Abstract

   Acute kidney injury (AKI) manifests a hallmark pathological feature of
   extensive and severe DNA damage in renal tubules, primarily induced by
   the excessive of toxic reactive oxygen species (ROS) from the
   mitochondrial electron transport chain. The kidney's complex intricate
   physiological architecture and the heterogeneous intracellular
   environment pose significant challenges for effective sequential and
   high‐resolution drug delivery—an urgent issue that remains unresolved.
   To address this, a hierarchical‐targeting antioxidant nanodrug has been
   developed with a folic acid moiety (HAND) designed for high‐resolution
   drug delivery in AKI treatment. For the first time, HAND enables
   sequential targeting from the kidney to the most severely damaged
   proximal tubular epithelial cells (PTECs), ultimately concentrating in
   the DNA‐rich mitochondria and nucleus. As a result, HAND effectively
   scavenges ROS in situ, protecting both mitochondria and nuclei along
   with their vital genetic material. This action restores mitochondrial
   function, mitigates DNA oxidation and fragmentation, reduces apoptosis,
   and inhibits cGAS/STING‐mediated sterile inflammation. Consequently,
   HAND demonstrates remarkable efficacy in safeguarding injured kidneys
   during AKI. Overall, this work pioneers a hierarchical, high‐resolution
   antioxidant strategy, providing innovative guidance for the development
   of AKI therapies.

   Keywords: AKI, antioxidative nanomedicine, mitochondria, nucleus,
   oxidative stress, subcellular organelle targeting
     __________________________________________________________________

   This study developed a hierarchical‐targeting antioxidant nanodrug
   (HAND) for precise AKI treatment, achieving targeted delivery from
   kidney to severely damaged proximal tubular epithelial cells, and
   concentrating in DNA‐rich mitochondria and nucleus. HAND effectively
   scavenges ROS in situ, protecting vital genetic material and
   demonstrating significant efficacy in preserving kidney function during
   AKI.

   graphic file with name ADVS-12-2411254-g005.jpg

1. Introduction

   Acute kidney injury (AKI) refers to the sudden deterioration of kidney
   function, primarily due to acute dysfunction of proximal tubular
   epithelial cells (PTECs).^[ [48]^1 ^] Globally, AKI affects over 13
   million individuals annually and is particularly prevalent among
   hospitalized patients, with incidence rates reaching 30%−60% in
   critical care settings.^[ [49]^2 ^] Unfortunately, no pharmacological
   interventions have been successfully demonstrated to provide renal
   protection in AKI. Supportive care and renal replacement therapy are
   currently the only clinical treatment options,^[ [50]^3 ^] but their
   effectiveness is limited, and they impose a significant burden on
   healthcare systems. If untreated or inadequately managed, AKI can lead
   to renal failure and progress toward kidney fibrosis, driven by
   sustained oxidative stress and chronic inflammation, ultimately
   resulting in severe end‐stage renal disease, multi‐organ failure, or
   death.^[ [51]^4 ^] With an unacceptably high mortality rate of 1.7
   million deaths annually, AKI presents a significant clinical challenge
   and underscores the urgent need for novel drug development.^[ [52]^5 ,
   [53]^6 ^]

   AKI is marked by a defining pathological feature: extensive and severe
   DNA damage in renal tubules.^[ [54]^7 , [55]^8 ^] DNA, the repository
   of genetic information, is located exclusively in the nucleus and
   mitochondria of human cells, orchestrating cellular structure,
   function, and responses to stimuli through gene expression
   regulation.^[ [56]^9 ^] Damage to DNA disrupts this regulatory function
   and collapses the cellular command center, directly resulting in cell
   death.^[ [57]^10 ^] Moreover, irreparable DNA fragments released into
   the cytoplasm or extracellular space act as critical signals that
   activate innate DNA sensing pathways, triggering inflammatory cascades
   and cytokine storms.^[ [58]^11 ^] Consequently, the loss of functional
   cells combined with persistent inflammation significantly impairs renal
   function, leading to acute manifestations such as electrolyte
   imbalances, fluid overload, and acidosis, as well as the malignant
   progression of fibrosis.^[ [59]^12 ^] The primary source of DNA damage
   in AKI is endogenous oxidative stress, particularly from the production
   of toxic reactive oxygen species (ROS) by the mitochondrial electron
   transport chain (ETC).^[ [60]^13 ^] The kidneys, as one of the most
   mitochondria‐rich organs, receive 20% of cardiac output and utilize 10%
   of the body's oxygen.^[ [61]^14 ^] To support the intense transport and
   reabsorption functions in PTECs, mitochondria, serving as cellular
   power factories, are densely concentrated within these cells.^[ [62]^15
   ^] Stimulants of AKI, such as rhabdomyolysis, systemic inflammation, or
   ischemia/reperfusion, trigger electron leakage from the mitochondrial
   ETC in PTECs, leading to a surge in mitochondrial ROS (mtROS).^[
   [63]^16 ^] Critically, the high mitochondrial density within PTECs
   allows mtROS to rapidly diffuse through the mitochondrial pool,
   creating a storm that engulfs neighboring mitochondria and the nucleus,
   the latter of which is closely and fully surrounded by these
   mitochondria.^[ [64]^17 ^] As highly nucleophilic species with unpaired
   electrons, ROS engage in intense chemical reactions with electrophilic
   biomolecules, including DNA.^[ [65]^18 ^] These interactions result in
   substantial oxidative double‐stranded DNA damage, which subsequently
   activate cell death pathways and inflammatory signaling mediated by the
   cGAS/STING axis.^[ [66]^19 ^] Thus, the damage inflicted by ROS on the
   two crucial DNA‐rich organelles in PTECs—mitochondria and the
   nucleus—propagates rapidly, akin to a cascading domino effect,
   ultimately compromising the entire kidney.

   Therefore, effectively neutralizing excessive toxic ROS represents a
   crucial therapeutic objective to mitigate the source of damage and
   protect PTECs, ultimately improving renal function. However, clinically
   utilized antioxidant molecules, such as amifostine and N‐acetylcysteine
   (NAC), offer limited protection against AKI due to their lack of
   targeting capability and poor bioavailability. In recent years,
   emerging nanomedicine has demonstrated a remarkable trajectory of
   advancement,^[ [67]^20 , [68]^21 ^] with several mitochondria‐specific
   targeting nanostrategies under active investigation.^[ [69]^22 ,
   [70]^23 ^] Nonetheless, these approaches face significant challenges in
   achieving effective and precise treatment for AKI. First, the presence
   of positive charges and/or hydrophobic structures in
   mitochondria‐targeting compounds limits their stability and in vivo
   applicability, as negatively charged enzymes or proteins in circulation
   may interact with them, thereby diminishing the targeting efficacy.^[
   [71]^24 ^] Additionally, complex encapsulation or modification can
   result in increased particle size, complicating passage through the
   glomerular filtration barrier (GFB) and raising safety concerns related
   to residual materials.^[ [72]^25 ^] Second, some nanomaterials that are
   appropriately sized for the GFB often fail to effectively target PTECs,
   the primary site of injury in AKI, leading to a rapid loss of
   therapeutic efficacy as they are swiftly excreted in the urine. The
   nucleus, serving as the cell's primary command center, is often
   overlooked by researchers, leading to inadequate protection. This
   neglect is particularly concerning, given the close spatial and
   functional interconnections between mitochondria and the nucleus,^[
   [73]^26 ^] both of which are highly susceptible to ROS. Thus,
   developing a hierarchical targeting strategy for the precise delivery
   of therapeutic agents to kidney‐PTECs‐mitochondria and the
   nucleus—facilitating targeted ROS clearance at the tissue, cellular,
   and subcellular levels—holds significant promise for innovative and
   effective treatment of AKI. However, the unique physiological structure
   of the kidney and the complex, heterogeneous intracellular environment
   present formidable challenges to achieving such sequential and
   high‐precision targeting. As a result, no practical applications from
   this perspective have been reported to date.

