perfusion pressure, and this impairment has been shown in part to be the result of increased p66Shc expression in renal microvessels as p66Shc-KO rats have restored contractility and are protected from progressive proteinuria and renal injury (Miller et al., 2016). Furthermore, recent evidence suggests acute and chronic treatment with SHetA2 restores contractility in renal microvessels of hypertensive SS rats expressing p66Shc (Miller et al., 2025). By 6 weeks of STZ-induced hyperglycemia, afferent arterioles of SS rats lose myogenic tone, i.e., contraction, in response to higher intraluminal pressure. Interestingly, acute treatment of afferent arterioles with 2.5 µM SHetA2 restored contractility in response to increased pressure (Figure 1A), an effect also seen in SS rats with hypertensive nephropathy. Although knockout of p66Shc also restored contraction of vessels in rats with both HN and DN, acute treatment with SHetA2 did not provide an additive improvement of tone, suggesting that the action of SHetA2 may involve a p66Shc-dependent mechanism (Figure 1B).

As shown in Figure 1, addition of SHetA2 to isolated renal afferent arterioles was sufficient to partially restore myogenic tone and reduce vessel dilation. Accordingly, we tested whether chronic SHetA2 administration would prevent p66Shc-induced vascular dysfunction in our rat model of Type 1 diabetes. The oral formulation and weekly dosing of SHetA2 required for maintaining the predicted effective drug dose were determined by us previously (Miller et al., 2025). Rats were maintained on 0.4% salt diet for 9 weeks prior to single STZ injection. To determine if SHetA2 was able to improve myogenic tone in vivo, SHetA2 was given to diabetic SS rats by oral gavage over 11 weeks (Figure 2). Blood glucose levels were checked to confirm the development hyperglycemia and following 9 weeks of SHetA2 treatment the perfused juxtamedullary nephron preparations were used to evaluate microvascular reactivity. Myogenic tone was improved upon treatment, as constriction of afferent arterioles in response to increased pressure was restored by SHetA2 treatment [Figure 3A, *P <0 .0001]. Purinergic signaling via P2X receptors is thought to be a mediator of renal resistance vessel constriction in response to increased intraluminal pressures to maintain proper blood flow to glomerular capillaries. In agreement, vessel contraction to acute ATP treatment was restored in diabetic SS rats treated chronically with SHetA2 (Figure 3B, F1, 13) = [14.10], *P = 0.002).

We have previously reported that p66Shc-mediated loss of vascular dysfunction in rats with hypertension-induced nephropathy is associated with inhibition of calcium influx in renal vascular smooth muscle cells (SMC) (Miller et al., 2016). To test whether SHetA2 effect on myogenic tone of rats with induced DN is caused by modulating calcium influx, we have determined the effects of SHetA2 on calcium mobilization in renal vascular SMCs. When SMCs were stimulated with vasoactive peptide endothelin-1 (ET-1), treatment of cells with 2.5 µM SHetA2 reduced calcium entry compared to the DMSO control (Figures 4A,B). In addition, the effect of SHetA2 on calcium was independent of p66Shc, as 2.5 µM SHetA2 had the same inhibitory effect on p66Shc-KO SMCs (Figures 4C,D). For both strains of SMC, 0.25 and 1 µM SHetA2 did not have a significant impact on calcium mobilization. Our data suggest that SHetA2-mediated restoration of renal vascular tone cannot be explained by increased calcium mobilization (Figure 4).

