Increased Circulating and Urinary Levels of Soluble TAM Receptors in Diabetic Nephropathy

Increased Circulating and Urinary Levels of Soluble TAM Receptors in Diabetic Nephropathy

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 ...
Download PDF
2MB Sizes 0 Downloads 21 Views
Report

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62

The American Journal of Pathology, Vol. -, No. -, - 2017

ajp.amjpathol.org

Increased Circulating and Urinary Levels of Soluble TAM Receptors in Diabetic Nephropathy Q21

Peter Ochodnicky,* Lionel Lattenist,* Mohamed Ahdi,y Jesper Kers,* Melissa Uil,* Nike Claessen,* Jaklien C. Leemans,* Sandrine Florquin,*z Joost C.M. Meijers,x{ Victor E.A. Gerdes,yx and Joris J.T.H. Roelofs* From the Departments of Pathology* and Experimental Vascular Medicine,x Academic Medical Center, University of Amsterdam, Amsterdam; the Department of Internal Medicine,y Slotervaart Hospital, Amsterdam; the Department of Plasma Proteins,{ Sanquin Research, Amsterdam; and the Department of Pathology,z Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands Accepted for publication May 2, 2017.

Q3

Q5

Address correspondence to Joris J.T.H. Roelofs, Department of Pathology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, the Netherlands. E-mail: [email protected].

TAM receptors (Tyro3, Axl, and Mer) have been implicated in regulation of innate immunity. Circulating levels of TAM receptor soluble forms (sTyro3, sAxl, sMer) are related to autoimmune disorders. We investigated levels of TAM and their ligand protein S in patients with diabetes. Urinary and plasma levels of protein S, sTyro3, sAxl, and sMer were determined by enzyme-linked immunosorbent assay in 126 patients with diabetes assigned to a normoalbuminuric or macroalbuminuric (urinary albumin excretion <30 mg/24 hours and >300 mg/24 hours, respectively) study group and 18 healthy volunteers. TAM and protein S immunostaining was performed on kidney biopsy specimens from patients with diabetic nephropathy (n Z 9) and controls (n Z 6). TAM expression and shedding by tubular epithelial cells were investigated by PCR and enzyme-linked immunosorbent assay in an in vitro diabetes model. Patients with macroalbuminuria diabetes had higher circulating levels of sMer and more urinary sTyro3 and sMer than normoalbuminuric diabetics. Increased clearance of sTyro3 and sMer was associated with loss of tubular Tyro3 and Mer expression in diabetic nephropathy tissue and glomerular depositions of protein S. During in vitro diabetes, human kidney cells had down-regulation of Tyro3 and Mer mRNA and increased shedding of sTyro3 and sMer. Renal injury in diabetes is associated with elevated systemic and urine levels of sMer and sTyro3. This is the first study reporting excretion of sTAM receptors in the urine, identifying the kidney as a source of sTAM. (Am J Pathol 2017, -: 1e13; http://dx.doi.org/10.1016/j.ajpath.2017.05.004)

Diabetic nephropathy (DN) is the leading cause of chronic kidney disease, reported in up to 45% of end-stage renal disease.1,2 Despite significant progress in understanding the DN pathophysiology, the complex sequence of events leading to glomerular and tubular damage is far from clear, and current treatment strategies still provide inadequate renoprotection. Therefore, identification of novel mediators of disease and potential therapeutic targets is warranted to reduce the enormous health and economic burden of DN. Glomerular injury associated with an alteration of glomerular filtration barrier and development of microalbuminuria attributable to podocyte and endothelial dysfunction constitute key early pathologic features of DN.3e5 These events are followed by the development of overt proteinuria, glomerulosclerosis, and tubulointerstitial injury, leading eventually to nephron loss and renal function