   To address this challenge, a portable and elegant
   hierarchical‐targeting antioxidant nanodrug (HAND) derived from folic
   acid (FA) and curcumin (Scheme [74]1i) has been developed for precise
   delivery to the mitochondria and the nucleus in PTECs, which enabled
   the specific and highly efficient clearance of toxic mtROS and
   mitigation of DNA damage. Curcumin, a lipophilic polyphenol compound
   extracted from turmeric rhizomes, has demonstrated a wide range of
   pharmacological activities, including antioxidant, antimicrobial, and
   anti‐inflammatory properties. Moreover, curcumin was recognized as a
   “generally regarded as safe” compound by the U.S. Food and Drug
   Administration. However, its therapeutic efficacy in treating AKI is
   limited by poor bioavailability, rapid metabolism, and low water
   solubility when used alone. Fortunately, the chemical structure of
   curcumin, characterized by abundant sp2 and sp3 carbon atoms along with
   antioxidant functional groups such as phenolic hydroxyl and methoxy
   groups, makes it an ideal precursor for synthesizing carbon dots with
   antioxidant properties. In this study, we have integrated curcumin with
   the targeting molecule FA into integrated carbon dots──HAND, not only
   enhancing curcumin's bioavailability but also promoting its precise
   delivery to AKI lesions. The ultrasmall HAND is abundant in FA‐like
   groups and possesses a strong negative charge on its surface.
   Therefore, HAND was less susceptible to sequestration by the hepatic
   reticuloendothelial system, facilitating its accumulation in the
   kidneys and enabling it to penetrate the GFB to reach the renal tubular
   lumen. Furthermore, PTECs expressed high levels of FA receptors (FR) on
   their surface, granting them a high affinity for HAND and facilitating
   efficient endocytosis. Notably, we have identified, for the first time,
   that FA demonstrates a strong affinity for translocases of the outer
   and inner mitochondrial membrane (TOM complex and TIM23 complex) and FG
   nucleoporins (FG Nups) within nuclear pore complexes. This property is
   effectively inherited by HAND, which shows pronounced co‐localization
   with both the nucleus and mitochondria in human renal tubular
   epithelial cells (HK‐2 cells). Therefore, HAND can actively target
   PTECs and preferentially accumulate in DNA‐rich regions
   (Scheme [75]1ii). By leveraging its abundant antioxidant groups, HAND
   efficiently scavenged toxic mtROS, thereby protecting the mitochondria
   and nucleus, restoring mitochondrial function, mitigating apoptosis,
   and attenuating cGAS/STING‐mediated sterile inflammation
   (Scheme [76]1iii,iv). This multifaceted approach effectively
   safeguarded PTECs and facilitated the restoration of renal function in
   AKI mouse models. Moreover, HAND, derived from natural products
   (curcumin) and essential nutrients (FA), exhibited commendable
   biocompatibility and was effectively eliminated from the body through
   renal clearance within 72 h. In conclusion, this study introduces a
   high‐resolution, hierarchical targeting antioxidant therapeutic
   strategy, providing a new paradigm for the development of therapeutic
   agents for renal diseases, including but not limited to AKI.

Scheme 1.

   Scheme 1
   [77]Open in a new tab

   Hierarchical Targeting Nanodrug with Holistic DNA Protection for
   Effective Treatment of Acute Kidney Injury. i) HAND was synthesized
   using curcumin and FA as precursor compounds through a hydrothermal
   method. Infrared spectroscopy results indicated that HAND successfully
   inherited the surface groups of FA. ii) Upon intravenous injection,
   ultrasmall HAND could passively transverse the GFB and be actively
   taken up by PTECs through FR‐mediated endocytosis. Furthermore, HAND
   could localize in the nucleus and mitochondria due to the high affinity
   of FA for their surface membrane proteins. iii) HAND effectively
   protected mitochondrial structure and function via scavenging mtROS,
   subsequently inhibiting damage‐induced mitophagy and intrinsic
   apoptosis. iv) Through effectively scavenging mtROS, HAND could reduce
   the release of dsDNA, thereby suppressing sterile inflammation caused
   by cGAS/STING pathway activation. v) TEM image of HAND (a). Insert:
   Size distribution of HAND. XPS N1s spectrum of HAND (b). UV absorption
   spectra of O[2] ^·− reacting with different concentrations of HAND.

2. Results

2.1. Synthesis and Characterization of HAND

   In this study, we synthesized HAND and CND (without FA) by inducing a
   carbonization process using FA and curcumin as carbon source precursors
   at 240 °C (Scheme [78]1i–a). Transmission electron microscopy (TEM)
   confirmed that HAND and CND were monodisperse, spherical nanoparticles
   with an average diameter of ≈11 nm (Scheme [79]1v–a; Figure [80]S1,
   Supporting Information), well below the particle size threshold for GFB
   (≈14 nm). The surface charges of HAND and CND were −34.5 and −23.5 mV,
   respectively, supporting their stable dispersion in circulation (Figure
   [81]S2, Supporting Information). We subsequently used X‐ray
   photoelectron spectroscopy (XPS) to analyze the composition of HAND.
   The XPS spectrum of HAND showed three main peaks: C1s (285 eV), N1s
   (400 eV), and O1s (534 eV) (Figure [82]S3, Supporting Information),
   while CND exhibited only two peaks: C1s and O1s (Figure [83]S4,
   Supporting Information), indicating that HAND inherited nitrogen from
   FA. As illustrated in Scheme [84]1v–b, the high‐resolution XPS N1s
   spectrum of HAND identified three distinct peaks attributed to
   pyridinic nitrogen (400.5 eV), amino nitrogen (401.6 eV), and graphitic
   nitrogen (402.6 eV). Concurrently, the XPS C1s spectrum (Figure [85]S5,
   Supporting Information) of HAND displayed five distinct peaks
   corresponding to C─C, C═C, C─N/C─O, C═O, and π–π^* transitions,
   suggesting a nitrogen‐doped carbon framework with FA‐derived surface
   groups. Fourier‐transform infrared (FT–IR) spectroscopy further
   supported these findings, revealing N‐containing absorption peaks such
   as ─NH, C═N, and C─N, consistent with FA, alongside typical carbon dot
   peaks for ─OH, C─H, C═C, and C─O (Scheme [86]1i–b). In contrast, CND
   showed only characteristic carbon dot peaks for ─OH, C─H, C═O
   (1638 cm⁻¹), C═C, and C─O (Figure [87]S6, Supporting Information).
   UV–vis spectroscopy of HAND and CND revealed two major absorption bands
   at 277 and 300–400 nm, corresponding to π–π^* transitions of polycyclic
   aromatic chromophores (C═C) and n‐π^* transitions of C─N/C═O,
   respectively (Figure [88]S7, Supporting Information). These results
   confirmed the successful synthesis of HAND, which exhibited an N‐doped
   conjugated carbon framework with an abundant surface group derived from
   FA and curcumin as expected. Consequently, these surface groups,
   including methoxy, phenolic hydroxyl, and amino groups inherited from
   FA and curcumin, endowed HAND with exceptional and broad‐spectrum ROS
   scavenging capacity. For example, HAND effectively neutralized O[2] ^·⁻
   in vitro, counteracting the primary ROS generated by mitochondrial ETC
   electron leakage, thus potentially limiting mtROS propagation
   (Scheme [89]1v–c). As a reductive sacrificial agent, HAND also
   effectively detoxified H₂O₂, a longer‐lived ROS, and secondary radicals
   derived from O[2] ^·⁻, such as ·OH and ONOO^⁻ (Figure [90]S8,
   Supporting Information). Similarly, CND demonstrated comparable ROS
   scavenging activity due to the antioxidant groups inherited from
   curcumin (Figure [91]S9, Supporting Information). Thus, HAND's
   broad‐spectrum antioxidant activity as a reductive sacrificial agent
   was robustly validated. In conclusion, HAND was successfully
   synthesized and comprehensively characterized, confirming its expected
   structure and potent antioxidant properties. With its ultra‐small size
   and potential for active targeting to PTECs, HAND holds considerable
   promise for renal accumulation, which is evaluated in subsequent animal
   models.