K_ATP channels are present in SMC, and by modulating vascular responses to diverse stimuli, they contribute to physiological regulation of vascular tone (Brayden, 2002). Afferent arteriolar dilation and glomerular hyperfiltration in diabetes are due to increased K_ATP channel availability and activity. We have previously reported that inactivation of p66Shc decreases afferent arteriolar K_ATP channel activity and decreases renal damage in diabetic SS rats (Miller et al., 2018). The antidiabetic drug glibenclamide binds and inhibits K_ATP inhibitory regulatory subunit resulting in cell membrane depolarization and an increase in intracellular calcium and vessel contraction. We show here that while the dose-dependent inhibition of K_ATP channels by glibenclamide results in constriction of afferent arterioles of SS STZ-treated rats, there is no effect upon SS STZ-treated rats that received SHetA2 every other day (Figure 5 F1 12) = [27.31], *P <0 .001). This result indicates that SHetA2 treatment reduces K_ATP channel activity implicated in hyperfiltration of the glomerulus during diabetes. This is in agreement with our previous finding in STZ-treated p66Shc-KO rats (Mousavi et al., 2022).

Progressive renal decline is the major feature of DN in type 1 diabetes (Krolewski et al., 2014). The most characteristic marker associated with DN progression is proteinuria (Lin et al., 2018). We evaluated the effect of chronic treatment with SHetA2 upon proteinuria in rats with induced DN. Despite the improvement of myogenic tone, we did not detect the inhibition of progressive proteinuria seen in DN with chronic treatment using SHetA2 up to 12 weeks (Figure 6A F2 28) = [0.03], P = 0.97). Urinary Neutrophil gelatinase-associated lipocalin (NGAL) has been associated with tubular injury in the progression of DN (Alter et al., 2012; Liu et al., 2015). In agreement with our proteinuria results, SHetA2 treatment of diabetic rats did not modify the levels of NGAL in the urine as assessed by Western blot analysis (Figure 6B).

The existence of an imbalance between prooxidant and antioxidant processes results in an increase in reaction oxygen species (ROS), which have been shown to act as a trigger and modulator of pathological events, that occur in DN (Miranda-Diaz et al., 2016). Generation of ROS by the mitochondrial electron transport system is partially responsible for kidney damage associated with DN (Miranda-Diaz et al., 2016; Brownlee, 2001). It is generally accepted that p66Shc shuttles to mitochondria to contribute to ROS production (Natalicchio et al., 2011; Pellegrini and Baldari, 2009). We tested whether SHetA2 could inhibit p66Shc-mediated ROS production. Treatment with SHetA2 resulted in statistically significant decrease of ROS production induced by both recombinant human p66Shc and CH2CB domain of human p66Shc (Figure 7). CH2CB is the N-terminal CH2 domain plus the small extension connecting into the phosphotyrosine binding domain (PTB). The current literature supports an argument that ROS produced by p66Shc is derived from the CH2CB domain and this domain is ROS active as a distinct protein from the full-length p66Shc. Our data suggest that there is an interaction between SHetA2 and the N-terminal domain of p66Shc.

Animal use and welfare procedures were followed according to the NIH Guide for the Care and Use of Laboratory Animals, and study protocols were reviewed and approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee. The generation of genetically modified p66Shc-KO on the genetic background of Dahl SS was described previously (Slaughter et al., 2013). Male rats were weaned at 3 weeks of age and provided diet (Teklad 7034) and water ad libitum. At 9–10 weeks of age, rats were induced for hyperglycemia by single intraperitoneal injection of STZ (55 mg/kg). After 1 week, hyperglycemia was confirmed using nonfasting blood glucose measured using a meter and test strips (Contour; Bayer Healthcare). Only animals in which glucose levels exceeded 250 mg/dL throughout the 12-week induction period were used for analysis. To assess proteinuria, spot urine was collected from rats housed in metabolic cages for 1–2 h, 24 h after previous treatment. Urine was frozen at −80°C until analysis of total protein (Bradford assay, Bio-Rad, United States) and creatinine (BioAssay Systems, United States).