Q4

decline.6,7 Although diabetes mellitus has been traditionally viewed as a nonimmune disease, emerging evidence suggests that activation of innate immune mechanisms plays an important role in the development of diabetic renal injury.8,9 Infiltration of inflammatory monocytes or macrophages and subsequent cytokine release are prominent features of DN10,11 and are promoted by a proadhesive and proinflammatory phenotype of dysfunctional diabetic endothelium. Identification and characterization of novel mediators

Supported by the Dutch Kidney Foundation grants C09.2287 and KJPB 10.017 (J.J.T.H.R.), the Foundation for Clinical Research in Slotervaart Hospital (V.E.A.G.), and the Netherlands Organisation for Scientific Q1 Research grants 91712386 (J.C.L.) and 90713480 (J.J.T.H.R.). Q2 Disclosures: None declared.

Copyright ª 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2017.05.004

FLA 5.4.0 DTD  AJPA2648_proof  28 June 2017  10:49 pm  EO: 2017_39

63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124

125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186

Ochodnicky et al involved in the regulation of renal inflammation might improve our understanding of DN pathology. Tyrosine kinase receptors of the TAM family, including the three subtypes Tyro3, Axl, and Mer, have recently emerged as novel crucial regulators of innate immunity.12 Although serving as classic tyrosine kinase membrane receptors activating proliferation and survival, cell adhesion, and migration in malignant cells, all three subtypes are also expressed on macrophages, dendritic cells, and natural killer lymphocytes.13,14 Activation of TAM receptors limits the cytokine release after macrophage activation and mediates clearance of apoptotic cells by macrophages.15,16 Consequently, triple TAM knockout mice have the phenotype that resembles autoimmune disorder.17,18 A natural anticoagulant, protein S, has been identified as a ligand for Tyro3 and Mer receptors.19,20 Originally described as a cofactor for activated protein Cedependent inactivation of coagulation factors Va and VIIIa,21,22 protein S induces multiple coagulation-independent effects in a TAM receptoredependent manner. Interestingly, in addition to regulation of macrophage and other immune cell function, protein S may activate TAM receptor subtypes expressed on nonimmune cell types, including endothelial and epithelial cells or neurons. For instance, protein S protects against various types of central nervous system or lung injury through activation of the Tyro3 receptor.23e25 Protein S has much lower affinity toward Axl, which preferentially binds to the growth arrestespecific protein (Gas-6), a noncoagulant soluble protein that shares a significant homology with protein S.19,20 Gas-6/Axl axis has been recently extensively studied in the kidney, and Gas-6 activation of the mesangial Axl receptor has been implicated in the development of glomerular damage in several glomerulopathies, including DN.26,27 In contrast to Gas-6/Axl, little is known about the role of protein S, Tyro3, and Mer in the development of renal disease. Interestingly, extracellular domains of TAM receptors can be proteolytically cleaved by metalloproteases to yield soluble receptor forms designated sTyro3, sMer, and sAxl.28e30 All three soluble TAM (sTAM) receptors can be found in plasma, although their role has not been characterized yet. Such soluble forms might function as decoy receptors to inactivate their respective circulating ligands28 or even possess specific effects antagonistic to those of the canonical ligands.31 Excessive circulating levels of sMer, indicating increased systemic shedding, have been recently related to the severity of nephritis in patients with lupus30 and to the severity of renal function decline in patients with chronic kidney disease of variable origin.32 However, systemic levels of sTAM receptors in patients with diabetes have not been studied so far. Although highmolecular-weight proteins, such as sTAM receptors (approximately 150 kDa), are unlikely to be filtered under normal conditions, we hypothesize that specific subtypes of TAM receptors might be expressed in the normal and in the diabetic kidney, and eventually, the soluble forms of the

2

receptors might be released to urine. Furthermore, we propose that on metabolic and inflammatory injury in DN, renal expression and urinary and plasma levels of sTAM receptors might be altered.

