NOP2/Sun RNA methyltransferase family member 4 (NSUN4)-mediated 5‑methylcytosine (m5C) RNA modification has been implicated in diabetes‑related disorders. The authors sought to define the role of NSUN4 in diabetic nephropathy (DN) and to elucidate the molecular mechanism by which NSUN4 and m5C contribute to renal injury and mitochondrial dynamics in DN.
The study combined in vivo and in vitro approaches. A streptozotocin (STZ)‑induced DN mouse model was used to study renal injury in vivo. An in vitro cellular DN model used human proximal tubular HK-2 cells exposed to high glucose (HG). The investigators measured expression of NSUN4, SMURF1, CAMK1, and mitochondrial proteins using qRT‑PCR, western blot, and immunohistochemistry. Global m5C levels were assessed by LC‑MS/MS and dot blot. Renal injury endpoints included serum BUN, creatinine, urinary NAG, and albumin/creatinine ratio, plus histological scoring (H&E, PAS). Mitochondrial function and cell state were evaluated with TUNEL, DHE, MitoSOX Red, EdU, JC‑1, ATP assay, and TEM. RNA immunoprecipitation, co‑immunoprecipitation, and ubiquitination assays were applied to define interactions among SMURF1, ALYREF, and CAMK1.
The authors report increased m5C modification in DN patient samples, STZ‑induced DN mouse kidneys, and HG‑treated HK-2 cells. Among m5C writer enzymes examined, NSUN4 expression was elevated in DN contexts as measured by qRT‑PCR and western blot. Representative sample sizes reported in figure legends included DN patients (n = 10), STZ model mice (n = 8), and cell experiments (n = 3).
Conditional knockout of NSUN4 (NSUN4‑cKO) in mice attenuated STZ‑induced renal injury. NSUN4‑cKO mice showed reduced serum BUN and creatinine, lower urinary NAG and albumin/creatinine ratio, and improved histological scores for tubular and glomerular damage compared with STZ‑treated wild‑type controls. NSUN4 deletion reduced TUNEL‑positive cell apoptosis and reactive oxygen species (ROS) production (DHE staining) in renal tissue. Transmission electron microscopy demonstrated fewer fragmented mitochondria in NSUN4‑cKO kidneys. Western blot analyses indicated shifts in mitochondrial dynamics proteins consistent with decreased fission and increased fusion (changes in Mfn1, Mfn2, Fis1, p‑Drp1 Ser616, and Drp1).
In HG‑treated HK-2 cells, shRNA‑mediated silencing of NSUN4 reduced NSUN4 mRNA and protein, decreased global m5C, and mitigated mitochondrial dysfunction and cell death. NSUN4 knockdown increased markers of mitochondrial fusion (Mfn1, Mfn2), decreased fission markers (Fis1, p‑Drp1 Ser616), stabilized mitochondrial membrane potential (JC‑1), lowered mitochondrial ROS (MitoSOX Red), preserved cellular ATP, increased cell proliferation (EdU), and reduced apoptosis (TUNEL). These findings indicate that reducing NSUN4 restrains HG‑induced mitochondrial fragmentation and tubular cell injury.
Mechanistic experiments connected NSUN4‑dependent m5C modification to posttranscriptional regulation of SMURF1. NSUN4‑mediated m5C increased SMURF1 mRNA stability through interaction with the m5C reader ALYREF (demonstrated by RNA immunoprecipitation). Thus, elevated NSUN4 and m5C lead to higher SMURF1 expression in DN models. The authors used molecular assays to show that altering NSUN4 levels changes SMURF1 transcript stability consistent with m5C/ALYREF‑dependent regulation.
SMURF1 was identified as a critical downstream effector. Overexpression of SMURF1 promoted mitochondrial fission and apoptosis, whereas SMURF1 depletion produced protective effects. Biochemically, SMURF1 facilitated ubiquitination and degradation of CAMK1, linking SMURF1 upregulation to reduced CAMK1 protein levels. Loss of CAMK1 was associated with increased mitochondrial fragmentation and cell death in the DN context.
Functional rescue studies supported the pathway: SMURF1 overexpression reversed the protective effects of NSUN4 knockdown on mitochondrial fission and apoptosis in HK-2 cells. Conversely, knockdown of CAMK1 (sh‑CAMK1) eliminated the protective effects of SMURF1 depletion, demonstrating that CAMK1 is a necessary downstream mediator of SMURF1’s impact on mitochondrial dynamics. In vivo, NSUN4 downregulation alleviated STZ‑induced renal injury by reducing mitochondrial fission via the SMURF1/CAMK1 axis.
The study concludes that NSUN4‑dependent m5C modification promotes DN progression by stabilizing SMURF1 mRNA (via ALYREF), increasing SMURF1 protein, and thereby enhancing ubiquitination and degradation of CAMK1, which leads to increased mitochondrial fission and renal tubular cell apoptosis. Genetic or molecular reduction of NSUN4 disrupts this axis, diminishes mitochondrial fragmentation, and attenuates renal injury in STZ mice and HG‑treated HK-2 cells. The NSUN4/SMURF1/CAMK1 pathway provides mechanistic insight into DN pathogenesis and identifies nodes that could be explored for therapeutic targeting.
Note: All findings, experimental approaches, and reported sample sizes are drawn from the source article. The original figures and figure legends contain additional experimental detail and specific quantitative results.