Acta Diabetologica

https://doi.org/10.1007/s00592-021-01736-4

ORIGINAL ARTICLE

Klotho protects against diabetic kidney disease via AMPK‑

and ERK‑mediated autophagy
Meng Xue1 · Feng Yang2 · Ying Le1 · Yanlin Yang2 · Bingsen Wang1 · Yijie Jia2 · Zongji Zheng2 · Yaoming Xue2

Received: 9 March 2021 / Accepted: 6 May 2021

© Springer-Verlag Italia S.r.l., part of Springer Nature 2021

Abstract
Background Diabetic kidney disease (DKD) is a serious complication of diabetes mellitus and results in serious public
health problems. Although a great number of studies have been performed to elucidate the mechanisms of this disease, these
mechanisms remain largely unknown.
Methods Cell and animal models were first constructed using human renal proximal tubule cells stimulated by high glucose
(HG) and mice induced by streptozotocin (STZ). After Klotho overexpression, Klotho expression was assessed by RT-PCR
and western blot, immunofluorescence; autophagy and AMPK/ERK proteins were confirmed using western blot or immu-
nohistochemical assay; the autophagosomes were observed by transmission electron microscope; the pathological structure,
fibrosis, polysaccharides and glycogen of kidney were evaluated by H&E staining, Masson staining and PAS staining.
Results We first confirmed that Klotho expression and autophagic activity were reduced in DM mice and HG-induced
human renal proximal tubule cells. Besides, overexpression of Klotho could significantly enhance autophagy and AMPK
and ERK1/2 activities in vivo and in vitro, which also could be abolished by selective AMPK inhibitor and ERK activator.
Moreover, we proved that Klotho could inhibit hyperglycemia-induced renal tubular damage.
Conclusion In summary, our results proved that Klotho improved renal tubular cell autophagy via the AMPK and ERK path-
ways and played a role in renal protection. These findings provide new insight into the mechanism of Klotho and autophagy
in DKD.

Keywords Diabetic kidney disease · Klotho · Autophagy · AMPK · ERK

Introduction pathophysiology of DKD occurrence and development is

Diabetic kidney disease (DKD) is a microvascular compli-
cation that develops in approximately 30–40% of patients
with type 1 or 2 diabetes mellitus (DM) [1]. With increas-
ing prevalence and a lack of effective therapies, DKD has
become the leading cause of end-stage renal disease and a
serious public health problem worldwide [2, 3]. Since the
Managed by Giuseppe Pugliese.
* Yaoming Xue
yaomingxue@126.com
1
Department of Endocrinology and Metabolism, The Second
Clinical Medical College, Shenzhen People’s HospitalJinan
UniversityThe First Affiliated Hospital, Southern University
of Science and Technology), Shenzhen 518020, China
2
Department of Endocrinology and Metabolism, Nanfang
Hospital, Southern Medical University, Guangzhou 510515,
China
largely unknown, standard therapies can slow DKD progres-
sion but cannot arrest or reverse it. Therefore, studies to
elucidate the pathogenesis of DKD are crucial in developing
new targeted treatments for the disease.
Autophagy is an evolutionarily conserved cellular self-
consumption process and a crucial recycling system that
degrades aggregated proteins and dysfunctional organelles.
Cellular activity occurs at baseline levels in various cells to
preserve cytoplasmic homeostasis and plays an important
role in numerous physiological activities, including apopto-
sis, necrosis, nutrient metabolism, development, immunity
and aging [4]. On the other hand, autophagy dysregulation
contributes to the pathogenesis of a variety of diseases, such
as cancers, infectious and inflammatory diseases, metabolic
diseases, cardiovascular diseases and kidney diseases [5]. In
the context of both type 1 and type 2 diabetes, autophagy is
attenuated in tubular cells, which is associated with hyper-
trophy [6]. In studies with streptozotocin (STZ)-induced

