12
3
4
5

Inhibition of amino acids influx into proximal tubular cells improves

6

lysosome function in diabetes.

7
8
9

Yuzuki Kano, Satoshi Yamaguchi, Koki Mise, Chieko Kawakita, Yasuhiro Onishi,

10

Naoko Kurooka, Ryosuke Sugawara, Haya Hamed Hassan Albuayjan,

11

Atsuko Nakatsuka, Jun Eguchi, and Jun Wada

12
13

Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama

14

University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,

15

Okayama, Japan

16
17
18

Correspondence:19

Jun Wada, M.D., Ph.D.

20

Department of Nephrology, Rheumatology, Endocrinology and Metabolism,

21

Okayama University Graduate School of Medicine,

22

Dentistry and Pharmaceutical Sciences, Okayama, Japan23

2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan

24

Tel: +81-86-235-7232

25

FAX: +81-86-222-5214

26

E-mail: junwada@okayama-u.ac.jp

27
28
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Key Points

31



32
33

Collectrin (CLTRN) serves as a chaperone for the trafficking of neutral amino acidtransporters in the apical membranes of proximal tubular cells (PTCs).



Cltrn knockout reduced amino acids influx into PTCs, inactivated mTOR, activated

34

TFEB, improved lysosome function, and ameliorated vacuolar formation of PTCs

35

in diabetic mice treated with streptozotocin and high fat diet.

36
37



The inhibition of neutral amino acid transporter, such as B0AT1 (SLC6A19), and
TFEB activator is a new therapeutic strategy against diabetic kidney disease.

38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
2

56

Abstract

57

Background/aims: Inhibition of glucose influx into proximal tubular cells (PTCs) by

58

sodium-glucose co-transporter 2 (SGLT2) inhibitors revealed prominent therapeutic

59

impacts on diabetic kidney disease (DKD). Collectrin (CLTRN) serves as a chaperone

60

for the trafficking of neutral amino acid transporters in the apical membranes of61

proximal tubular cells. We investigated the beneficial effects of reduced influx of amino

62

acids into proximal tubular cells in diabetes and obesity model of Cltrn-/y mice.

63

Methods: Cltrn+/y and Cltrn-/y mice at 5 weeks of age were assigned to standard diet-

64(STD) and streptozotocin and high fat diet-treated (STZ-HFD) groups.

65

Results: At 22-23 weeks of age, body weight and HbA1c levels significantly increased66

in STZ-HFD-Cltrn+/y compared to STD-Cltrn+/y; however, they were not altered in

67

STZ-HFD-Cltrn-/y compared to STZ-HFD-Cltrn+/y. At 20 weeks of age, urinary albumin

68

creatinine ratio (UACR) was significantly reduced in STZ-HFD-Cltrn-/y compared to69

STZ-HFD-Cltrn+/y. Under the treatments with STZ and HFD, the Cltrn gene deficiency

70

caused significant increase in urinary concentration of amino acids such as Gln, His,

71

Gly, Thr, Tyr, Val, Trp, Phe, Ile, Leu and Pro. In proximal tubular cells in STZ-HFD-

72

Cltrn+/y, the enlarged lysosomes with diameter of 10 μm or more were associated with73

reduced autolysosomes, and the formation of giant lysosomes was prominently

74

suppressed in STZ-HFD-Cltrn-/y. Phospho-mTOR and inactive form of phospho-TFEB

75

were reduced in STZ-HFD-Cltrn-/y compared to STZ-HFD-Cltrn+/y.

76

Conclusions: The reduction of amino acids influx into proximal tubular cells

77

inactivated mTOR, activated TFEB, improved lysosome function, and ameliorated

78

vacuolar formation of PTCs in STZ-HFD-Cltrn-/y mice.

79
80
81
3

82

Introduction

83

Collectrin is a 222-amino acid (AA) transmembrane glycoprotein and angiotensin

84

converting enzyme 2 (ACE2) homologue. Although ACE2 shares collectrin domain at

85

C-terminal end, collectrin lacks the active dipeptidyl carboxypeptidase catalytic

86

domains located at N-terminal of ACE and ACE21. Collectin is highly expressed in an

87

apical brush border of PTCs2, 3, collecting duct cells1, and pancreatic β cells4. Collectrin

88

is one of the downstream target genes of hepatocyte nuclear factor-1α (HNF-1α) and89

the overexpression of a dominant-negative mutation form (HNF1α-P291fsinsC) derived

90

from the patients with type 3 form of maturity-onset diabetes of the young (MODY3) in91

INS-1 pancreatic β cells resulted in the reduced expression of collectrin5. In the92

patients with MODY3, hyperglycemia increases over time, resulting in the need for93

treatment with oral hypoglycemic drugs or insulin. Collectrin binds to soluble N-

94

ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex

95

consisting of synaptosomal-associated protein, 25kDa (SNAP25), vesicle associated96

membrane protein 2 (VAMP2), and syntaxin 1 via direct interaction with snapin and97

facilitates the glucose-stimulated insulin exocytosis. In rat insulin promoter (RIP)-

98

collectrin transgenic mice, the reduced blood glucose levels associated with enhanced

99

insulin secretion of in the glucose tolerance tests were reported4. The patients with100

MODY3 also develop Fanconi’s syndrome and manifest glucosuria and generalized

101

aminoaciduria. The collectrin knockout mice develop severe generalized aminoaciduria

102

accompanied with reduced expression of AA transporters, such as SLC6A19 (B0AT1;

103

XT2s1) for neutral/aromatic AAs, SLC6A18 (B0AT3; XT2; Xtrp2) for Gly/Gln, SLC6A20

104(IMINO; SIT1; XT3; Xtrp3) for imino acids (Pro) at the apical membrane of PTCs,

105

suggesting collectrin serves as a chaperone for the trafficking of amino acid106transporters in the apical membranes of PTCs2, 3.