Materials and Methods Patients The study was approved by the local ethical committee of the Slotervaart Hospital, Amsterdam, the Netherlands. A total of 126 patients from a larger, previously described33 cohort of patients with type 2 diabetes were included in the study. The cohort was recruited between May 2009 and December 2010 and consisted of patients visiting the Department of Internal Medicine, Slotervaart Hospital, for their annual comprehensive diabetes evaluation. Patients with urinary albumin excretion (UAE) levels >300 mg/24 hours (macroalbuminuria, indicating presence of a diabetic renal injury, n Z 67) and with available blood and 24-urine samples were identified and assigned to a DN study group. A similar number of patients with UAE levels <30 mg/24 hours (normoalbuminuria, patients with diabetes without DN, diabetic control group, n Z 59) was randomly selected. A minimal sample size of 52 patients was calculated to detect a 25% difference in plasma level of sMer with a power of 80%. In addition to UAE and glucose biochemistry, the patients were characterized in terms of plasma lipid profile and serum and urinary creatinine by standard biochemical assays, as described previously.33 Both blood and urine samples were centrifuged at 2000  g to obtain EDTA plasma and urine supernatant, respectively. To characterize normal values for sTAM receptors in body fluids, plasma and urine were collected from 18 healthy volunteers (nonobese, without diabetes mellitus, metabolic syndrome, hypercholesterolemia, hypertension, cardiovascular, or renal disease, age >40 years). In addition, histopathologic examination was performed using normal control tissue (n Z 6) or a separate set of archived kidney biopsy specimens (Department of Pathology, Academic Medical Center, Amsterdam, the Netherlands) from patients with DN (n Z 9), which were not part of the cohort described above. For all patients with diabetes, a follow-up serum creatinine and urinary albuminecreatinine ratio (UACR) sample was collected. We used a doubling in the serum creatinine on follow-up compared with baseline or a doubling of UACR on follow-up compared with baseline as a clinical outcome measure. One patient with diabetes was lost to follow-up.

ELISAs for TAM Receptor Subtypes DuoSet enzyme-linked immunosorbent assay (ELISA) kits for human Tyro3 or Axl or Mer receptor (R&D Systems, Minneapolis, MN) were used to determine plasma and urine levels of sTAM receptor subtypes, as well as sTAM receptor

ajp.amjpathol.org

-

The American Journal of Pathology

FLA 5.4.0 DTD  AJPA2648_proof  28 June 2017  10:49 pm  EO: 2017_39

187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248

249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310

sTAM Receptors in Diabetic Nephropathy

Q6

concentrations in cell culture supernatant (see below). After the coating of 96-well ELISA plates with the respective monoclonal capture antibodies, the plate was blocked with 3% fish gelatin (Sigma-Aldrich, St. Louis, MO) in trisbuffered saline and Tween 20 buffer (20 mmol/L Tris, 150 mmol/L NaCl, pH 7.4, 0.1% Tween 20). Washing steps with phosphate-buffered saline and Tween 20 (3.2 mmol/L Na2HPO4, 0.5 mmol/L KH2PO4, 1.3 mmol/L KCl, 135 mmol/L NaCl, 0.1% Tween 20) were performed after the blockade and each subsequent incubation. EDTA plasma was diluted 1:10 in all cases, whereas 24-hour urine samples were diluted by factors 8, 20, and 4 for sTyro3, sAxl, and sMer, respectively. The diluted samples and serial dilutions of standards (recombinant Tyro3, recombinant Axl, and recombinant Mer, respectively) were added to the plate and subsequently incubated with the respective biotinylated polyclonal detection antibodies. After the addition of horseradish peroxidaseeconjugated streptavidin, the color reaction was initiated using hydrogen peroxide and tetramethylbenzidine, and the absorbance was measured using automated plate reader. The concentrations were calculated using regression analysis to the respective standard curves. Renal clearance of specific sTAM receptors was calculated as [urinary concentration (nanograms per milliliter)  daily urine volume (milliliters)]/[plasma concentration (nanograms per milliliter)  24 hours  60 minutes].