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diabetic mice, Wistar fatty rats with type 2 diabetes and
kidney biopsies from type 2 diabetic patients, consistent
autophagy impairment was observed [5].
It is well known that autophagy is regulated by signaling
pathways that sense nutrients [7, 8]. It has been suggested
that dysregulation of these nutrient sensing pathways is
associated with the pathogenesis of DKD [9]. As a mas-
ter regulator of cellular metabolism that integrates diverse
pathways associated with energy and metabolism, the evo-
lutionarily conserved heterotrimeric protein complex AMP-
activated protein kinase (AMPK) is activated under condi-
tions of nutrient/energy depletion [10]. Therefore, alterations
in the AMPK signaling pathway under diabetic conditions
may inhibit autophagy, resulting in DKD and subsequently
exacerbating renal injury [11]. As a critical member of the
mitogen-activated protein kinase (MAPK) family, extracel-
lular signal-regulated kinase (ERK) is well recognized for
its regulatory role in cell proliferation, survival and differ-
entiation [12]. Recently, ERK signaling pathways have been
shown to play important roles in energy homeostasis, such
as regulating glycolysis and adipogenesis. Moreover, there
is increasing evidence that the ERK pathway is an important
contributor to type 2 diabetes and the subsequent complica-
tion of diabetic cardiomyopathy [13].
Klotho was originally discovered as an antiaging factor
[6] and is primarily expressed in kidneys, particularly in
renal distal tubular cells [14]. This protein acts in an endo-
crine/paracrine manner and plays multiple roles in oxidation
suppression, lifespan extension, insulin sensitivity regula-
tion and stem cell preservation [15]. Klotho expression has
been shown to be significantly decreased in renal tubules of
both humans and animals with DKD [12]. Additionally, a
decrease in plasma Klotho was suggested to be indicative
of DKD progression in type 2 diabetic patients [16]. These
findings have led to increasing interest in the role of Klotho
in DKD.
In the present study, we investigated the role of Klotho
in DKD and further explored the underlying mechanism.
For the first time, we noted a close relationship between
Klotho and autophagy in diabetic nephropathy. Specifically,
an increase in Klotho expression promoted autophagy via
AMPK activation and ERK pathway inhibition. Our find-
ings reveal new insight into the mechanism of Klotho and
autophagy in DKD.
Materials and methods
Animal experiment
Male C57BL/6 J mice aged 7 weeks were purchased from
the Guangdong Medical Laboratory Animal Center. All
experimental animal procedures in this study were approved
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Acta Diabetologica
by the Institutional Animal Care and Use Committee of the
Laboratory Animal Center, Nanfang Hospital, Southern
Medical University, Guangzhou, China (certificate number:
SYXK2015-0056). One week before the experiment, all
mice were housed in a specific pathogen-free environment
with controlled temperature (20–26 °C), humidity (30%-
70%) and light (12-h light/dark cycle).
Animal groups
The mice were randomized into two treatment groups: con-
trol and high-fat diet (HFD)/STZ. The mice had free access
to water and were continuously fed either a normal chow
diet or an HFD containing 60% fat. After 4 weeks of an HFD
to induce obesity and a prediabetic status, HFD/STZ mice
were intraperitoneally administered 35 mg/kg STZ. Concur-
rently, control mice received the sodium citrate vehicle. One
week after STZ treatment, blood glucose >16.7 mmol/L was
defined as successful modeling. Then, each treatment group
was randomly subdivided into 3 groups (n = 6/group): the
1-week, 6-week and 18-week groups. The mice were eutha-
nized at 1, 3 and 18 weeks after STZ or vehicle administra-
tion. pCMV and pCMV-Klotho plasmids were purchased
from Qiagen. Klotho overexpression was induced by inject-
ing the pCMV-Klotho plasmid via the tail vein in the HFD/
STZ group, and pCMV was injected as a control. After an
additional 18 weeks of HFD feeding, the mice were eutha-
nized, and the kidneys were harvested. During the experi-
ment, the health and certain behaviors of the mice were
monitored daily, and a 20% loss of body weight was con-
sidered to be the humane end point for euthanasia. Then,
the mice were put into the euthanasia chamber containing
­CO2 at a rate of 20% chamber volume/min. When the mice
lost consciousness and stopped breathing, the CO2 flow
continued for 1 min. The mice were then killed by cervical
dislocation [17].
Cell culture and transfection
Human renal proximal tubule cells (Neuromics, Cat. No.
SC00A1-PTEC) were obtained from an agent (Biopike,
Beijing, China) and maintained in DMEM/F12 (Gibco, cat.
no. 11330–032) with 10% fetal bovine serum (FBS; Gibco).
Human renal proximal tubule cells were seeded in 12-well
plates (5 × ­104 cells/well). Following overnight incubation,
the cells were exposed to normal (5 mM) or high (30 mM)
concentrations of glucose for 24 h. Some of the cells were
harvested and stored at -80 °C until analysis.
Real‑time PCR (RT‑PCR)
Total RNA was extracted from human renal proximal tubule
cells using TRIzol reagent (Invitrogen, CA, USA), and the
Acta Diabetologica
quality was examined by a NanoDrop ND-1000 spectropho-
tometer. Then, cDNA was generated using 1 μg of RNA
with a reverse transcription kit (Takara, Dalian, China). RT-
PCR was performed with a Roche 480 Thermal Cycler using
a SYBR Green qPCR kit (Takara). The following primer
sequences were used: human Klotho: forward 5’-GAA​
AAA​TGG​CTT​CCC​TCC​TT-3’ and reverse 5’-ACA​ACT​
CCC​CAA​GCA​AAG​TC; murine Klotho forward 5’-TTG​
GGT​CAC​TGG​GTC​AAT​CTC-3’ and reverse 5’-CCG​GCA​
CGA​TAG​GTC​ATG​TT-3’; human β-actin: forward 5’-GCA​
AGT​GCT​TCT​AGG​CGG​AC-3’ and reverse 5’-AAG​AAA​
GGG​TGT​AAA​ACG​CAGC-3’; and murine β-actin: forward
5’-GGC​TGT​ATT​CCC​CTC​CAT​CG-3’ and reverse 5’-CCA​
GTT​GGT​AAC​AAT​GCC​ATGT-3’. The relative mRNA lev-
els were calculated by the ­2−ΔΔCt method.
Western blotting
Using RIPA lysis buffer (Beyotime, Nantong, China), the