4

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Since collectrin was up-regulated in 5/6 nephrectomized mice and Wistar-Kyoto rats

108

fed with high-salt diet, it suggested possible role of collectrin in salt-sensitive

109

hypertension. In the initial research, on a mixed genetic background of 129/SvEv ×

110

C57BL/6J, a statistically significant difference in arterial pressure compared to wild-type

111(WT) mice was not detected6. The 129/SvEv strain is more susceptible to the112

development of hypertension than the C57BL/6J and collectrin deficiency resulted in113

severe hypertension associated with augmented salt sensitivity and impaired pressure

114

natriuresis7. Collectrin is also expressed in endothelial cells, maintains plasma

115

membrane levels of cationic amino acid transporter 1 (CAT1) and L-type amino acid116

transporter 1 (y(+)LAT1), facilitates L-arginine uptake and NO production by endothelial

117

nitric oxide synthase. Recently, Le TH et al. reported loss of collectrin in the proximal

118

tubule is sufficient to induce hypertension and proximal tubule specific knockout mice

119

exhibited hypertension associated with increased sodium-hydrogen exchanger 3

120(NHE3) expression and compensatory enhanced endothelium-mediated dilatation

121

without exacerbation of salt sensitivity8.

122

Here, we investigated Cltrn-/y mice backcrossed to C57BL/6JJcl, the strain resistant123

to salt-sensitive hypertension and sensitive to development of diabetes and obesity, by

124

inducing strepotozotocin (STZ) and high fat diet (HFD) model of diabetes. We aimed to125

focus on the beneficial effects of reduced influx of amino acids into proximal tubular126

cells in Cltrn-/y mice by using this animal model characterized by increased influx of127

nutrients into proximal tubules.

128
129

Methods

130

Animal Models

131

We obtained Cltrn-/y male and Cltrn+/- female mice (B6;129S5-Cltrntm1LexMmucd,132

MGI:5007377) from MMRRC (Mutant Mouse Regional Resource Centers) at UC Davis

5

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(Davis, CA, USA). Cltrn gene consists of 7 exons and exons 1 and 2 were targeted

134(NM_020626.1) by homologous recombination. Lexicon cell line derived from135

129S5/SvEvBrd was used as donor strain and the mice were mated with C57BL/6JJcl

136

to produce F5 mice and used for following experiments. The PCR genotyping was

137

performed using wild-type-specific primers (3: 5’-TGCAGGGGCACTAGTACAGATC-‘3

138

and 12: 5’-CTCATTCTATCAGTGACTCCC-‘3) and mutation-specific primers (Neo3a:139

5’-GCAGCGCATCGCCTTCTATC-3’ and 2: 5’-CTCATTCTATCAGTGACTCCC-‘3), and140

size of predicted PCR products were 365 bp and 300 bp, respectively.

141

C57BL/6 male mice fed high fat diet (HFD) and single dose of streptozotocin (STZ), a

142

model of type 2 diabetes and diabetic kidney disease with increased Urinary

143

albumin/creatinine ratio (ACR) were used9. Five-week-old mice were randomly

144

assigned to standard diet (STD) group (D12450 [10 kcal% fat, 7 kcal% sucrose],

145

Research Diets, New Brunswick, NJ) or STZ + HFD group (D12492 [60 kcal% fat, 7146kcal% sucrose], Research Diets), the latter received single injection of 100 mg/kg of

147

STZ in 0.1 M citrate buffer, pH 4.5 (Sigma-Aldrich). The 13-week-old mice (n=4 in each148

experimental group) were fasted for 16 hours in glucose tolerance test (GTT) and for 3

149

hours in insulin tolerance test (ITT). They were then intraperitoneally injected with150

glucose solution (1 mg/g body weight) and human insulin (1 unit/kg in STZ+HFD and

151

0.75 unit/kg in STD groups) for GTT and ITT, respectively.

152

All animal experiments were approved by the Animal Care and Use Committee of the153

Department of Animal Resources, Advanced Science Research Center, Okayama

154

University (OKU-2017218, 2017222, 2018208, 2018469, 2018470, 2018545, 2018666,

155

2021519, 2021579, and 2021699).

156
157

Western Blot Analysis

6

158

The kidney tissues from 35-week-old mice were homogenized in RIPA lysis and159

Extraction buffer plus Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher

160

scientific). The samples were boiled in Laemmli Sample buffer, separated on 4-20%

161

Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad), and transferred to a PVDF

162

Blotting Membrane (cytiva). After blocking with 5% nonfat milk for 1 hour at room163

temperature (RT), the blots were incubated with primary antibodies diluted with Can

164

Get Signal Solution 1 (TOYOBO); Phospho-mTOR (Ser2448) (D9C2) XP Rabbit

165

Monoclonal Antibody (Cell Signaling Technology Cat# 5536, RRID: AB_10691552),

166

mTOR (7C10) Rabbit Monoclonal Antibody (Cell Signaling Technology Cat# 2983,

167

RRID: AB_2105622), Anti-phospho TFEB (Ser142) (Sigma-Aldrich Cat# ABE1971-I,

168

RRID:AB_2928101), ATG13 antibody (Proteintech Cat# 18258-1-AP,

169

RRID:AB_2130658), Anti-p62 (SQSTM1) Polyclonal Antibody (MBL International Cat#170

