Hypertension in children is an increasingly recognized public health concern, with an estimated prevalence ranging from 2% to 5% in the pediatric population, and elevated blood pressure affecting approximately 13%–18% of children and adolescents globally (Yang et al., 2021). Recent systematic reviews indicate that the pooled prevalence of hypertension among children and adolescents is about 4.00%, with notable increases observed over the past two decades, primarily associated with rising rates of obesity (Song et al., 2019). The causes of juvenile hypertension can be categorized into primary (essential) and secondary hypertension. Primary hypertension is more common in older children and is often linked to genetic factors and lifestyle choices, such as obesity, physical inactivity, and dietary habits. In contrast, secondary hypertension can arise from identifiable medical conditions, including renal disease or endocrine disorders, but it is less prevalent, accounting for about 10%–14% of cases in the pediatric population (Hsu et al., 2021). The development of juvenile hypertension is recognized to be a potential precursor of hypertension in adulthood (Meng et al., 2024). Early identification and successful treatment of juvenile hypertension may prevent the development of multiple organ damage (Hinton et al., 2020). Childhood-onset hypertension is linked to subclinical cardiovascular disease and persists throughout adulthood (Myette and Flynn, 2024; Robinson et al., 2024). Children with hypertension have an increased risk of stroke, hospitalization for myocardial infarction or unstable angina, coronary intervention, and congestive heart failure. The risk of adult cardiovascular disease may be decreased by managing childhood hypertension (Robinson et al., 2024).

Alpha-1 antitrypsin (AAT) is a multifunctional protein that is known to have anti-inflammatory effects and protective effects in various organ systems and tissues. AAT administration has been shown to prevent type 1 diabetes in a non-obese diabetic mouse model (Song et al., 2004), reduce cigarette smoke-induced emphysema in mice (Pemberton et al., 2006), lower blood pressure in diabetic mice (Lugo et al., 2022), and delay the development of arthritis in mice (Grimstein et al., 2011). The role of AAT in mitigating diabetic kidney disease and hypertension secondary to diabetes has not been thoroughly studied.

Several blood pressure regulating proteins have been shown to be affected by protein kinases C (PKC) activity. Free radical superoxide has been shown to stimulate PKC and increase apical plasma membrane expression of the Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2) (Haque and Ortiz, 2019). PKC-mediated phosphorylation was shown to regulate the activity and function of the with-no-lysine kinase 4 (WNK4), which in turn phosphorylates the kinase Ste20-related proline alanine-rich kinase (SPAK) and oxidative stress-responsive kinase (OSR1), leading to the phosphorylation and activation of the renal Na-Cl cotransporter (NCC) (Castaneda-Bueno et al., 2017). Multiple studies have shown PKC negatively regulates renal epithelial sodium channels (ENaC). Bao et al. showed ENaC activity is elevated in native split-open cortical collecting ducts of PKC alpha knockout mice (Bao et al., 2014). Other studies have shown PKC negatively regulates ENaC activity and its density at the luminal membrane by phosphorylating the myristoylated alanine-rich C kinase substrate (MARCKS)/MARCKS like protein 1 (MLP1) protein, causing its translocation to the cytoplasm (Alli et al., 2012; Montgomery et al., 2017; Song et al., 2020).

Diacylglycerols (DAGs) are bioactive lipids that are known to activate specific isoforms of PKC. The availability of DAG to function as a second messenger depends on its regulation by other proteins. For example, diacylglycerol kinase (DGK) terminates DAG signaling by converting DAG to phosphatidic acid, which by itself is involved in signal transduction (Cai et al., 2009).

Here we investigated for the first time the efficacy of administering human AAT (hAAT) to mitigate salt-induced hypertension in juvenile non-obese 129Sv mice and investigated whether a potential mechanism for this protective effect of hAAT could be due to the suppression of PKC activity as a result of decreased levels of the bioactive lipid DAG. Mechanistically, we investigated whether a decrease in DAG and PKC is associated with a decrease in surface expression of renal NKCC2 to presumably mitigate sodium retention and alleviate salt-induced hypertension.

Our study demonstrates for the first time that hAAT treatment alters the concentrations of multiple classes of lipids in the membranes of kidneys from hypertensive juvenile 129Sv mice. A previous study from our group showed hAAT attenuated ENaC and MARCKS protein expression along with ceramide metabolism to hexosylceramides in renal epithelial cells to alleviate salt-induced hypertension in adult diabetic db/db mice (Lugo et al., 2022). However, this is the first study to investigate the efficacy and mechanism of hAAT administration to mitigate hypertension in juvenile mice.

In an attempt to address this knowledge gap, we designed a study to investigate changes in the concentration of membrane lipids from salt-induced hypertensive juvenile 129Sv mice treated with hAAT or vehicle. Our lipidomics data identified several bioactive lipids that were downregulated in the kidney cortex membrane fractions of hypertensive 129Sv mice that received hAAT compared to vehicle. Diacylglycerols represented the largest class of bioactive lipids that were downregulated in the kidney membranes after administration of hAAT. Importantly, diacylglycerols play an important role in the activation of specific PKC isoforms including PKC alpha, beta, delta, and epsilon.