collected cells were lysed, and the kidneys were homog-
enized for protein extraction. After determining the protein
concentrations using a BCA assay (Beyotime), the proteins
were denatured at 95 °C. Equal amounts of protein were
loaded into 10% SDS-PAGE gels, electrophoresed, trans-
ferred to PVDF membranes, blocked with 5% nonfat milk
and then incubated with primary antibodies against Klotho,
GAPDH, LC3B, AMPK, p-AMPK, ERK1/2 and p-ERK1/2
(Abcam, Cambridge, UK) at 4 °C overnight. After being
washed with 0.2% TBST, the membranes were incubated
with peroxidase-conjugated secondary antibody at room
temperature for 1 h. The images were acquired using an
Odyssey infrared imaging system. Gel-Pro Analyzer soft-
ware was used for densitometric analysis. The immunore-
activity was visualized with enhanced chemiluminescence
and calculated with ImageJ software (NIH).
Histology and immunohistochemical staining
The collected kidneys were fixed in 10% paraformaldehyde
(PFA). After being dehydrated with increasing concentra-
tions of ethanol and hyalinized with xylene, the tissues were
embedded in paraffin and cut into serial sections at a thick-
ness of 5 μm. Then, the sections were dewaxed in xylene
and rehydrated in graded ethanol solutions. Some sections
were stained with hematoxylin and eosin (H&E) for histo-
pathological examination. Some sections underwent Mas-
son’s trichrome and periodic acid-Schiff (PAS) staining for
morphological assessments. Some sections were subjected
to immunohistochemistry (IHC). Briefly, the sections were
heated in a citrate solution for antigen retrieval and blocked
with 2% bovine serum albumin (BSA) at room temperature
for 30 min. Then, the sections were incubated with primary
antibodies against LC3B (Abcam, Cambridge, UK) at room
temperature for 1 h. After being incubated with secondary
antibodies (Beyotime) at room temperature for 30 min, the
sections were exposed to DAB and observed under a light
microscope (× 200, Olympus Corporation, Tokyo, Japan).
The positive cell rate and degree of immunohistochemical
staining were assessed by two pathologists. The positive
cells are brown-yellow in color or granular and brown. The
score was calculated based on the percentage of positive
cells and the staining intensity. A score of 0–3 was consid-
ered negative, and a score of > 3 was considered positive.
Transmission electron microscope (TEM)
The ultrastructure of human renal proximal tubule cells
was observed with TEM to assess autophagy. After treat-
ment, the cells were harvested and fixed in 0.1 M cacodylate
buffer containing 2.5% glutaraldehyde (Sigma) and post-
fixed in 1% osmium tetroxide. After treatment with 0.5%
tannic acid and 1% sodium sulfate, the cells were cleared in
2-hydroxypropyl methacrylate, embedded in Ultracut (Leica,
Wetzlar, Germany), sliced into sections with a thickness of
60 nm and stained with uranyl acetate and lead citrate. The
ultrathin sections were then examined using a JEM-1230
TEM (JEOL, Tokyo, Japan).
Immunofluorescence analysis
After treatment, the cells were fixed in 4% PFA for 15 min
at room temperature and blocked with 5% BSA for 30 min
at room temperature. The cells were incubated with anti-
Klotho antibodies at 4 °C overnight. Subsequently, the cells
were incubated with secondary antibodies for 1 h. The cells
were counterstained with DAPI (5 μg/ml; Sigma-Aldrich)
for 5 min and observed under a laser confocal microscope
(Leica, Germany).
Statistical analysis
All results are presented as the mean ± SD. A two-tailed Stu-
dent’s t test was used for comparisons between two inde-
pendent groups. One-way ANOVA was used to compare
three or more independent groups. P values less than 0.05
were considered significant.
Results
Expression of Klotho in the kidneys of DM mice
To confirm the role of Klotho in the pathogenesis of DKD,
we first evaluated Klotho expression in kidney samples from
DM mice. As shown in Fig. 1, Klotho expression was sig-
nificantly decreased at the transcriptional and translational
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Fig. 1  Klotho expression was decreased in the kidneys of DM mice.
(a) Levels of Klotho mRNA and (b) Klotho protein were assessed in
healthy mice and DM mice treated for different times. ImageJ soft-
levels in DM mice compared to control mice. Furthermore,
the protein levels decreased in a time-dependent manner
(Fig. 1b). These data indicated that the changes in Klotho
expression might be associated with the development of
DKD.
Autophagic activity in the kidneys of DM mice
LC3 conversion is a well-accepted biomarker of
autophagic activity. In the present study, we determined
the levels of LC3 by western blotting and IHC. Western
blotting was used to examine the LC3 expression and
Fig. 2  LC3 conversion was increased in the kidneys of DM mice. (a)
Western blot analysis of LC3-II/I expression in the kidney cortex at
different treatment times. ImageJ software was used to quantify the
relative protein expression. (b) Immunohistochemical staining of
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Acta Diabetologica
ware was used to quantify the relative protein expression. The data
are the means ± SEM. n ≥ 3 per group. *p < 0.05, **p < 0.01 and
***p < 0.001 compared to the control group
conversion in the samples from mice after 1, 6 or 18 weeks
of treatment. As shown in Fig. 2a, the LC3-II/LC3-I ratio
was significantly higher in DM mice after 1 week of
treatment than in control mice. In contrast, the LC3-II/
LC3-I ratio was significantly decreased after both 6 and
18 weeks of STZ treatment. The samples from the mice
after 18 weeks of treatment were additionally analyzed
by IHC. As expected, we observed a consistent decrease
in LC3 in the renal cortex in DM mice (Fig. 2b). These
results indicated that autophagy declined in DM mice after
long-term treatment with STZ.
LC3 in the kidney cortex of healthy and DM mice after 18 weeks.
Original magnification: × 400, scale bar = 100 μm. The data are the
means ± SEM; n ≥ 3 per group; *p < 0.05; and **p < 0.01 compared to
the control group
Acta Diabetologica