PM045, RRID:AB_1279301), Anti-LC3 Polyclonal Antibody (MBL International Cat#

171

PM036, RRID:AB_2274121), Raptor (24C12) Rabbit mAb (Cell Signaling Technology

172

Cat# 2280, RRID: AB_561245), p70 S6 Kinase (49D7) Rabbit mAb (Cell Signaling

173

Technology Cat# 2708, RRID: AB_10694087), p70 S6 Kinase (49D7) Rabbit mAb (Cell174

Signaling Technology Cat# 2708, RRID: AB_10694087) overnight at 4°C. GAPDH

175(D16H11) XP Rabbit mAb (HRP Conjugate) (Cell Signaling Technology Cat# 8884,

176

RRID: AB_11129865) was used as a loading control. After washing three times with

177

Tris-buffered saline (TBS), the blots were incubated with ECL Donkey Anti-Rabbit IgG,

178

HRP-Conjugated Antibodies (NA934V, GE healthcare Life science, 1:100,000) diluted

179with Can Get Signal Solution 2 (TOYOBO) at RT for 1 hour. The blots were developed

180

with Pierce ECL Western Blotting Substrate (TE261327, Thermo Fisher Scientific). The

181

chemiluminescence was analyzed using ImageQuant LAS-4000 mini (FUJIFILM).

182
183

Statistical Analysis

7

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All values were represented as the mean ± standard deviation (SD). Statistical

185

analyses were conducted using GraphPad Prism (version 8.0). Unpaired t tests, One-186

way and Two-way ANOVA with Tukey tests were used to determine the differences.

187

p<0.05 was considered statistically significant.

188
189

Results

190

Glucose metabolism in STZ+HFD-Cltrn-/y mice

191

Under STD, there are no significant differences in body weight between STD-Cltrn+/y

192(34.6±2.5 g) and STD-Cltrn-/y (31.7±2.1 g) at 35 weeks of age. By STZ injection and193

HFD feeding, body weight significantly increased in STZ-HFD-Cltrn+/y (51.4±1.7 g)

194

compared to STD-Cltrn+/y (34.6±2.5 g) at 35 weeks of age (p=0.0041). There were no195

significant differences in body weight between STZ-HFD-Cltrn+/y (51.4±1.7 g) and

196

STZ-HFD-Cltrn-/y (44.1±8.1 g) at the final observation point of 35 weeks of age (Figure

1971A). The weight of white adipose tissues (WATs) and liver significantly increased in

198

STZ-HFD-Cltrn+/y compared to STD-Cltrn+/y at 35 weeks of age; however, there were199

no significant differences in weight of various organs between STZ-HFD-Cltrn+/y and

200

STZ-HFD-Cltrn-/y (Figure 1B). There were no significant differences in systolic blood

201

pressure in any timepoints (Figure 1C).

202

Ad libitum blood glucose levels were significantly reduced in STZ-HFD-Cltrn-/y203(191±41 mg/dL) compared to STZ-HFD-Cltrn+/y (343±5 mg/dL) at 23 weeks of age

204(p=0.0034) (Figure 2A), and there was significant reduction in HbA1c levels in STZ-

205

HFD-Cltrn-/y (5.0±0.45 %) compared to STZ-HFD-Cltrn+/y (6.2±0.40 %) (p=0.0346) at

206

23 weeks of age (Figure 2B). In GTT, there were no significant differences in blood

207

glucose and serum insulin levels between STZ-HFD-Cltrn+/y and STZ-HFD-Cltrn-/y at

208

13 weeks of age (Figure 2C and 2D). In ITT, blood glucose levels were significantly

209

reduced in STZ-HFD-Cltrn-/y (129±71 mg/dL) compared to STZ-HFD-Cltrn+/y (272±42

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mg/dL) at 15 minutes after intraperitoneal injection of glucose (p=0.0474) (Figure 2E),

211

and area under the curve (AUC) wad significantly reduced in STZ-HFD-Cltrn-/y212(214±122 hr*mg/dL) compared to STZ-HFD-Cltrn+/y (433±127 hr*mg/dL) (p=0.0495)

213(Figure 2E). Overall, there were metabolic improvements in insulins sensitivity in

214

STZ+HFD-Cltrn-/y compared to STZ+HFD-Cltrn+/y mice.

215
216

Oxygen consumption rate and locomotor activities in STZ+HFD-Cltrn-/y mice

217

Respiratory quotient was significantly reduced in the mice treated with STZ and HFD;

218

however, there were no significant differences in STZ-HFD-Cltrn+/y (0.757±0.004 and219

0.739±0.011) and STZ-HFD-Cltrn-/y (0.765±0.006 and 0.755±0.011) during light and220

dark periods, respectively (Figure 3A and 3B). Oxygen consumption rate significantly

221

increased during dark periods in STZ-HFD-Cltrn-/y (0.080±0.009 mL/g/min) compared222

to STZ-HFD-Cltrn+/y (0.066±0.005 mL/g/min) (p=0.0458) (Figure 3C and 3D). By the223

treatment of STZ and HFD, locomotor activities were significantly reduced in STZ-HFD-

224

Cltrn+/y (1.9±0.3 counts/min) compared to STD-Cltrn+/y (5.6±1.9 counts/min)

225(p<0.0001). Lowered locomotor activities were significantly and partially reversed by226

the deficiency of Cltrn in STZ-HFD-Cltrn-/y (3.7±0.9 counts/min) compared to STZ-

227

HFD-Cltrn+/y (1.9±0.3 counts/min) (p=0.0209) (Figure 3E and 3F).