2.2. Hierarchical Targeting of HAND: from Kidney to PTECs, and Ultimately to
Mitochondria and Nucleus

   To investigate the hierarchical targeting properties of HAND, a
   rhabdomyolysis‐induced AKI (RM‐AKI) mouse model was established by
   injecting equal volumes of 50% glycerol into both hind limbs. RM‐AKI,
   commonly encountered in trauma and perioperative contexts, represents
   one of the most prevalent forms of AKI in clinical practice. Following
   intravenous administration of HAND to the AKI mice, TEM images of the
   glomerulus were captured to assess the passage of HAND through the GFB.
   As shown in Figure [92]1A, HAND could be directly observed in the
   glomerular capsule, confirming its successful traversal of the GFB. To
   further track their distribution in vivo, HAND and CND were labeled
   with fluorescein isothiocyanate to form FITC‐HAND and FITC‐CND, which
   showed comparable particle size and surface charge to HAND and CND
   (Figure [93]S10, Supporting Information). The labeling process involved
   the reaction of the isothiocyanate group of FITC with the amino and
   hydroxyl functional groups present in HAND and CND, forming stable
   covalent bonds (Figure [94]S11, Supporting Information). The resulting
   conjugates were then visualized through stereo‐fluorescence microscopy
   to assess their distribution and localization in biological systems. As
   shown in Figure [95]1B and Figures [96]S12,S13 (Supporting
   Information), FITC‐HAND and FITC‐CND demonstrated significant renal
   accumulation in both Control and AKI mice, with minimal distribution in
   the liver and lungs, and negligible presence in the heart and spleen.
   Such in vivo pharmacokinetic behavior of HAND and CND can be attributed
   to their negative charge, ultra‐small size, and excellent water
   solubility. We then further analyzed the temporal variation in renal
   concentrations of HAND and CND. Within 1 h post‐injection, a distinct
   green fluorescence was observed in the kidneys of the AKI+FITC‐HAND and
   AKI+FITC‐CND groups, with fluorescence intensity increasing over time,
   peaking ≈6 h before gradually declining due to drug excretion
   (Figure [97]1C; Figures [98]S14,S15, Supporting Information). Notably,
   the peak fluorescence intensity (6 h) and fluorescence duration (24 h)
   of FITC‐HAND in the kidneys were significantly greater than those of
   FITC‐CND (Figure [99]S16, Supporting Information). This sustained
   accumulation highlighted the superior of HAND in actively targeting the
   focus, ensuring more specific and prolonged ROS scavenging in PTECs, as
   opposed to merely retaining in the tubular lumen or being rapidly
   excreted in urine. The enhanced specific and sustained accumulation of
   HANDs in the kidneys compared with CNDs may be attributed to surface
   groups inherited from FA, which specifically bind to FR‐rich PTECs.
   Meanwhile, HAND was labeled with a Cy7 probe and administered to mice
   for in vivo imaging. The results showed that once in circulation, HAND
   rapidly accumulated in the kidneys, demonstrating time‐dependent uptake
   that peaked at 6 h before gradually declining. This accumulation
   pattern is well‐aligned with stereo fluorescence imaging results,
   providing strong evidence of the targeted renal distribution of HAND.
   Notably, the distribution of HAND in other organs, such as the liver,
   was relatively low, further supporting its kidney‐specific targeting
   capability (Figure [100]S17, Supporting Information).

Figure 1.

   Figure 1
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   The hierarchical targeting property of HAND. A) TEM images of GFB in
   different groups. B) Representative fluorescence images of different
   organs in AKI mice 6 h after intravenous injection of HAND. C)
   Representative fluorescence images of kidneys in AKI mice at various
   time points (1‐72 h) after intravenous injection of HAND. D)
   Representative IF staining images of FITC‐HAND with glomerular
   (synaptopodin) and tubular (AQP1) markers in mice kidney tissues, with
   yellow circles indicating glomeruli. E) Representative IF staining
   image of FITC‐HAND with FR in mice kidney tissues. F,G) Representative
   fluorescence images (F) and quantitative fluorescence analysis (G) of
   HK‐2 cells incubated with FITC‐HAND or FITC‐CND in different groups. H)
   Colocalization fluorescence images and correlation coefficient analysis
   of FITC‐HAND with different organelles in HK‐2 cells. I–L) Molecular
   docking results of FA with TOM comple, TIM23, and FG Nups. Data are
   presented as mean ± SD. One‐way ANOVA followed by the
   Student–Newman–Keuls (SNK) test was used for analysis. n = 3, ^*** p
   < 0.001, ^**** p < 0.0001; ^## p < 0.01, ^### p < 0.001; ns, not
   significant (p > 0.05).