Klotho expression in human renal proximal tubule
cells after high‑glucose stimulation
Due to their high sensitivity to diabetes-associated metabolic
factors, renal tubules are important in the pathogenesis and
development of DKD [18]. Human renal proximal tubule
cells were used in this study to investigate tubular expression
of Klotho after glucose stimulation. As shown in Fig. 3a,
the mRNA levels of Klotho were significantly decreased in
human renal proximal tubule cells exposed to high glucose
for 48 h. Consistently, western blotting and immunofluo-
rescence staining showed a decrease in the protein level of
Klotho (Fig. 3b–c). These data suggested a potential cor-
relation between aberrant Klotho levels and tubular changes.
proximal tubule cells compared to vector-transfected human
renal proximal tubule cells (Fig. 4b). Moreover, the p-AMPK
level was increased, but p-ERK expression was decreased in
human renal proximal tubule cells treated with high glucose
after transfection with pcDNA3.1-Klotho compared to those
of human cells treated with high glucose after transfection
with pcDNA3.1-Vecto (Fig. 4c). These data indicated that
under high-glucose conditions, decreased expression of
Klotho could contribute to the suppression of autophagic
activity in human renal proximal tubule cells.
Role of Klotho in autophagy and AMPK and ERK1/2
activation in diabetic mouse kidneys