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229

Urinary albumin excretion and urinary amino acids profile

230

Urinary albumin/creatinine ratio (ACR) significantly increased in STZ-HFD-Cltrn+/y231(424±65 and 571±18 μg/mg) compared to STD-Cltrn+/y (193±48 and 193±40 μg/mg) at

232

14 and 20 weeks of age (p=0.0308 and p=0.0028), respectively (Figure 4A). ACR was

233

significantly reduced in STZ-HFD-Cltrn-/y (276±78 μg/mg) compared to STZ-HFD-

234

Cltrn+/y (571±18 μg/mg) at 20 weeks of age (p=0.0024) (Figure 4A). At 33 weeks of235

age, Kidney injury molecule-1 (Kim-1)/creatinine ratio (KCR) increased in STZ-HFD-

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Cltrn+/y (18.3±5.19 pg/mg) compared to STD-Cltrn+/y (9.51±1.72 pg/mg) (p=0.0533).

237

KCR was significantly reduced in STZ-HFD-Cltrn-/y (8.59±4.47 pg/mg) compared to238

STZ-HFD-Cltrn+/y (18.3±5.19 pg/mg) (p=0.0219) (Figure 4B). Cltrn deficiency resulted

239

in amino acids uptake defects because of down regulation of apical amino acids

240transporters in PTCs in previous studies. We next investigated the urinary amino acids

241

profile and influence of the treatments with STZ and HFD. By the multiple unpaired t

242

tests between STD-Cltrn+/y and STD-Cltrn-/y, 17 amino acids significantly increased in

243

STD-Cltrn-/y except Arg, Cys, and Lys (Table 1). In contrast, any amino acids

244

concentrations were not altered by the treatment of STZ and HFD in the comparison

245

between STD-Cltrn-/y and STZ+HFD-Cltrn-/y (Table 1). In the comparison between

246

STZ+HFD-Cltrn+/y and STZ+HFD-Cltrn-/y, the Cltrn gene deficiency caused significant

247

increase in urinary amino acids concentration of neutral amino acids such as Gln, His,

248

Gly, Thr, Val, Trp, Phe, Ile, Leu and Pro by unpaired t tests (Table 1) and Gln, Gly, Thr,

249

and Ala in Two-way ANOVA with Tukey tests (Figure 4B).

250
251

Morphological changes in mouse kidney tissues treated with STZ and HFD

252

Under feeding with STD, there were no discernible morphological changes in kidney253

tissues of STD-Cltrn+/y and STD-Cltrn-/y (Figure 5A and 5B) by light microscopic

254

observations with PAS stain. However, prominent vacuolations were observed in255

tubular cells of STZ-HFD-Cltrn+/y (Figure 5C, red arrows) and they were mostly

256

suppressed in STZ-HFD-Cltrn-/y (Figure 5D). The lysosome/total area % was

257

significantly reduced in STZ-HFD-Cltrn-/y compared to STZ-HFD-Cltrn+/y (Figure 4D).

258

The PTCs characterized by prominent apical microvilli formation and richness of259

mitochondria, and they were investigated by electron microscopy. In PTCs in STZ-260

HFD-Cltrn+/y, enlarged giant lysosomes with diameter of 10 μm or more were261

observed (Figure 6A, red arrows). The regular size of lysosomes with diameter of 1 μm

10

262

or less were also observed (Figure 6A, red arrowheads). Furthermore, various forms263

of autophagosomes were observed in higher magnification (Figure 6A, yellow

264

arrowheads); however, autolysosomes were not observed in the section of single

265

proximal tubular cell showing giant lysosomes. The proximal tubular cell sections

266

without giant lysosomes were also observed in STZ-HFD-Cltrn+/y (Figure 6C and D),

267

where many autolysosomes (Figure 6D, blue arrows) and lipid autolysosomes (Figure

268

6D, orange arrows) were observed. Electron microscopic observation suggested the269

lysosome stress, enlargement of lysosome, and the stagnation of the autolysosome

270

formation in proximal tubules in STZ-HFD-Cltrn+/y. In STZ-HFD-Cltrn-/y, the formation271

of giant lysosomes was prominently suppressed (Figure 6E) as observed in light272

microscopy (Figure 5D). In higher magnification, the formation of both273

autophagosomes (Figure 5F, yellow arrowheads) and autolysosomes (Figure 5F, blue

274

arrows) were observed in STZ-HFD-Cltrn-/y.

275
276

mTOR signaling pathways in STZ+HFD-Cltrn-/y mice

277

Since amino acids activate mammalian target of rapamycin complex 1 (mTORC1) and278

it also regulates autophagy by controlling lysosome biogenesis through279

phosphorylation of transcription factor EB (TFEB), we further investigated the kidney

280

expression of phospho-mTOR, phospho-TFEB, autophagy related 13 (ATG13), p62,281

Microtubule-associated protein light chain 3 (LC3), Raptor, p70 S6 Kinase (S6K) and282

glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by Western blot analyses

283(Figure 7A). Both phospho-mTOR/mTOR and phospho-TFEB/GAPDH ratios were284

significantly reduced in STZ-HFD-Cltrn-/y compared to STZ-HFD-Cltrn+/y (Figure 7B).