   Thereafter, we further delved into the influence of FA‐derived groups
   on the more microcosmic targeting properties of HANDs at the cellular
   and organellar levels. FA, an essential micronutrient, is primarily
   regulated by the kidneys to maintain systemic homeostasis.^[ [102]^27
   ^] Upon entering circulation, FA is freely filtered through the
   glomerulus, with most being reabsorbed by PTECs to prevent excretion.
   The reabsorption process relies heavily on the FR located on the brush
   border of PTECs.^[ [103]^28 ^] Our immunofluorescence (IF) staining of
   human kidney samples confirmed that PTECs express high levels of FR,
   establishing a foundation for the cell‐specific targeting of
   FA‐functionalized HAND (Figure [104]S18, Supporting Information). As
   expected, the ultrasmall FITC‐HAND and FITC‐CND were able to cross the
   GFB, primarily distributing within renal tubules (marked by AQP1)
   rather than glomeruli (marked by synaptopodin) in mouse kidney tissues
   (Figure [105]1D; Figure [106]S19, Supporting Information). Importantly,
   FITC‐HAND derived from FA showed a high degree of colocalization with
   FR on the surface of PTECs, which was significantly greater than that
   of FITC‐CND, highlighting the active targeting capability of HAND
   toward FR (Figure [107]1E; Figure [108]S20, Supporting Information). We
   also verified HAND's FR‐targeting ability at the cellular level by
   comparing HAND uptake across various cell lines with differing FR
   expression. As shown in Figure [109]S21 (Supporting Information),
   FITC‐HAND was effectively and abundantly taken up by FR‐high‐expressing
   HK‐2 cells, while its uptake by FR‐low‐expressing cardiomyocytes (H9c2)
   and hepatic stellate cells (LX2) was lower under same conditions. In
   contrast, FITC‐CND exhibited no significant differences in uptake among
   these cell lines. Notably, pre‐treatment with FA significantly
   inhibited FITC‐HAND uptake in HK‐2 cells, evidenced by a marked
   decrease in intracellular fluorescence (Figure [110]1F,G). This
   competitive inhibition indicates that HAND internalization is primarily
   FR‐mediated. Conversely, under the same conditions, FITC‐CND exhibited
   markedly lower fluorescence intensity in HK‐2 cells compared to
   FITC‐HAND, and FA pre‐treatment did not affect its uptake. Together,
   these findings underscore the FR‐dependent targeting capability of
   HAND. Furthermore, FA is a pivotal component of intracellular
   one‐carbon metabolism, exhibiting a compartmentalized pattern of
   utilization, predominantly within the mitochondria and nucleus. Such
   compartmentalization likely drives the selective intracellular
   localization of HAND. Specifically, HAND exhibited a pronounced
   dual‐targeting specificity for the nucleus and mitochondria in HK‐2
   cells, with Pearson's correlation coefficient of 0.85 and 0.68,
   respectively, corresponding to the primary compartments involved in
   FA‐mediated one‐carbon metabolism (Figure [111]1H). To further
   elucidate the targeting and adhesion mechanisms of HAND, we employed
   molecular docking to assess FA's affinity for mitochondrial and nuclear
   proteins. TOM and TIM are critical macromolecular complexes embedded in
   the outer and inner mitochondrial membranes, respectively, facilitating
   the localization and import of nuclear‐encoded proteins into
   mitochondria.^[ [112]^29 ^] The nuclear pore complex (NPC) serves as
   the principal gateway for macromolecular transport between the nucleus
   and cytoplasm. NPC is highly conserved and intricate, with FG Nups in
   its central channel mediating the interactions with cargo.^[ [113]^30
   ^] Molecular docking results indicated that FA achieved high docking
   scores with TOM, TIM, and FG Nups due to its molecular flexibility,
   stable conformation, and effective non‐bonded interactions, with
   calculated free binding energies of −6.15, −7.10, and
   −6.76 kcal mol^−1, respectively (Figure [114]1I–L). To further validate
   the affinity of FA‐mediated HAND for TOM, TIM, and FG Nups, we
   pre‐treated HK‐2 cells with FA before incubation with FITC‐HAND and
   examined its the colocalization of FITC‐HAND with various organelles.
   As illustrated in Figure [115]S22 (Supporting Information),
   pre‐treatment with FA significantly reduced the distribution of HAND in
   the mitochondria and nucleus, with co‐localization coefficients
   decreasing to 0.37 and 0.42, respectively. In contrast, there were no
   significant changes in the co‐localization of HAND with the endoplasmic
   reticulum (ER), Golgi apparatus, and lysosomes (Lyso), confirming that
   the dual‐targeting capability of HAND is mediated by FA. These findings
   provided compelling validation and mechanistic insights into the
   distinct dual‐targeting affinity of HAND for both mitochondria and the
   nucleus.

   In summary, the above evidence strongly demonstrated the hierarchical
   targeting properties of HAND: sequentially from the kidney to PTECs,
   and ultimately to the mitochondria and nucleus, which is characterized
   by high and persistent accumulation in the kidneys, specific binding to
   PTECs, and subsequent precise localization targeting to mitochondria
   and nucleus.

2.3. HAND Protected the Kidneys of AKI Mice by Suppressing Oxidative Stress

   Given its hierarchical targeting (Figure [116]2A) and potent
   antioxidant properties, the in vivo therapeutic effect of HAND on AKI
   was further explored in RM‐AKI mice. Renal tubular injury scores and
   renal function indicators are standard criteria for assessing the
   extent of renal damage in AKI. The renal tubular injury score, ranging
   from 0 to 5, reflects the severity of renal damage based on renal
   tubule morphology, with higher scores indicating more severe injury.
   Renal function is evaluated through serum creatinine (CRE) and urea
   nitrogen (BUN) levels, both of which increase with declining kidney
   function. In addition, neutrophil gelatinase‐associated lipocalin
   (NGAL) is a sensitive biomarker for early kidney injury, providing
   important information about renal damage. We first optimized the dosage
   of HAND for the treatment of AKI. As shown in renal function indicators
   detection results (Figure [117]S23, Supporting Information), HAND could
   increase the therapeutic effects of AKI in a dose‐dependent manner,
   with the optimal efficacy observed at a dose of 2 mg kg^−1. We then
   compared the effects of HAND, CND, and the clinical antioxidant NAC on
   the treatment of AKI were compared at the same dosage. Renal cortex
   morphology was observed by HE staining and the degree of renal tubular
   injury was scored accordingly. As shown in Figure [118]2B,C, RM induced
   severe renal tubular injury in the AKI group, characterized by obvious
   dilation and deformation of the renal tubules, loose arrangement
   between tubules, deposition of substances in the tubular lumen (casts),
   loss of the brush border of PTECs, PTECs swelling, necrosis, and
   sloughing, with a renal injury score as high as 4.5. Treatment with
   2 mg kg^−1 HANDs effectively alleviated the pathological damage of the
   kidneys, restoring tubule morphology and structure to near‐normal
   conditions. In contrast, the pathological damage remained severe in the
   CND group (lacking PTECs targeting) and the NAC group
   (Figure [119]2B,C), with renal injury scores of 3.2 and 3.8,
   respectively, which was primarily due to their lower targeting and
   accumulation at PTECs. Moreover, the serum CRE (Figure [120]2D) and BUN
   (Figure [121]2E) levels in the AKI group rose sharply, reaching 4.67
   and 3.59 times those of the Control group, meeting the criteria for
   stage 3 AKI. Similarly, NGAL levels in serum and urine also surged
   (Figure [122]2F; Figure [123]S24, Supporting Information), confirming
   the severity of renal injury. HAND treatment provided superior renal
   protection compared to the CND and NAC groups, with CRE, BUN, and NGAL
   levels returning to normal. During the experimental period, mice in the
   AKI group exhibited significant weight loss, whereas those in the HAND
   group displayed normal growth patterns (Figure [124]S25, Supporting
   Information). HO‐1 and KIM‐1, specific biomarkers of renal injury, are
   minimally expressed in normal renal tissues but are markedly
   upregulated in PTECs during renal impairment. Compared with the AKI
   group, levels of HO‐1 (Figure [125]2G) and KIM‐1 (Figure [126]2H) in
   the HAND group returned to normal, demonstrating far superior
   therapeutic effects compared to the CND group, while the NAC group
   showed negligible therapeutic benefit.

Figure 2.

   Figure 2
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   HAND protected the kidneys of AKI mice by suppressing oxidative stress.
   A) Schematic illustration of HAND crossing the GFB and actively
   targeting PTECs. B) Representative H&E‐stained images of kidney tissues
   from different treatment groups. Arrows indicate damaged PTECs, and
   asterisks indicate casts. C–E) Kidney injury scores (C), levels of
   serum CRE (D), and BUN (E) in different treatment groups. F–H) Levels
   of NGAL (F), HO‐1 (G), and KIM‐1 (H) in kidney tissues of different
   treatment groups. I) Representative DHE fluorescence images of kidney
   tissues in different treatment groups. J) IHC results of 8‐OHdG in
   mouse kidneys from different treatment groups. Data are presented as
   mean ± SD. One‐way ANOVA followed by the SNK test was used for
   analysis. n = 6, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001; ns,
   not significant (p > 0.05).