Effects of Klotho on autophagy and AMPK
and ERK1/2 activation in high‑glucose‑treated
human renal proximal tubule cells
To investigate the role of Klotho in tubular autophagy,
Klotho was overexpressed in human renal proximal tubule
cells by pcDNA3.1 Klotho transfection. The protein levels of
LC3-1 and II were examined, and we observed that Klotho
overexpression significantly enhanced LC3-II conversion
in cells after 48 and 72 h compared to that of control cells
(Fig. 4a). Additionally, we examined autophagy by TEM,
and the results indicated that the number of autophagosomes
was notably increased in Klotho-overexpressing human renal
To further confirm the role of Klotho in autophagy and
AMPK and ERK1/2 activation, we examined the kidneys of
diabetic mice. After intravenously injecting pCMV-Klotho
into the mice, the renal levels of LC3-II/LC3-I were signifi-
cantly increased (Fig. 5a). Consistently, immunohistochemi-
cal analysis showed an increase in LC3 in the kidneys of
these mice (Fig. 5b). These results indicated that the level
of autophagy was significantly increased after Klotho over-
expression. Additionally, western blot analysis showed that
AMPK phosphorylation was increased, whereas ERK phos-
phorylation was decreased in pCMV-Klotho-treated mice,
indicating that AMPK was activated, but ERK was inhibited
(Fig. 5c).