285

Decreased autophagic activity is reflected by the reduction of ATG13/GAPDH and LC3-

286

II/LC-I accompanied with the accumulation of p62/GAPDH. There were no significant

287

differences between STZ-HFD-Cltrn+/y and STZ-HFD-Cltrn-/y in ATG13/GAPDH, LC3-

11

288

II/LC-I, p62/GAPDH, Raptor/GAPDH, and S6K/GAPDH (Figure 7B). The data289

suggested that reduction of amino acids influx into proximal tubular cells activated290

mTOR and TFEB, which resulted in the improved lysosome function, formation of291

autolysosome and reduction of giant lysosomes in STZ-HFD-Cltrn-/y.

292
293

Discussion

294

The PTCs play a central role in the regulation of reabsorption, degradation, and295

production of the nutrients such as glucose, free fatty acids, and amino acids by296

sensing the nutrient signals from urinary space under the physiological conditions10. In

297

the patients with diabetes and obesity, the excess of food intake results in the overload298

of glucose, free fatty acids and amino acids to PTCs and the enhanced activation of

299

mTORC1 and AMP-activated protein kinase (AMPK) links the subsequent induction300

and progression of tubular injury, inflammation, and fibrosis10. Neutral amino acids are

301

actively transported from lumen to cytoplasm by SLC6A19 (B0AT1; XT2s1), SLC6A18

302(B0AT3; XT2; Xtrp2), and SLC6A20 (IMINO; SIT1; XT3; Xtrp3) clustered in the apical303

membranes of PTCs2, 3. Collectrin binds to snapin, facilitates the exocytosis of insulin in304

pancreatic β cells and could form the heterodimers with SLC6A19, SLC6A18, and305

SLC6A20, suggesting collectrin is responsible for the clustering of neutral amino acids

306

transporters on the apical membrane of PTCs11, 12. In STZ-HFD-Cltrn-/y, urinary307

excretion of many of neutral amino acids significantly increased but acidic (Asp and

308Glu) and basic (Arg and Lys) amino acids were unaltered compared to STZ-HFD-

309

Cltrn+/y. It enabled us to investigate the effects of reduced influx of most of the neutral

310

amino acids into the PTCs under the state of diabetes and obesity.

311

mTOR acts as a serine/threonine kinase by interacting with multiple protein

312

complexes such as mTORC1 and mTORC2. mTORC1 is consisted of DEPTOR,

313

PRAS40, Raptor, mLST8/GbL, Tti/Tel2, and mTOR and its activation is initiated by

12

314

amino acid-mediated formation of active RagA/B-GTP and RagC/D-GDP heterodimers

315

binding to Raptor followed by recruitment of mTORC1 to lysosome where Rheb

316

activator locates, suggesting a dominant role of amino acids in the regulation of

317

mTORC113. The mechanism how the cells sense amino acids and transduce signals to

318

mTORC1 is particularly investigated in Leu and Arg. Under the Leu starvation, Sestrin2319

binds to GATOR2 and the complex inhibits the GTPase-activating protein activity of

320

GATOR1, which results in the hydrolysis of RagA/B-GTP to GDP, dissociation of

321

mTORC1 from lysosome, and its inactivation. Leu is recognized by Sestrin2, cytosolic

322

sensor, dissociates from GATOR2, and induces the activation of mTORC114. Cytosolic

323

arginine sensors for mTORC1 subunit 1 (CASTOR1) regulates mTORC1 activity

324

through a mechanism of action like Sestrin2. CASTOR1 functions as a homodimer or325

heterodimer with CASTOR2, which is required for arginine binding, GATOR2

326

sequestration and mTORC1 inactivation15. Gln is an important amino acid for327

activation of mTORC1 depending on α-ketoglutarate production in a Rag GTPases-

328

dependent manner16. Gln also activates mTORC1 via v-ATPase and Arf1 in a Rag

329

GTPases-independent manner17. In our animal model with diabetes and obesity,

330

significant increase in urinary excretion of Gln and Leu in STZ-HFD-Cltrn-/y suggesting331

reduced influx Gln and Leu into PTCs contribute the ameliorated activation of mTOR,

332

i.e. phosphorylation of mTOR, compared with STZ-HFD-Cltrn+/y. To support this333

notion, synergistic promotive effect of Gln on Leu-mediated mTORC1 activation has

334

been reported18.

335

STZ-HFD-Cltrn+/y demonstrated the increased phosphorylation of mTOR and we336

observed reversed activation of mTOR in STZ-HFD-Cltrn-/y associated with increased

337amino acids excretion into urine. We further investigated the status of autophagy

338

downstream of mTORC1 in the kidney tissues. In STZ-HFD-Cltrn+/y, the PTCs

339

demonstrates enlargement of lysosome associated with impairment of autolysosomes

13

340

formation. In Western blot analyses, ATG13 (autophagy initiation), p62 (autophagy

341

substrate and marker for autophagy activity), and LC3 (autophagosome marker)

342

demonstrated the impairment of autophagy process in STZ-HFD-Cltrn+/y; however,343

they were not reduced in STZ-HFD-Cltrn-/y. The activation of mTORC1 by amino acids

344

induces phosphorylation of TFEB and remains in the cytoplasm with inactivated form.

