Diet-induced obesity contributes to chronic inflammation, mitochondrial dysfunction, and progressive kidney injury, while increased dietary acid load may further impair renal homeostasis. In our previous studies, we showed that ammonium hydroxide enhancement (AHE) of dietary protein, prior to incorporation into the complete diet, increases dietary alkalinity and has improved longevity, decreased secretion of adipocytokines, and beneficially affected the microbiome in high-fat diet fed C3H/HeJ mice. However, its effects on renal molecular and structural responses remain unknown. In this study, kidney tissues from male C3H/HeJ mice fed control casein (CC), ammonium hydroxide-enhanced casein (CCN), high-fat casein (HFC), ammonium hydroxide-enhanced high-fat casein (HFCN), high-fat beef (HFB), or ammonium hydroxide-enhanced high-fat beef (HFBN) diets were analyzed using quantitative PCR, Western blotting, and histological morphometry. Results showed that AHE reduced renal inflammatory signaling, evidenced by decreased TLR4, TNFα, Il1-β, IL6, and NFκB expression. Apoptosis-associated signaling was attenuated through reduced Caspase-3 and p53 expressions and increased BCL2 levels. AHE also modulated mitochondrial-associated markers. Increased markers of mitochondrial biogenesis and fusion, and decreased fission-associated markers were observed, along with increased PINK1 expression, while Parkin remained unchanged. In addition, expression levels of the ammonia transporters, RHBG and RHCG, were elevated, indicating altered renal ammonia transporter expression. Histological and morphometric analyses demonstrated AHE-associated changes in tubular areas, while glomerular and Bowman’s capsule areas remained largely unchanged. Collectively, these findings suggest that long-term dietary AHE is associated with alterations in renal inflammatory and apoptotic signaling, mitochondrial-associated pathways, ammonia transporter expression, and renal histomorphometry in male C3H/HeJ mice.

Breast cancer remains a leading cause of cancer-related mortality worldwide, highlighting the need for novel therapeutic strategies that selectively target tumor cells while minimizing systemic toxicity. Dietary ammonium hydroxide enhancement (AHE) has previously been shown to modulate metabolic pathways in animal studies. However, its potential as a localized anticancer therapy has not been investigated. In the present study, we evaluated the antitumor efficacy and systemic safety of NH₄OH using complementary in vitro and orthotopic breast cancer xenograft models. MDA-MB-231 breast cancer cells and non-tumorigenic MCF10A mammary epithelial cells were treated with increasing concentrations of NH₄OH (2.5–225 µM) to assess dose-dependent effects on cell proliferation, viability, and apoptosis. Following this, MDA-MB-231 cells were orthotopically xenografted into female athymic nude mice and treated by intratumoral injection with NH₄OH using a stepwise dose-escalation regimen (0.01%, 0.1%, and 0.5%; total volume of 20 μL per tumor divided between two injection sites) or phosphate-buffered saline (PBS). Differences between treatment groups of mammary tumors and kidney tissues were analyzed molecularly and histologically. NH₄OH significantly suppressed MDA-MB-231 cell growth and metabolic activity, with minimal effects on MCF10A cells, and induced apoptosis in MDA-MB-231 cells without detectable apoptotic induction in MCF10A cells. In vivo, although tumor volume only showed a non-significant downward trend, histological and molecular analyses demonstrated substantial alterations in tumor biology. NH₄OH treatment induced molecular changes consistent with an antitumor response, including increased Caspase-3 and p53 expression, reduced BCL2 and Ki-67 expression, and attenuation of TNFα, IL-6, and TLR4 inflammatory signaling. Furthermore, the tumor architecture in T-NH tumors displayed increased pale eosinophilic regions and reduced cellular density, suggestive of treatment-associated tumor tissue disruption. Histological analysis of kidney tissue showed no evidence of overt renal toxicity. Indeed, localized NH₄OH administration was associated with reduced renal inflammatory and apoptotic signaling, preserved renal morphology, and increased expression of the ammonia transporters RHBG and RHCG. Cross-sectional morphometric measurements showed decreased area for the distal convoluted tubules in NH₄OH-treated samples. Although this initial preclinical study was limited by a relatively small sample size, further studies are warranted to validate these findings and define the molecular mechanisms underlying NH₄OH-mediated antitumor activity. Collectively, these findings suggest that intratumoral NH₄OH modulates tumor metabolic, inflammatory, and apoptotic pathways associated with a less aggressive tumor phenotype while showing no overt molecular or histological evidence of renal injury, supporting further investigation as a localized metabolic intervention targeting molecular and histological drivers of breast cancer progression.