Fig. 3  Klotho expression in

human renal proximal tubule
cells after high-glucose stimula-
tion. (a) Levels of Klotho
mRNA and (b) Klotho protein
were assessed in human renal
proximal tubule cells treated
with low glucose and high glu-
cose. (c) Morphological obser-
vations of Klotho expression
by confocal microscopy. Scale
bar: 20 μM. The data are the
means ± SEM. n ≥ 3 per group.
**p < 0.01 and ***p < 0.001
compared to the control group

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Fig. 4  Klotho overexpression increased autophagy via the AMPK/
ERK pathway in human renal proximal tubule cells stimulated with
high glucose. LC3 protein expression in human renal proximal tubule
cells transfected with the vector or Klotho and treated with high
glucose for 24 h, 48 h and 72 h. (b) Changes in autophagosomes in
human renal proximal tubule cells cultured in high glucose for 72 h
after transfection with Klotho. Red arrows indicate autophagosomes
Role of Klotho in renal injury in diabetic mice
DM mice were injected with pCMV-Klotho, and we col-
lected kidneys to evaluate the association between pathologi-
cal changes in the kidney and Klotho. H&E staining showed
that DM mice with Klotho overexpression exhibited the
densest tissue arrangement without obvious damage, such as
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with damaged organelles and other cytoplasmic components. Origi-
nal magnification: × 13,500. (c) p-AMPK, AMPK, p-ERK and ERK
protein expression in human renal proximal tubule cells cultured in
high-glucose conditions for 72 h after transfection with the vector or
Klotho. The data are the means ± SEM. n ≥ 3 per group. *p < 0.05 and
**p < 0.01 compared to the control group
a thicker glomerular basement membrane and the accumu-
lation of extracellular matrix or mesangial matrix (Fig. 6).
Masson’s trichome staining was used to examine fibrosis.
DM mice injected with pCMV-Klotho exhibited significant
decreases in fibers with only small collagen fibers in the
kidney cortex. PAS staining showed significantly decreased
expression of polysaccharides and glycogen in the kidneys
Acta Diabetologica
Fig. 5  Klotho increased renal autophagy via AMPK activation and
ERK1/2 pathway inhibition in diabetic mice. (a) Western blot show-
ing LC3-I/II protein expression. (b) Immunohistochemical staining
of LC3 in the kidney cortex after intravenous injection of the vec-
tor or Klotho. Original magnification: × 400, scale bar = 100 μm. (c)
of DM mice injected with pCMV-Klotho. These results indi-
cated that Klotho overexpression suppressed renal tubular
damage in diabetic mice.
Role of AMPK and ERK1/2 in the effect of Klotho
on human renal proximal tubule cell autophagy
after high‑glucose stimulation
To examine whether AMPK and ERK1/2 were involved
in Klotho-mediated regulation of autophagy in human
renal proximal tubule cells, we first determined the lev-
els of these factors in the cells transfected with Klotho.
As shown in Fig. 7, the AMPK and ERK1/2 levels were
p-AMPK, AMPK, p-ERK and ERK protein expression in the kid-
ney cortex after injection of the vector or Klotho. The data are the
means ± SEM; n ≥ 3 per group; **p < 0.01 and ***p < 0.001 com-
pared to the vector group
comparable between cells with or without Klotho over-
expression. However, AMPK phosphorylation was sig-
nificantly increased, while ERK1/2 phosphorylation was
inhibited. After curcumin and compound C treatment, the
changes in the phosphorylation of AMPK and ERK1/2
were restored. LC3-I and LC3-II expression was exam-
ined and showed that the increased conversion of LC3 was
abolished by curcumin or compound C. These results indi-
cated that in tubule cells exposed to high glucose, Klotho
regulated AMPK and ERK1/2 phosphorylation, which in
turn played a critical role in Klotho-mediated regulation
of autophagy in these cells.
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Fig. 6  Klotho protected against DKD in mice. Photomicrographs
of kidney sections from the vector and Klotho groups stained with
hematoxylin and eosin (H&E), showing tubulointerstitial collagen
Fig. 7  AMPK and ERK1/2 regulated autophagy in human renal
proximal tubule cells simulated with high glucose. (a) After transfec-
tion with Klotho and culture under high-glucose conditions, human
renal proximal tubule cells were further treated without or with
compound C (AMPK inhibitor) or curcumin (ERK inhibitor). The
Discussion
With the recent increase in diabetes cases, DKD has
become an extreme threat to human life [19]. Despite
dramatic developments in renoprotective drugs, DKD
treatment remains a formidable challenge for clinicians
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deposition (Masson’s trichome staining) and glycogen or polysaccha-
ride (periodic acid-Schiff (PAS) staining) expression. (Original mag-
nification: × 400)
expression of AMPK, p-AMPK, ERK and p-ERK in cells was inves-
tigated. (b, c and d) ImageJ software was used to quantify the relative
protein expression. The data are the means ± SEM. n ≥ 3 per group.
**p < 0.01: compared to the control group. &&p < 0.01: compared to
the Klotho group
[20]. Therefore, it is important to understand the potential
pathological mechanism of DKD for the development of
novel therapeutic strategies.
An animal model of DM is the main method for study-
ing the mechanism of disease and drug therapy. Studies
have shown that HFD can lead to insulin resistance and DM
induction, and low-dose STZ treatment can cause moderate
Acta Diabetologica
impairments in β-cell function and incomplete insulin
secretion, which leads to impaired glucose metabolism. It
has been reported that HFD combined with low-dose STZ
induces insulin resistance and elevates blood lipids in mice.
The pathogenesis and progression of DM in mice induced by
an HFD and STZ were similar to those of human DM. This
animal model can simulate the disease process in human DM
patients and has been used in research to study the mecha-
nism of DM and develop drugs to treat DM. Therefore, the
DM mouse model was induced by an HFD combined with
low-dose STZ in this study. Previous studies also revealed
that the level of Klotho was significantly decreased in DKD
patient serum; thus, Klotho was identified as a potential bio-