   The pharmacological basis of HAND is its ability to inhibit oxidative
   stress. To investigate this, we conducted a thorough examination of
   HAND's in vivo antioxidant activity as a meticulously designed
   reductive scavenger. Dihydroethidium (DHE) probe staining
   (Figure [128]2I; Figure [129]S26, Supporting Information) revealed that
   the ROS level in the renal tissues of AKI mice was significantly
   elevated compared to the Control group. After treatment with HAND, the
   level of ROS markedly decreased to baseline, demonstrating its
   effectiveness in alleviating oxidative stress in the kidneys during
   AKI. In comparison, both CND and NAC exhibited weaker antioxidant
   activity at the same dosage, primarily due to their limited capacity
   for active targeting of PTECs. As highly destructive molecules, ROS
   inflict extensive cellular oxidative damage by aggressively attacking
   biomacromolecules, particularly DNA and membrane lipids in mitochondria
   and the nucleus.^[ [130]^31 ^] As shown in the IHC results of the DNA
   oxidation marker, 8‐OHdG, DNA was severely damaged and widely diffused
   throughout the PTECs, affecting both the nucleus and mitochondria in
   the AKI group (Figure [131]2J). Elisa's results further corroborated a
   significantly elevated level of 8‐OHdG in the kidney tissues of the AKI
   group (Figure [132]S27, Supporting Information). Thanks to the precise
   targeting of mitochondria and the nucleus, HAND offered comprehensive
   protection for the DNA by significantly reducing DNA oxidative damage,
   as evidenced by the decreased levels of 8‐OHdG in both IHC and Elisa
   results. In comparison, CND and NAC exhibited lower DNA protection at
   the same dosage. Moreover, the γ‐H2AX staining results provided
   additional confirmation of HAND's efficacy in mitigating DNA damage. In
   the AKI group, a marked increase in γ‐H2AX foci was observed in PTECs,
   indicating widespread DNA double‐strand breaks (DSBs) (Figure [133]S28,
   Supporting Information). However, treatment with HAND significantly
   reduced the number of γ‐H2AX‐positive cells. In contrast, CND and NAC
   treatments resulted in higher γ‐H2AX levels, underscoring their
   comparatively weaker protection against DSBs. These results
   collectively validate the superior capability of HAND in safeguarding
   DNA integrity under oxidative stress conditions in AKI. Moreover, the
   antioxidant effects of HAND are further underscored by the reduction in
   damage to lipids. As shown in Figure [134]S29 (Supporting Information),
   levels of the lipid peroxidation product products malondialdehyde (MDA)
   and thiobarbituric acid reactive substances (TBARS) in the kidneys of
   the AKI group were significantly elevated, reaching two to three times
   those of the Control group. In the HAND group, these levels returned to
   normal, indicating that the heightened oxidative stress in kidney
   tissues was effectively mitigated by HAND. However, the levels of lipid
   peroxidation products in the NAC group showed no significant reduction,
   and the antioxidant effect of CND was notably weaker compared to the
   HAND group. To sum up, owing to its high‐precision hierarchical
   targeting and outstanding antioxidant properties, HAND demonstrated
   superior therapeutic benefits over CND and NAC in RM‐AKI mice, which
   are underscored by its effective suppression of severe oxidative stress
   in AKI kidneys.

2.4. RNA Sequencing Excavated the Therapeutic Mechanism of HAND

   To further explore the pathological mechanisms of AKI and the
   pharmacological effects of HAND, we conducted RNA sequencing analysis
   (RNA‐Seq) on kidney samples from Control, AKI, and HAND groups. The
   quality and reliability of the data were evaluated through correlation
   analysis (Figure [135] 3A) and principal component analysis (Figure
   [136]S30, Supporting Information). These analyses revealed complete
   separation among samples from different groups, indicating significant
   intergroup differences and high intragroup correlation. Subsequently,
   we screened for differentially expressed genes (DEGs) using the
   criteria of |log[2]FC| > 1 and Q < 0.05. The Venn diagram
   (Figure [137]3B) and volcano plot (Figure [138]3C) revealed 1246
   overlapping DEGs between the AKI group and the other two groups
   (Control group and HAND group). These DEGs underwent enrichment and
   clustering analyses, leveraging the Gene Ontology (GO) and Kyoto
   Encyclopedia of Genes and Genomes (KEGG) databases to uncover gene
   functions and pathways pertinent to our study. The GO enrichment
   analysis indicated that the DEGs between the AKI and HAND groups were
   primarily associated with the regulation of cellular processes such as
   ROS metabolism, ROS response, inflammation response, DNA damage
   response, apoptosis process, mitophagy, and mitochondrial ETC activity
   (Figure [139]3D). KEGG pathway enrichment analysis further revealed
   that these genes were involved in the regulation of pathways related to
   ROS, oxidative phosphorylation, cell cycle, apoptosis, TNF signaling,
   NF‐κB signaling, mitophagy, cytokine‐cytokine receptor interactions,
   cytosolic DNA sensing, and the citric acid cycle (Figure [140]3E).
   These findings underscored the therapeutic potential of HAND in
   alleviating oxidative stress, protecting mitochondria and DNA, and
   suppressing excessive inflammation during AKI, which benefited from its
   precise targeting capabilities and broad‐spectrum antioxidant activity.
   We further illustrated gene expression variations across samples
   through visual clustering heatmaps. As shown in Figure [141]3F, the
   overall gene expression pattern of the HAND group closely resembled
   that of the Control group, whereas the AKI group showed significant
   divergence from the other two, suggesting that HAND effectively
   reversed the gene expression profile alterations induced by AKI.
   Additionally, we constructed clustering heatmaps for five key
   categories—oxidative stress, apoptosis, mitophagy, inflammation, and
   DNA sensing—highlighted from the above enrichment analysis. As depicted
   in Figure [142]3G–K, the gene expression patterns in the HAND group
   more closely aligned with those in the Control group, further
   confirming the regulatory efficacy of HAND on these critical gene
   categories, which will be further explored in detail in the following
   sections.

Figure 3.

   Figure 3
   [143]Open in a new tab

   RNA‐seq revealed the therapeutic mechanisms of HAND. A) Heatmap showing
   the correlation of gene expression from different groups in RNA‐seq of
   mouse kidney tissues. B) Venn diagram of DEGs in RNA‐seq of kidney
   tissues. C) Volcano plot of DEGs between the AKI group and the AKI+HAND
   group. D,E) GO enrichment chord diagram (D) and KEGG pathway enrichment
   bubble chart (E) for DEGs between the AKI and AKI+HAND groups. F–K)
   Cluster heatmap of gene expression across different groups (F) and for
   five functional categories: oxidative stress (G), apoptosis (H),
   mitophagy (I), inflammation (J), and DNA sensing (K). n = 3, |log[2]FC|
   > 1, Q < 0.05.

2.5. HAND Comprehensively Protected Mitochondria

   Mitochondria, as prime examples of endosymbiotic integration,
   coordinate critical cellular and organelle signaling pathways essential
   for adaptive responses and evolutionary processes, making them pivotal
   signaling hubs that determine cell fate.^[ [144]^32 , [145]^33 ^]
   During AKI, electron leakage from the mitochondrial ETC turns
   mitochondria into a primary source of ROS. Simultaneously, these
   mitochondria become the main victims of the resulting toxic ROS, which
   leads to severe mitochondrial dysfunction, disrupted energy metabolism,
   and heightened ROS production, ultimately triggering a ROS storm that
   engulfs the entire PTECs.^[ [146]^34 , [147]^35 ^] Consequently,
   mitochondrial health emerges as a crucial target in AKI therapy,
   serving as a direct indicator of the effectiveness of therapeutic
   interventions, a theme we have examined comprehensively in this
   section.