345

By forming mTORC1-TFEB-Rag-Regulator megacomplex19, TFEB is critically involved346

in the maintenance of structural integrity of lysosomes by promoting biosynthesis of347

lysosomes20 and autophagy. The phosphorylated inactive form TFEB increased in

348

STZ-HFD-Cltrn+/y and it was reduced in STZ-HFD-Cltrn-/y associated with349

disappearance of giant lysosomes in the PTCs. In diabetes animal models such as

350

C57BL/Ks db/db mice, TFEB overexpression or pharmacological activation of TFEB

351

alleviates the tubular epithelial cell injuries by enhancing lysosomal clearance,

352

promoting lysosomal biogenesis, and formation of autophagosomes21, 22.

353

The current investigation provides the insights into drug discovery related to354

diabetes, obesity, and diabetic kidney disease. β amyloid precursor protein cleavage

355

enzyme 1 (BACE1) cleaved amyloid β (Aβ) and the development of guanidine-based356

novel BACE1 inhibitors for the treatment and maintenance of Alzheimer’s disease was357

attempted; however, most of the studies were discontinued23, 24. Collectrin is a358

substrate for BACE2 and it cleaves and release the collectrin ectodomain outside the359

cells25. Since transgenic mice overexpressing collectrin in pancreatic β cells resulted in360

increased secretion of insulin4 and collectrin deficiency in whole body or proximal

361

tubules causes hypertension in mice7, 8, 26. BACE2 inhibitor may the candidate for the362

treatment of type 2 diabetes and hypertension27, 28. However, the current study

363

provides the evidence that the increased influx of amino acids into PTCs and activation

364

of mTORC1 pathway may deteriorate the progression of diabetic and obesity-related365

kidney disease. In the model of diet-induced obesity, BACE2 knockout mice leads to

14

366

exacerbated body weight gain, hyperinsulinemia, and insulin resistance29. In addition,

367

BACE2 cleaves the amyloid β protein precursor within the Aβ domain that accordingly

368

prevents the generation of Aβ42 peptides associated with aggregation of the Aβ30 and

369

BACE2 loss-of-function mutation (BACE2G446R) showed greater apoptosis and370

increased levels of Aβ oligomers in human pluripotent stem cell (hPSC)-derived brain

371

organoids31. Further investigation is required to confirm the relevance of BACE2

372

inhibitor in the treatment of diabetes and obesity. In addition to BACE2, the inhibition of373

neutral amino acid transporter, B0AT1 (SLC6A19), is another therapeutic approach.

374

The inhibition of B0AT1 activity was shown to improve the glycemic control by375

upregulating glucagon like peptide (GLP1) and fibroblast growth factor (FGF21) in376

mice32. B0AT1 inhibitor is also beneficial for the diabetic kidney disease by reducing the377

neutral amino acids influx such as Gln and Leu and ameliorating the mTORC1 activity.

378

Finally, the reduced phosphorylated inactive form of TFEB in STZ-HFD-Cltrn-/y379

suggested the beneficial effects of TFEB activator and this notion was suggested in the380

previous studies21, 22.

381

The limitation of current investigation is that it is inconclusive whether the382

improvement of renal tubular lesions is due to reduced amino acid influx or systemic

383

metabolic improvements. To further confirm the results, the investigation of the effects384

of altered amino acids influx on mTORC1 activity and autophagy-lysosomal function by385

using proximal tubule-specific Cltrn knockout mice or primary cultured tubular cells

386

isolated from wild and Cltrn knockout mice.

387
388

Disclosures

389

JW receives speaker honoraria from Astra Zeneca, Bayer, Boehringer Ingelheim,

390

Daiichi Sankyo, Kyowa Kirin, Novo Nordisk Pharma, and Tanabe Mitsubishi, and

15

391

receives grant support from Bayer, Chugai, Kyowa Kirin, Otsuka, Shionogi, Sumitomo

392

Pharma, and Tanabe Mitsubishi.

393
394

Funding395This work was supported by Grant-in-Aid for Scientific Research (B) (22H03088),

396

Japan Agency for Medical Research and development (AMED, grant no:397

22ek0109445h0003).

398
399

ACKNOWLEDGEMENTS

400

We acknowledge excellent technical supports from Ms. Masumi Furutani at Central

401

Research Laboratory, Okayama University Medical School to perform morphological402

analyses by electron microscopy.

403
404

Author contributions

405

YK, KM, CK and JW designed the project and experiments and wrote the manuscript.

406

YK, SY, KM, CK, YO, NK, RS, and HHHA performed animal experiments and analyzed

407

and interpreted data. SY, AN and JE performed Western blotting, genotyping and other408

molecular biology experiments.

409
410

Data Sharing Statement

411

Data of Cltrn knockout mice (B6;129S5-Cltrntm1LexMmucd) and targeted Cltrn gene are412

available at MGI:5007377 and NM_020626.1, respectively.