Western Blot Analysis Molecular weights of the urinary sTAM receptors were determined by Western blot analysis in urine samples of 12 randomly chosen study participants (four healthy, four with normoalbuminuria, and four with macroalbuminuria). Protein concentrations were determined by the bicinchoninic acid method, and equal amounts of protein were resuspended in Laemmli sample buffer and boiled for 10 minutes at 95 C. The proteins were separated by SDS-PAGE on polyacrylamide gels (Thermo Fisher Scientific, Landsmeer, the Netherlands) and transferred to Immobilon-P nitrocellulose membranes by blotting for 2 hours (BioRad, Transblot SD, Hercules, CA). The membranes were blocked for 1 hour in tris-buffered saline and Tween 20 (20 mmol/L Tris, 150 mmol/L NaCl, 1% Tween, pH 7.5)/5% nonfat dry milk and incubated overnight at 4 C with the primary antibodies mouse anti-Tyro3 monoclonal and rabbit anti-Mer monoclonal (both LifeSpan Biosciences, Seattle, WA) and goat anti-Axl polyclonal (R&D Systems). Membranes were washed and incubated with the corresponding secondary antibody, and immunoreactivity was visualized using ECL PLUS Western blotting detection reagent (GE Healthcare Europe, Diegen, Belgium).

Total and Free Protein S ELISA Total protein S levels were determined as described previously34 with an in-house ELISA (using antibodies from

The American Journal of Pathology

-

Dako, Glostrup, Denmark). Free protein S was measured by precipitating the C4b-binding proteinebound fraction with polyethylene glycol 8000, followed by the measurement of the concentration of free protein S in the supernatant.

Immunohistochemistry Paraffin-embedded sections of the kidney biopsy specimens from healthy individuals and patients with diabetes were stained for TAM receptor subtypes using mouse anti-Tyro3 monoclonal and rabbit anti-Mer monoclonal (both LifeSpan Biosciences), goat anti-Axl polyclonal (R&D Systems), rabbit anti-protein S polyclonal (Sigma-Aldrich), and mouse anti-CD68 monoclonal (Dako) primary antibodies. After incubation with the respective peroxidase-conjugated secondary antibodies, the staining was developed with diaminobenzidine kit. The specificity of the anti-TAM antibodies was confirmed by a characteristic staining pattern in testicular epithelium. All sections were coded and scored by two blinded investigators simultaneously by means of Q7 consensus. Intensity and abundance of glomerular, tubular, and interstitial signal were scored on a semiquantitative scale from 0 to 3 (absent, weak, moderate, or strong, respectively). For each section, a mean score was calculated from at least seven high-power fields. For double staining of individual TAM receptors with the macrophage marker CD68, co-incubation of the individual primary antibodies was followed by the corresponding species-compatible secondary antibodies and visualized by Nova Red peroxidase and Vector Blue alkaline phosphatase kits, respectively (Vector Laboratories, Burlingame, CA).

Cell Culture Human tubular epithelial HK-2 cells (ATCC, Manassas, VA) were grown to subconfluence in Dulbecco’s modified Eagle’s medium F12 medium (Invitrogen, Carlsbad, CA) (3:1) con- Q8 taining 10% fetal bovine serum, 2 mmol/L L-glutamine, 100 U/ mL penicillin, and 100 mg/mL streptomycin in a humidified 5% CO2 atmosphere at 37 C. Cells were serum starved for 24 hours and then stimulated with 30 mmol/L of glucose (or 30 mmol/L of mannitol as osmotic control) and 10 ng/mL of transforming growth factor (TGF)-b to model the diabetic environment for 48 hours as described previously,35 after which supernatant was collected and cells were harvested after washing with phosphate-buffered saline. Cell viability was assessed by the standard trypan blue exclusion test.36 Cellular TAM receptor mRNA expression was studied by real-time PCR. First, RNA was extracted from the cells using the Trizol reagent (Life Technologies), according to the protocol recommended by the manufacturer. RNA concentration and purity were determined by the Nanodrop spectrophotometer (Thermo Fisher Scientific), and cDNA was synthesized using oligo dT primers and reverse transcription mix (Roche Applied Science, Penzberg, Germany). Specific primers for the PCR were designed using the