   HAND exhibited precise mitochondrial targeting and sustained
   antioxidant activity, enabling it to effectively eliminate mtROS in
   situ and protect mitochondria from oxidative damage and its detrimental
   consequences (Figure [148]4A). We conducted a series of experiments to
   validate the comprehensive protective effects of HAND on mitochondrial
   structure, function, oxidative stress status, and homeostatic
   regulation. First, we analyzed the mitochondrial morphology of PTECs
   across different groups using TEM. In contrast to the intact
   mitochondria observed in the control group, those in the AKI group
   exhibited significant structural damage, including swelling, rupture,
   and cristae fragmentation (Figure [149]4B). Following HAND treatment,
   mitochondrial morphology in the AKI mice showed notable restoration,
   while the CND group continued to display morphological abnormalities.
   Next, we investigated the mitochondrial protective mechanisms of HAND
   in HK‐2 cells. DCFH‐DA (ROS probe) detection results showed that HAND
   significantly alleviated the overall increase in intracellular ROS
   levels induced by H[2]O[2] (Figure [150]4C,D). Additionally, mitoSOX
   (mtROS‐specific probe with red fluorescence) fluorescence imaging and
   flow cytometry analysis further revealed the potent mtROS clearance of
   HAND under H[2]O[2] stimulation, underscoring its targeted
   mitochondrial protection (Figure [151]4E,F; Figure [152]S31, Supporting
   Information). Furthermore, mitochondria maintain an electrochemical
   gradient across their inner membrane via proton pumps in the ETC, which
   drives ATP synthesis. Thus, mitochondrial membrane potential (MMP) and
   ATP levels are key indicators of mitochondrial function. We evaluated
   the effect of HAND on MMP under H[2]O[2] stimulation using the JC‐1
   fluorescent dye. As shown in Figure [153]4G,H; Figure [154]S32
   (Supporting Information), H[2]O[2] administration led to a significant
   decrease in MMP, which was indicated by the predominant green
   fluorescence corresponding to JC‐1 monomers in the mitochondria.
   Co‐incubation with HAND effectively mitigated the loss of MMP,
   resulting in the formation of JC‐1 aggregates that emitted red
   fluorescence in mitochondria. Consequently, the restoration of MMP
   ensured the efficiency and integrity of the mitochondrial ETC, leading
   to a notable recovery of ATP production in the HAND group compared to
   the H[2]O[2] group (Figure [155]4I). Moreover, mitophagy is another
   indicator of mitochondrial damage, aiding in the removal of impaired
   mitochondria to prevent further damage.^[ [156]^36 ^] Mitophagy is
   tightly regulated by various signaling pathways and proteins, with the
   PINK1‐Parkin pathway being the most important.^[ [157]^37 ^] PINK1,
   which contains a mitochondrial targeting sequence at its N‐terminus, is
   normally located in mitochondria, where it is cleaved and degraded by
   the proteasome. However, disruptions such as MMP loss disrupt this
   process, leading to PINK1 accumulation on the outer mitochondrial
   membrane. Activated PINK1 recruits Parkin from the cytosol to the
   mitochondria, where Parkin, an E3 ubiquitin ligase, ubiquitinates outer
   mitochondrial membrane proteins, attracting autophagic vesicle proteins
   like LC3B II. During this process, P62/SQSTM1 acts as a bridging
   protein, linking LC3B II with ubiquitinated proteins on the
   mitochondrial surface, promoting the formation of autophagosome1^[
   [158]^12 ^] (Figure [159]4A). TEM images showed that during AKI, high
   levels of mtROS caused severe mitochondrial damage and activated
   mitophagy, resulting in the presence of many autophagosomes in PTECs
   (Figure [160]4J). Western blot (WB) analysis further revealed that the
   expression levels of mitophagy‐related proteins, including PINK1,
   Parkin, P62, and LC3BII/I, were significantly elevated in AKI kidneys,
   indicating high mitophagy activation. HAND treatment reduced the number
   of autophagosomes in PTECs and downregulated the expression of
   mitophagy‐related proteins compared to the AKI group, suggesting
   reduced mitochondrial damage (Figure [161]4K; Figure [162]S33,
   Supporting Information). In summary, HAND provided exceptional,
   comprehensive protection for mitochondria due to its high‐resolution
   mitochondrial localization and remarkable antioxidant capacity, thereby
   preserving the most critical organelles in PTECs during AKI.

Figure 4.

   Figure 4
   [163]Open in a new tab

   Comprehensive mitochondrial protection of HAND. A) Schematic
   illustration of the mitochondrial protecting ability of HAND. B) TEM
   images of mitochondria in PTECs from different treatment groups of
   mice. C,D) Representative DCFH‐DA fluorescence images (C) and
   corresponding fluorescence intensity (D) of HK‐2 cells in different
   treatment groups. E,F) Mito‐SOX fluorescence staining images and
   corresponding fluorescence intensity (F) of HK‐2 cells from different
   treatment groups. G,H) JC‐1 fluorescence staining images (G) and
   corresponding fluorescence intensity (H) of HK‐2 cells from different
   treatment groups. I) ATP production of HK‐2 cells from different
   treatment groups. J) TEM images of mitophagy in PTECs from different
   treatment groups of mice. K) The grayscale analysis of PINK1, Parkin,
   P62, and LC3BII/I of Western blot results from kidney tissues in
   different treatment groups. Data are presented as mean ± SD. One‐way
   ANOVA followed by the SNK test was used for analysis. n = 3, ^* p
   < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001.