413
414

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20

Table 1. Urinary concentrations of amino acids (mmol/mL) (unpaired t tests).
STD-Cltrn+/y

STD-Cltrn-/y

STZ+HFDCltrn+/yMean ± SD
208.6 ± 98.0
30.7 ± 9.3
672.6 ± 202.4
382.3 ± 43.5
5622.0 ± 2169.1
122.3 ± 24.4
8326.0 ± 4945.0
32.3 ± 23.5
68240.0 ±101.0 ± 36.1
27578.54814.0 ± 1760.5
6.7 ± 5.532500.0 ±453.0 ± 90.1
14452.3

Asp
Glu
Asn
Ser

19.0 ± 7.0
429.7 ± 20.0
70.0 ± 17.3
18.0 ± 1.0

Gln26.0 ± 8.7

His

1.7 ± 2.1

Gly

329.0 ± 82.5

Thr

16.0 ± 10.4

18140.0 ± 5838.9

40.7 ± 4.5

Arg19.3 ± 14.6
337.3 ±
136.1
18.0 ± 3.6
n.d.
27.0 ± 10.1
383.0 ± 59.0
53.7 ± 5.9
83.3 ± 8.6
112.0 ± 20.0
177.7 ± 26.7
n.d.
19.0 ± 8.2

207.2 ± 162.4
27740.0 ±10444.3
5726.0 ± 2340.2
n.d.
8582.0 ± 2425.8
3208.0 ± 1298.0
855.8 ± 357.8
2394.0 ± 797.1
4602.0 ± 1346.4
10080.0 ± 2815.2
993.0 ± 934.9
5202.0 ± 1303.0

66.7 ± 57.8

Ala
Tyr
Cys
Val
Met
Trp
Phe
Ile
Leu
Lys
Pro

676.7 ± 163.3
64.3 ± 31.3
n.d.
47.7 ± 12.9
584.0 ± 125.4
87.7 ± 33.5
131.3 ± 24.1
183.7 ± 53.9
215.0 ± 45.0
8.0 ± 13.9
66.7 ± 15.0

STD-Cltrn/y vs
STZ+HFDCltrn-/y

STZ+HFD-Cltrn-/y

STDCltrn+/y vs
STD-Cltrn/y326.2 ± 276.3
1016.2 ± 609.0
7234.0± 5013.0
10708.4 ± 9005.7

0.017682*
0.091432
0.005135**
0.030451*

STDSTZ+HFDCltrn+/y vs Cltrn+/y vs
STZ+HFD- STZ+HFDCltrn+/y
Cltrn-/yP values
0.123083
0.136779
0.131901
0.162092
0.054834
0.038999*
0.093964
0.349997

88500.0 ± 1493.0

0.006024**

0.011743*

0.024938*

0.389769

7624.0 ± 3028.0

0.003754**

0.005569**

0.215285

0.110577

41120.0 ± 1727.6

0.009701**

0.007551**

0.153708

0.416976

0.002004**

0.029506*

0.01983*0.859181

0.100972

0.247159

0.241099

0.280215

0.004569**

0.062056

0.050621

0.879596

0.006425**n.d.
0.00104**
0.01073*
0.009405**
0.00282**0.00139**0.001054**
0.125150.000549***

0.005786**
n.d.
0.037427*
0.111115
0.009196**
0.045072*
0.032507*
0.041272*
0.246622
0.035265*

0.063728
n.d.
0.094623
0.065872
0.158395
0.031543*
0.097166
0.28433
0.373901
0.008418**

0.59856n.d.
0.621012
0.454476
0.156133
0.632449
0.553424
0.685529
0.867022
0.632302

19182.0 ±11296.9
534.4 ± 610.5
26240.0 ±18731.3
6586.0 ± 2613.8
n.d.
10148.0 ± 6363.3
4536.4 ± 3548.9
1310.6 ± 542.3
2830.4 ± 1793.6
5580.0 ± 3269.2
11558.0 ± 7347.7
1127.2 ± 1462.2
6080.0 ± 3725.9526
21

0.395936
0.265491
0.527849
0.618135

527

FIGURE LEGENDS

528

Figure 1. The metabolic phenotypes of Cltrn+/y and Cltrn-/y male mice treated with529

standard diet (STD) or streptozotocin and high fat diet (STZ-HFD). (A) Body weight of530

Cltrn+/y (n=3) and Cltrn-/y (n=5) mice treated with STD or STZ-HFD. (B) Organs

531

weight of Cltrn+/y (n=3) and Cltrn-/y (n=5) mice treated with STD or STZ-HFD at 35

532

weeks of age. (Epi, epididymal; Mes, mesenteric; Sub, inguinal; Brown, Brown adipose

533

tissues) (C) Systolic blood pressure of Cltrn+/y (n=3) and Cltrn-/y (n=5) mice treated534

with STD or STZ-HFD. There are no statistical differences. Data shown as mean ± SD535

and analyzed by One-way ANOVA with Tukey test at each time point in A and C.

536(*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

537

Figure 2. Glucose metabolism in Cltrn+/y and Cltrn-/y male mice treated with standard

538

diet (STD) or streptozotocin and high fat diet (STZ-HFD). (A) Ad labium blood glucose539levels in Cltrn+/y (n=3) and Cltrn-/y (n=5) mice treated with STD or STZ-HFD. (B)540

HbA1c levels in Cltrn+/y (n=3) and Cltrn-/y (n=5) mice treated with STD or STZ-HFD.

541(C) Glucose tolerance tests in Cltrn+/y (n=3) mice treated with STD or STZ-HFD and

542

Cltrn-/y mice (STD-Cltrn-/y n=7; STZ-HFD-Cltrn-/y n=5). (D) Serum insulin levels during543

glucose tolerance tests. (STD-Cltrn+/y, n=2; STD-Cltrn-/y, n=3; STZ-HFD-Cltrn+/y,544

n=3; STZ-HFD-Cltrn-y, n=4). (E) Insulin tolerance tests. (STD-Cltrn+/y, n=4; STD-

545

Cltrn-/y, n=3; STZ-HFD-Cltrn+/y, n=3; STZ-HFD-Cltrn+/y, n=5). Data shown as mean ±546

SD and analyzed by One-way ANOVA with Tukey test (A, C and E) and Mixed-effects

547

analysis due to missing values (B and D) at each time point separately. (*p<0.05;

548**p<0.01; ***p<0.001; ****p<0.0001).