It is known from previous studies that DAG serves as the primary stimulant of protein kinase C in biological processes (Ohanian and Ohanian, 2001), and acts as an allosteric activator of protein kinase C (PKC), while IP3 primarily regulates intracellular calcium levels by modulating calcium channels in both the plasma membrane and endoplasmic reticulum. This result is particularly important in relation to smooth muscle contraction (Berridge, 1987). At the same time Okumara et al. concluded that 1,2-diacylglycerol production by norepinephrine is elevated in the thoracic aorta of spontaneously hypertensive before the onset of hypertension (Okumura et al., 1990). In that study, 1,2-DAG content of spontaneously hypertensive rat hearts during the early stages appeared to be related to the initiation of cardiac hypertrophy in spontaneously hypertensive rats hearts before they developed hypertension (Okumura et al., 1990). Norepinephrine induces diacylglycerol production through its binding to alpha-1 adrenergic receptors, activating PLC and resulting in the generation of DAG (Hussain et al., 2024). In our study, DAG levels were found to be high in hypertensive mice. However, this study did not measure DAG levels in juvenile before inducing hypertension by salt-loading. But, the levels of several DAGs were found to be decreased after hAAT treatment (Figure 4). These studies show that DAGs via acting as a crucial regulator of protein kinase C activity, may have a role in the circulatory alterations associated with hypertension. Moreover, these changes have been shown to start before the onset of hypertension. Overall, the results from this study suggest that hAAT may have therapeutic potential to mitigate hypertension in juvenile mice. Johnsen et al. showed in their study that PKC levels were high in a mouse model of spontaneous hypertensive heart failure (Johnsen et al., 2005). Similarly, Osicka et al. demonstrated that the renal cortex in rat models of hypertension and hyperglycemia, both alone and when combined, exhibit an elevated expression of PKC isoforms (Osicka et al., 2003). In our study, basal levels of PKC activity was found to be higher in salt-loaded 129Sv mice treated with vehicle compared to mice treated with hAAT (Figure 5). Moreover, Wynne et al. showed that diastolic, systolic, and mean arterial pressure of PKC knockout mice were considerably lower compared to the control group (Wynne et al., 2018). PKC has effects beyond decreasing blood pressure. Activation of PKC-δ signaling pathway controls the contraction of the heart muscle and has a crucial function in the development of myocardial ischemia-reperfusion injury and cardiac fibrosis. Studies utilizing pig, rat, and PKC-δ gene deletion mice models demonstrated PKC-δ actively facilitates ischemia/reperfusion injury, and the suppression of PKC-δ can effectively prevent these detrimental effects. Peptide inhibitors administered during reperfusion have a specific effect on protecting the heart. This protection is achieved by decreasing the size of the damaged heart tissue (infarct area) and reducing the levels of troponin T, a protein associated with heart damage. Additionally, these inhibitors improve the recovery of heart function by reducing the activity of caspase-3, an enzyme involved in cell death, and decreasing the apoptosis (Lincoff et al., 2014; Miao et al., 2022). All of these findings indicate that inhibiting PKC could potentially prevent permanent damage to the heart during the restoration of blood flow not only during hypertension. Our data suggest that hAAT may lower PKC activity by reducing DAGs.

One limitation of our study is that we did not compare regional differences in lipids between the cortex and medulla. Another limitation is that we did not measure differences in membrane lipids between male and female mice. We used only male mice in this study since it is established that female sex hormones have a protective role in the development of hypertension (Xue et al., 2007). Third, we did not use other salt-sensitive animal models to corroborate the effects we observed in 129Sv mice. We and other groups have shown 129Sv mice are salt-sensitive and develop hypertension. In contrast, C57Bl6 mice are generally salt resistant to salt loading and are not the preferred model to study the development of hypertension due to dietary sodium. However, our group showed that db/db mice develop hypertension after salt-loading. Future studies will examine whether the lipids we found to be downregulated in 129Sv mice are also downregulated in juvenile salt-loaded hypertensive db/db mice. Another future direction relevant to the scope of this study to investigate whether hAAT administration affects the release and excretion of extracellular vesicles into the urine or the enrichment of bioactive lipids in urinary extracellular vesicles. A previous study showed several hexosylceramides and other classes of lipids are enriched in urinary extracellular vesicles after administration of hAAT in hypertensive diabetic db/db mice (Lugo et al., 2022). Another future direction is to investigate the mechanism by which hAAT is taken up by the thick ascending limb cells expressing NKCC2. Finally, we plan to investigate whether other effector proteins downstream in the DAG-PKC pathway are affected by hAAT treatment. DAG mediated activation of PKC has been shown to result in the activation of protein kinase D (PKD) (Wang, 2006).

Taken together, our descriptive lipidomic dataset identified several DAGs that were attenuated in kidney cortex membrane fractions of salt-loaded hypertensive juvenile 129Sv mice given hAAT compared to vehicle. A reduction in the availability of bioactive DAGs is expected to prevent high levels of PKC activity and subsequently reduce surface expression of renal NKCC2 (Figure 8). Renal NKCC2 plays an important role in regulating total body salt balance and blood pressure. A decrease in its expression and activity is expected to reduce sodium reabsorption in the kidney and mitigate salt-induced hypertension.