marker of DKD [21, 22]. Our results also confirmed that
Klotho was frequently diminished in the kidney cortex in
DM mice, and high glucose also suppressed the expression
of Klotho in human renal proximal tubule cells.
DKD is a complex disease that is associated with ultras-
tructural changes in all compartments in the kidney. These
changes are characterized by basement membrane thicken-
ing, glomerular and tubular hypertrophy, mesangial expan-
sion, glomerulosclerosis and tubulointerstitial fibrosis [23].
Although glomerular changes have attracted the most atten-
tion, renal tubules, an energy-consuming structure suscep-
tible to various metabolic and hemodynamic fluctuations,
is damaged before the occurrence of glomerulopathy, sug-
gesting that tubular injury critically contributes to the patho-
genesis and development of DKD [24, 25]. In the present
study, we used tubular cells to investigate the underlying
mechanism by which high glucose induces DKD. Our data
showed that a critical contributor to high glucose-induced
DKD might be a decrease in autophagy, which is consistent
with previous studies [26]. With the highest mitochondrial
content in the kidney, renal tubular cells are particularly sen-
sitive to mitochondrial dysfunction, which has been well
recognized as a pivotal factor that initiates DKD and is a
therapeutic target. By degrading damaged organelles, includ-
ing mitochondria, autophagy is essential for homeostasis
and cell survival [27]. Defective autophagy may ultimately
contribute to the accumulation of cellular damage, resulting
in the development of metabolic or age-related kidney dis-
eases [5]. In addition to autophagy, reactive oxygen species
(ROS) and apoptosis are also triggered by hyperglycemia
and lead to DKD [28]. Autophagy is upstream of these two
pathways and has been demonstrated to play an important
role in regulating their activities [5]. These findings further
suggest that inducing autophagy is a promising strategy for
treating DKD.
The core autophagy machinery is regulated upstream
by a complex signaling network. As an antiaging protein,
Klotho has been reported to be associated with autophagy
and a variety of diseases. For example, in Alzheimer’s dis-
ease and acute kidney injury, the upregulation of Klotho
expression could improve autophagic clearance and attenu-
ate cell injury, ultimately serving as a protective factor [29,
30]. In DKD, Klotho is aberrantly expressed and was sug-
gested to participate in this disease [31]. A clinical study
also explored the potential association of Klotho with DKD
and noted that Klotho was negatively associated with DKD
[32]. In STZ-induced diabetic mice, a study showed that
Klotho deficiency exacerbated early DKD [33]. Another
study also indicated that enhanced serum Klotho levels
might be a mechanism by which atrasentan inhibited renal
tubular injury in DKD [34]. Moreover, it was suggested that
autophagy is the convergent mechanism of Klotho pathways
and is central to DKD [35]. In this study, we observed a
protective effect of Klotho on renal tubular cells and against
DKD. Furthermore, our data are the first to demonstrate that
the induction of autophagy by Klotho critically contributes
to renoprotection.
Multiple pathways are involved in the biological function
of Klotho. Using a selective inhibitor or activator, we noted
that Klotho markedly induced the activation of AMPK and
the inactivation of ERK, ultimately regulating autophagic
activity and DKD. In some types of tumor cells, Klotho
overexpression or treatment with soluble protein consist-
ently increased AMPK phosphorylation [36]. In the aortas
of Klotho-expressing mice, AMPK activity was also reduced
[37]. In an additional study with db/db mice, Klotho ame-
liorated DKD via AMPK [38]. The role of Klotho in regu-
lating ERK signaling has been reported to vary according
to the pathways. When ERK phosphorylation was induced
by TGFβ1, AngII and phosphate, Klotho had no inhibitory
effects. However, Klotho inhibited ERK activity via FGF2
signaling [39]. These results raised the question of which
pathway Klotho regulates ERK phosphorylation through in
DKD.
In summary, our present study demonstrated the protec-
tive role of Klotho in DKD. In addition, we uncovered a
novel mechanism associated with the protective effect of
Klotho against DKD and found that the control of Klotho-
mediated autophagy might be a therapeutic strategy for
DKD. Therefore, we suggest that the increase in Klotho is a
promising strategy for DKD treatment. However, there are
limitations to current research. For example, models of both
type 1 and type 2 diabetes should be established to explore
the function and mechanism of Klotho in DKD, and the
phosphorylation of Klotho should also be further investi-
gated in DKD.
Acknowledgements This study was supported by the National Natural
Science Foundation of China (No. 81870570; No. 81900378).
Author contributions MX, FY, YL, YY, BW and YJ performed the
experiments and analyzed the data. MX and FY wrote the manuscript.
ZZ and YX revised the manuscript. All authors read and approved the
final manuscript.
13

Availability of data and materials The datasets used during the present
study are available from the corresponding author.
Declarations
Conflict of interest The authors declare that they have no conflict of
interests.
Patient consent for publication Not applicable.
Human and Animal Rights All procedures performed in studies involv-
ing animal participants were in accordance with the ethical standards
of the institutional or national research commitee and with the 1964
Heisinki declaration and its later amendments or comparable ethical
standards. This article does not contain any human experiments.
Informed consent All animal studies and procedures were approved
by the Institutional Animal Care and Use Committee of the Labora-
tory Animal Center, Nanfang Hospital, Southern Medical University,
Guangzhou, China (certificate number: SYXK2015-0056).
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diabetic nephropathy via LKB1-AMPK-PGC1 alpha-mediated jurisdictional claims in published maps and institutional affiliations.
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