SHetA2 (NSC 726189) was supplied to Dr. Doris Benbrook by the US National Cancer Institute RAPID Program and processed for use in animal studies as described (Brosius et al., 2009). Once hyperglycemia was established, rats were given either vehicle control (30% Kolliphor HS15 w/v in water, 0.2% volume of body weight) or SHetA2/30% Kolliphor at 60 mg/kg by oral gavage with equivalent volumes. Oral gavage was performed 3 times weekly every other day except on weekends for a period of 12 weeks. Each week, fresh drug was bulk homogenized, aliquoted and sonicated on the day of treatment, and kept in the dark at 4°C between uses.

The perfused juxtamedullary nephron preparation was used to assess renal microvascular reactivity in rats, as described (Imig et al., 2008). In experiments involving acute treatment with SHetA2, the drug was applied 30 min prior to diameter recordings at 60 and 120 mmHg perfusion pressure.

Isolated renal vascular SMCs (Miller et al., 2016) (passage 6, 20,000 cells/dish) were seeded on 35 mm glass microwell dishes and serum-starved (0.5% fetal bovine serum in DMEM) for 24 h prior to experiments. Cells were loaded with Fluo4-AM (2 µM) at room temperature for approx. 1 h in the dark. HEPES (10 mM) was added to medium for dye loading. After dye loading, cells were washed twice with PSS (140 mM NaCl, 1.2 mM MgCl₂, 4.5 mM KCl, 18 mM glucose, 10 mM HEPES, 2 mM CaCl₂) then incubated in PSS containing DMSO (0.3%) or SHetA2 (0.25 µM, 1 μM, or 2.5 µM) for 35 ± 5 min. Cells were imaged on a Nikon A1r confocal microscope using a ×20 objective lens (NA = 0.75) and excitation/emission settings of 488/500–550 nm. Time lapse experiments were performed at room temperature. Baseline fluorescence was recorded for 1 min prior to stimulation with ET-1 (100 nM). Changes in FL were recorded for approx. 7 min after stimulation. Images were analyzed using ImageJ FIJI software and calculations were performed in MS Excel. Changes in Ca⁺² are reported as changes in FL compared to baseline FL levels (ΔF/F₀). Total cytosolic Ca⁺² was calculated as the integral under each curve and is reported in arbitrary units (AU).

Urine collected from non-diabetic, diabetic and SHetA2-treated diabetic rats at 12-0weeks treatment was diluted in water and boiled in 1X sample buffer for 5 min. Sample volumes (up to 10 μL per lane) were normalized to urine creatinine levels and separated on 4%–15% SDS-Page gels and treansferred onto PVDF membranes (Bio-Rad). Membranes were blocked with Intercept TBS buffer (LICOR), probed using antibodies against rLipocain-2 (NGAL) (1:2000; R&D Systems, Minneapolis, MN). Blots were subsequently probed with donkey anti-goat horseradish peroxidase (HRP), secondary antibody (1:10,000, Santa Cruz). Chemiluminescence was measured using the Bio-Rad ChemiDoc MP system after exposure of the blots to SuperSignal West Femto ECL detection reagents (Thermo Scientific) for 5 min.

Protein expression (human p66Shc and CH2CB) and hydrdoethidine fluorescent assays using an ISS photon counting spectrofluorometer were performed as previously described (Haslem et al., 2022). SHetA2 was solubilized in 20 mM MES pH 5.5, 100 mM NaCl, and 50% w/v PEG 400 at a final concentration of 30 μM and added to reaction mixtures at a final concentration of 6 μM. When present, final protein concentrations were 10 uM. SHetA2 stocks were prepared fresh, and reaction mixtures protected from ambient light. Each assay condition was repeated 7 times using 3 different protein preparations for each target protein (first preparation for each protein has 3 technical replicates). Average values were used for statistical analysis.

For vessel reactivity to ATP a two-way repeated measures ANOVA was used. For vessel reactivity to pressure, a Mann-Whitney two-tailed t-test was performed. All tests were performed using GraphPad Prism 10.2.3. For total calcium measurements, Kruskal-Wallis tests and post-hoc Dunn’s Test were performed using R.