2.6. HAND Effectively Inhibited Apoptosis of PTECs

   As central arbiters of cellular function and integrity, mitochondria
   are crucial in mediating cell death signaling, particularly in
   intrinsic apoptosis (also known as the mitochondrial pathway of
   apoptosis)^[ [164]^38 ^] (Figure [165]5A). In the pathological state of
   AKI, mtROS damages mitochondrial lipids and proteins, leading to
   compromised membrane integrity and inducing mitochondrial outer
   membrane permeabilization (MOMP). MOMP, the initiating event of
   intrinsic apoptosis, is primarily mediated by pro‐apoptotic members of
   the Bcl‐2 family, such as Bax. Under oxidative stress, Bax accumulates
   on the mitochondrial outer membrane, forming pores that enable the
   release of essential apoptotic factors, such as cytochrome c (Cyt c),
   into the cytosol. Once released, Cyt c promptly activates the caspase
   cascade, with Caspase‐3 serving as the principal executor. Upon
   activation through cleavage, it degrades various cellular components,
   thereby driving apoptosis.^[ [166]^39 ^] As shown in the WB results
   (Figure [167]5B–G), expression levels of Bax and Cytosolic‐Cyt c (C‐Cyt
   c) were significantly upregulated in the kidneys of the AKI group,
   while the expression of the anti‐apoptotic protein Bcl‐2 was
   downregulated, indicating the activation of intrinsic apoptosis.
   Furthermore, the caspase cascade was effectively triggered, with
   Caspase‐3 being cleaved and downregulated, which corresponded to a
   significant increase in Cleaved‐Caspase‐3 levels, confirming the
   execution of intrinsic apoptosis. Importantly, following HAND
   treatment, the expression of mitochondrial pro‐apoptotic proteins,
   including Bax, C‐Cyt c, and Cleaved‐Caspase‐3, was significantly
   reduced in the kidneys, while Bcl‐2 levels were markedly increased
   compared to the AKI group. Moreover, DNA fragmentation induced by
   Caspase‐activated DNase or ROS is a critical hallmark of apoptosis,
   leading to chromatin fragmentation and condensation, ultimately
   resulting in irreversible cell death. We then validated the
   anti‐apoptotic effect of HAND through TUNEL staining, which identifies
   and labels DNA strand breaks. As shown in Figure [168]5H,I, the number
   of TUNEL‐positive cells in the renal tubules of the AKI group was
   significantly increased compared to the Control group, with an
   apoptotic cell rate of 37%. Notably, HAND treatment significantly
   reduced the proportion of TUNEL‐positive cells, demonstrating superior
   anti‐apoptotic efficacy compared to CND without PTEC targeting.
   Subsequently, the anti‐apoptotic effect of HAND was further validated
   in HK‐2 cells through Annexin V‐FITC/PI flow cytometry. Annexin V
   specifically binds to phosphatidylserines, which translocate to the
   outer leaflet of the cell membrane during early apoptosis, while
   propidium iodide (PI) penetrates only late apoptotic cells with
   compromised membranes to bind to DNA. Under H[2]O[2] stimulation, HK‐2
   cells exhibited a high apoptosis rate, with the proportion of early and
   late apoptotic cells reaching 49.2%, which was significantly reduced to
   14.9% following HAND treatment (Figure [169]5J; Figure [170]S34,
   Supporting Information). Additionally, WB analysis confirmed that HAND
   effectively down‐regulated the expression of pro‐apoptotic proteins in
   HK‐2 cells induced by H[2]O[2] (Figure [171]S35, Supporting
   Information). Collectively, these findings demonstrated the superior
   anti‐apoptotic protective effects of HAND in both in vivo and in vitro
   models.

Figure 5.

   Figure 5
   [172]Open in a new tab

   HAND effectively inhibited intrinsic apoptosis. A) Schematic
   illustration of HAND inhibiting intrinsic apoptosis. B–G) Western blot
   analysis of apoptosis‐related protein expression levels in mouse kidney
   tissues from different treatment groups and grayscale analysis of Bax
   (C), Bcl‐2 (D), Caspase‐3 (E), Cleaved‐Caspase 3 (F), and C‐Cyt c (G).
   (H‐I) TUNEL fluorescence staining results (H) and quantification of
   TUNEL‐positive cells (I) in mouse kidneys from different treatment
   groups. J) Annexin‐V/PI flow cytometry results in HK‐2 cells from
   different treatment groups. Data are presented as mean ± SD. One‐way
   ANOVA followed by the SNK test was used for analysis. n = 3, ^* p
   < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001.

2.7. HAND‐Inhibited cGAS‐STING‐Mediated Sterile Inflammation

   Excessive and unresolved sterile inflammation during AKI exacerbates
   tissue damage and accelerates the progression of renal fibrosis.^[
   [173]^40 ^] Because in the absence of pathogens, the indiscriminate
   attacks by immune cells are mainly directed against endogenous
   tissues.^[ [174]^41 ^] Notably, mitochondria and the nucleus, beyond
   their central role in mediating cell death during AKI, also serve as
   key regulators of the inflammatory response, significantly influencing
   inflammatory signaling through the release of damaged DNA. The precise
   targeting capability of HAND to mitochondria and the nucleus, along
   with its potent mtROS‐scavenging efficacy, offers HAND a significant
   advantage in modulating inflammation during AKI (Figure [175]6A). The
   activation of the cGAS‐STING DNA‐sensing pathway has been increasingly
   recognized as a contributor to AKI‐related sterile inflammation,
   initiating a cascade of biological effects by activating innate immune
   responses. cGAS, a cytosolic DNA sensor, detects double‐stranded DNA
   (dsDNA) and catalyzes the synthesis of the second messenger cGAMP.
   Thus, the aberrant presence of cytosolic dsDNA is amplified by cGAMP
   and transmitted to the ER membrane receptor STING. This interaction
   induces conformational changes in STING, triggering downstream
   signaling cascades, including the activation of IRF3 and the
   phosphorylation and nuclear translocation of NF‐κB.^[ [176]^11 ^] In
   eukaryotic cells, the nucleus and mitochondria are the exclusive
   compartments for DNA, both of which undergo intense oxidative stress
   during AKI, leading to the production of substantial oxidative‐DNA
   fragments.

Figure 6.

   Figure 6
   [177]Open in a new tab

   HAND inhibited cGAS‐STING mediated sterile inflammation. A) Schematic
   illustration of HAND inhibiting cGAS‐STING mediated sterile
   inflammation. B) Fluorescence staining results of dsDNA (green), TOM20
   (red), and DAPI (blue) in HK‐2 cells from different treatment groups.
   C) WB analysis of cGAS‐STING related protein expression in HK‐2 cells.
   D) Fluorescence staining results of dsDNA (green), TOM20 (red), and
   DAPI (blue) in kidneys from different treatment groups. E) Levels of
   2′‐3′cGAMP in kidneys from different treatment groups. F) IHC results
   of cGAS and STING in kidneys from different treatment groups. G–J)
   Levels of chemokines CXCL1 (G), CCL2 (H), and inflammatory cytokines
   TNF‐α (I), and IL‐6 (J) in kidneys from different treatment groups. K)
   IHC results of F4/80 and Ly‐6G in kidneys from different treatment
   groups. Data are presented as mean ± SD. One‐way ANOVA followed by the
   SNK test was used for analysis. n = 3, ^* p < 0.05, ^** p < 0.01, ^***
   p < 0.001, ^**** p < 0.0001.