549

Figure 3. Respiratory quotient, oxygen consumption rate and locomotor activities in

550

Cltrn+/y and Cltrn-/y male mice treated with standard diet (STD) or streptozotocin and551high fat diet (STZ-HFD). (A) Daily profile of respiratory quotient in Cltrn+/y (n=3) and

22

552

Cltrn-/y (n=5) mice treated with STD or STZ-HFD. Significant differences between STZ-

553

HFD-Cltrn+/y and STZ-HFD-Cltrn-/y are shown. (B) Respiratory quotient in light and554

dark periods. (C) Oxygen consumption rate in Cltrn+/y (n=3) and Cltrn-/y (n=5) mice555

treated with STD or STZ-HFD. Significant differences between STZ-HFD-Cltrn+/y and

556

STZ-HFD-Cltrn-/y are shown. (D) Oxygen consumption rate in light and dark periods.

557(E) Locomotor activities in Cltrn+/y (n=3) and Cltrn-/y (n=5) mice treated with STD or

558

STZ-HFD. Significant differences between STZ-HFD-Cltrn+/y and STZ-HFD-Cltrn-/y559

are shown. (F) Locomotor activities in light and dark periods. Data shown as mean ±560

SD and analyzed by Two-way ANOVA with Tukey test. (*p<0.05; **p<0.01; ***p<0.001;

561****p<0.0001).

562

Figure 4. Urinary albumin/creatinine ratio and urinary amino acids profiles in Cltrn+/y563

and Cltrn-/y male mice treated with standard diet (STD) or streptozotocin and high fat

564

diet (STZ-HFD). (A) Urinary albumin/creatinine ratio (ACR) in Cltrn+/y (n=3) and Cltrn-

565/y (n=5) mice treated with STD or STZ-HFD. (B) Urinary Kidney injury molecule-1

566(Kim-1)/creatinine ratio (KCR) in Cltrn+/y (n=3) and Cltrn-/y (n=4) mice treated with567

STD or STZ-HFD. (C) Concentrations of urinary amino acids in Cltrn+/y (n=3) and568

Cltrn-/y (n=5) mice treated with STD or STZ-HFD. Data shown as mean ± SD,

569

analyzed by One-way ANOVA with Tukey test at each time point in A and B. Significant

570

differences between STZ-HFD-Cltrn+/y and STZ-HFD-Cltrn-/y are shown in C.

571(*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

572

Figure 5. PAS (Periodic Acid Schiff) stain of kidney tissues in Cltrn+/y and Cltrn-/y573

male mice treated with standard diet (STD) or streptozotocin and high fat diet (STZ-574

HFD). (A and B) STD-Cltrn+/y, (C and D) STD-Cltrn-/y, (E and F) STZ-HFD-Cltrn+/y,575

and (G and H) STZ-HFD-Cltrn+/y. Red arrows in panels E and F show the vacuoles in

23

576

tubular cells. The red open squares in A, C, E, and G are areas for panel B, D, F, and577

H, respectively.

578

Figure 6. Electron micrographs of proximal tubules in Cltrn+/y and Cltrn-/y male mice579

treated with streptozotocin and high fat diet (STZ-HFD). (A) STZ-HFD-Cltrn+/y. The

580

enlarged lysosomes (red arrows) and regular sized lysosomes (red arrowheads) are581

seen. The white open square is area for panel B. (B) STZ-HFD-Cltrn+/y. Various forms582

of autophagosomes are seen (yellow arrowheads). (C) The proximal tubules without

583

giant lysosomes observed in STZ-HFD-Cltrn+/. The white open square is area for584

panel D. (D) STZ-HFD-Cltrn+/y. Various forms of autolysosomes (blue arrows) and lipid

585

autolysosomes (orange arrows) are observed. (E) STZ-HFD-Cltrn-/y. Regular sized586

lysosomes (red arrowheads) are seen. The white open square is area for panel F. (F)587

STZ-HFD-Cltrn-/y. Autophagosomes (yellow arrowheads) and autolysosomes (blue588arrows) are shown.

589

Figure 7. Western blot analyses in Cltrn+/y and Cltrn-/y male mice treated with590

standard diet (STD) or streptozotocin and high fat diet (STZ-HFD). (A) Western blot

591

analyses for phospho-mTOR, mTOR, phospho-TFEB, ATG13, p62, LC3, Raptor, S6K,

592

and GAPDH in STD-Cltrn+/y (n=4), STD-Cltrn-/y (n=5), STZ-HFD-Cltrn+/y (n=6), and

593

STZ-HFD-Cltrn-/y (n=7) mice. (B) Densitometric analyses of Western blots. Data

594

shown as mean ± SD and analyzed by Two-way ANOVA with Tukey test. (*p<0.05;595**p<0.01; ***p<0.001; ****p<0.0001). Significant differences between STZ-HFD-Cltrn+/y596

and STZ-HFD-Cltrn-/y are shown.

597

SUPPLEMENTARY MATERIAL

598

Supplementary Figure S1. A. Uncropped gel images for STD groups in Figure 7A. B.

599

Uncropped gel images for STZ-HFD groups in Figure 7A.

24