   As shown in Figure [178]6B, H[2]O[2] stimulation significantly reduced
   the colocalization of dsDNA with the nucleus and mitochondria in HK‐2
   cells. Further quantitative results showed that the level of mtDNA in
   the cytoplasm of the H[2]O[2] group increased significantly, while the
   mtDNA level in mitochondria decreased, which proved that the contents
   were released due to membrane rupture during mitochondrial injury
   (Figure [179]S36, Supporting Information). The released dsDNA then
   strongly sensitized the cGAS/STING pathway, evidenced by the
   significant upregulation of cGAS and STING in HK‐2 cells, as well as
   the activation of downstream IRF3 and phosphorylation of NF‐κB
   (Figure [180]6C). Notably, HAND treatment effectively mitigated dsDNA
   leakage and subsequent cGAS‐STING activation by mitigating DNA
   oxidative damage (Figure [181]6B,C; Figures [182]S36,S37, Supporting
   Information). We further validated the modulation of HAND on
   cGAS‐STING‐mediated inflammatory response in AKI mice. PTECs play a
   central role in AKI‐induced damage, serving as the initial site of
   injury that propagates signals to induce an inflammatory response. As
   shown in Figure [183]6D, the colocalization of dsDNA with TOM20 and
   DAPI in the renal cortex was significantly reduced in the AKI group,
   with dsDNA dispersed into the extracellular space. cGAS was then highly
   sensitized, generating a large amount of second messenger to activate
   STING, as evidenced by the high levels of 2′‐3′ cGAMP in renal tissue
   (Figure [184]6E) and the elevated expression of cGAS and STING in
   damaged proximal tubules (Figure [185]6F). Correspondingly, downstream
   pro‐inflammatory signals of the cGAS/STING pathway were activated
   (Figure [186]S38, Supporting Information), mediating the expression and
   secretion of various inflammatory cytokines (such as TNF‐α, IL‐1β,
   IL‐6) and chemokines (CXCL1, CXCL2, CCL2) (Figure [187]6G–J; Figure
   [188]S39, Supporting Information). Among these, CXCL1 and CXCL2
   primarily recruit neutrophils, while CCL2 mainly recruits macrophages
   and other monocytes. As a result, in contrast to the small number of
   resident immune cells in the Control group, the AKI group displayed
   extensive infiltration of neutrophils and macrophages in the renal
   tubular interstitium. These recruited immune cells further released
   inflammatory mediators such as cytokines, chemokines, ROS, and
   proteases, perpetuating a vicious cycle that fosters chronic
   inflammation. Such persistent and extensive damage to PTECs would
   ultimately deplete the functional epithelium, resulting in the
   formation of scar tissue formation and irreversible fibrosis.
   Fortunately, the potent ROS‐scavenging ability of HAND effectively
   disrupted the vicious cycle of oxidative stress, inflammation, and
   tissue damage, as evidenced by the significantly reduced levels of
   inflammatory mediators and decreased immune cell infiltration in the
   HAND group, demonstrating superior immunomodulatory effects compared to
   CND (Figure [189]6K). Furthermore, to substantiate these findings, we
   employed a conditioned medium model to investigate the interactions
   between injured PTECs and immune cells under oxidative stress. HK‐2
   cells were first treated with H[2]O[2] to induce oxidative damage and
   activation of the cGAS‐STING pathway, after which the conditioned
   medium from these HK‐2 cells was collected and used to stimulate RAW
   264.7 macrophages (Figure [190]S40 (A), Supporting Information). In
   this system, macrophages exhibited significant activation, as evidenced
   by the upregulation of COX‐2 and iNOS, along with elevated levels of
   pro‐inflammatory cytokines (TNF‐α, IL‐1β, IL‐6), which further
   exacerbated inflammation. However, HAND treatment to
   H[2]O[2]‐stimulated HK‐2 cells resulted in a marked reduction in COX‐2
   and iNOS expression in RAW 264.7 macrophages, as well as significantly
   lower levels of pro‐inflammatory cytokines compared to the CND group
   (Figure [191]S40 (B), Supporting Information). The enhanced
   anti‐inflammatory effect is primarily attributed to HAND's ability to
   protect HK‐2 cells from oxidative damage, underscoring the therapeutic
   advantage of HAND in mitigating oxidative stress‐induced inflammation
   by preserving cellular integrity and minimizing damage‐associated
   signals that drive excessive immune responses. Taken together, the
   above findings suggested that HAND could successfully halt the
   pathological progression of AKI by precisely eliminating the source of
   ROS and inhibiting the cGAS‐STING‐mediated sterile inflammation.

2.8. HAND Exhibited Excellent Biocompatibility

   Finally, we evaluated the biocompatibility of HAND in vivo (Figure
   [192]7A,B). Following the administration of a high dosage (10 mg kg^−1,
   5 times the therapeutic dose) for 24 h, the lungs, liver, heart,
   spleen, and kidneys were harvested for HE staining. Compared with the
   PBS group, HAND did not cause any damage to these vital organs
   (Figure [193]7C). Additionally, hematological parameters, including
   white blood cells, red blood cells, and platelets, showed no
   significant differences compared to the PBS group (Figure [194]7D;
   Figure [195]S41, Supporting Information). Liver function indicators
   (AST, ALT) (Figure [196]7E), kidney function markers (BUN, CRE)
   (Figure [197]7F), and inflammatory cytokine levels (TNF‐α, IL‐6, IL‐1β)
   (Figure [198]7G) also remained stable, indicating that HAND did not
   adversely affect liver or kidney function and did not provoke an
   inflammatory response. Moreover, during long‐term administration of
   HAND in mice (therapeutic dosage once a week for 4 weeks), no
   significant changes were observed in major organ histology
   (Figure [199]7C), hematological parameters (Figure [200]7H; Figure
   [201]S42, Supporting Information), liver and kidney function
   (Figure [202]7I,J), or inflammatory markers (Figure [203]7K). These
   results collectively demonstrated the excellent biosafety of the
   naturally derived HAND.

Figure 7.

   Figure 7
   [204]Open in a new tab

   HAND exhibited excellent biocompatibility. A,B) Schematic illustration
   of short‐term and long‐term treatment protocol for biosafety evaluation
   of HAND. C) Representative H&E‐stained images of major organs from KM
   mice after short‐term (left) and long‐term (right) administration of
   HAND or 1× PBS. D–G) Hematological parameters (D), liver function
   indicators (E), kidney function indicators (F), and inflammatory
   cytokine levels (G) of short‐term treated mice in different treatment
   groups. H–K) Hematological parameters (H), liver function indicators
   (I), kidney function indicators (J), and inflammatory cytokine levels
   (K) of long‐term treated mice in different treatment groups. Data are
   presented as mean ± SD. n = 6.

3. Discussion

   Over the past two decades, the proportion of deaths attributed to
   kidney disease has steadily increased, with its global prevalence
   surpassing that of any other major non‐communicable diseases, including
   cardiovascular diseases, cancer, chronic respiratory diseases, and
   diabetes.^[ [205]^42 ^] Patients with kidney disease are at high risk
   for comorbidities that contribute not only to elevated mortality rates
   but also to a significant symptom burden and substantial treatment
   costs.^[ [206]^43 ^] Given the urgent clinical demand, the development
   of therapeutics for kidney diseases holds significant promise but also
   faces substantial challenges. Recent advancements in genetics,
   bioinformatics, and pharmacodynamics have facilitated deeper
   exploration into g the pathophysiology of AKI and the development of
   new drugs. However, the diverse etiologies and heterogeneous
   pathological processes of AKI complicate efforts to create effective
   treatments targeting gene regulation or specific downstream molecules,
   which may fail to rapidly halt acute damage progression. Because the
   oxidative stress‐PTECs injury‐inflammation vicious cycle is a key
   factor in almost all types of AKI, which interferes with numerous
   cellular processes and spreads rapidly like a snowball to cause a swift
   decline in renal function.^[ [207]^16 ^] This cycle begins with the
   excessive production of ROS in response to various stressors, leading
   to oxidative stress that directly damages PTECs. Once injured, PTECs
   release pro‐inflammatory cytokines that intensify the inflammatory
   response and attract immune cells to the injury site. The influx of
   these inflammatory cells, in turn, generates additional ROS, creating a
   feedback loop that perpetuates cellular damage and inflammation. As the
   cycle continues, impairs vital cellular functions, including
   mitochondrial activity and signaling pathways, ultimately resulting in
   rapid renal function decline. Moreover, the increasing emphasis on
   precision medicine underscores the need for more organized, rational,
   and high‐resolution targeting strategies.^[ [208]^44 ^] Non‐selective
   drug distribution often necessitates higher therapeutic doses and leads
   to unavoidable off‐target toxic side effects.^[ [209]^45 ^] These
   challenges significantly complicate the development of AKI therapies,
   highlighting the urgent need for more precise and efficient treatment
   strategies.

   In this context, we specifically target the primary source of injury in
   AKI—excessive toxic ROS—and designed an FA‐optimized nanomedicine,
   HAND, that aligns with molecular preferences for targeted tissues