ajp.amjpathol.org

FLA 5.4.0 DTD  AJPA2648_proof  28 June 2017  10:49 pm  EO: 2017_39

3

311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372

373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434

Ochodnicky et al Q9

Primer-BLAST program (National Center for Biotechnology Information Tools). The primers sequences were as follows: Tyro3 forward: 50 -GCGAGCAGCACTGGATCG GC-30 , reverse: 50 -GTTCAGGGGGACCCACAGCCT-30 ; Axl forward: 50 -GCAAGCACAGCCAGTCCACCAG-30 , reverse: 50 -GCCAAGATGAGGACACAGGCAGC-30 ; Mer forward: 50 -ACGTGTGGCAGAGTGCAGGGA-30 , reverse: 50 -GCCCAACTCCCCCTCTGGTGA-30 ; housekeeping gene TBP forward: 50 -CCCATGACTCCCATGACC-30 , reverse: 50 -TTTACAACCAAGATTCACTGTGG-30 . For each PCR, a mastermix was prepared on ice, containing SensiFAST SYBR no-ROX mix (Bioline Reagents Ltd, London, UK), reverse and forward primers. The LightCycler 480 Real-Time PCR System (Roche Applied Science) was used with a 384-multiwell plate format. TAM receptor mRNA was normalized against TBP mRNA expression. Results presented represent at least three independent experiments.

Statistical Analysis Graphpad Prism version 7 (Graphpad Software, La Jolla, CA) was used for all statistical analyses. The R computation environment was used to plot the correlation matrices and to calculate the receiver operating characteristics curve analyses (c-indices). Comparisons between groups were performed using the Mann-Whitney test or Kruskal-Wallis test followed by Dunn post hoc comparisons, as appropriate. P < 0.05 was considered significant for all tests. Spearman

rank correlation coefficients were calculated to study the association (correlations) between sTAM receptors’ levels and clinical variables and to calculate multicollinearity in the correlation matrix. Renal clearance of specific sTAM receptors was calculated as follows: [urinary concentration (nanograms per milliliter)  daily urine volume (milliliters)]/[plasma concentration (nanograms per milliliter)  24 hours  60 minutes]. The fractional excretion of the sTAM receptors (ie, the renal clearance of the sTAMs relative to the renal clearance of creatinine) was calculated as follows: [100  (urinary sTAM concentration  plasma creatinine concentration)/ (plasma sTAM concentration  urinary creatinine concentration)]. To assess the performance of the measured parameters as markers to discriminate between patients with diabetes with and without albuminuria doubling of serum creatinine or a doubling of UACR on follow-up, receiver operating characteristics curve analysis was performed.

Results Cohort Characteristics The characteristics of both patient groups and healthy controls are given in Table 1. As expected, patients with mac- ½T1 roalbuminuria in DN group had significantly higher serum creatinine levels and reduced estimated glomerular filtration

Table 1 General Characteristics of the Patients with Type 2 Diabetes Mellitus and Macroalbuminuria and Normoalbuminuria Compared with Healthy Controls Type 2 diabetes mellitus Parameter

UAE <30 mg/24 hours (n Z 59)

UAE >300 mg/24 hours (n Z 67)

Healthy controls (n Z 18)

P value*

Males, n (%) Age, years Diabetes duration, years Body mass index Smoking history, n (%) Fasting glucose, mmol/L HbA1C, % Insulin therapy, n (%) Total cholesterol, mmol/L HDL-C, mmol/L LDL-C, mmol/L SBP, mm Hg DBP, mm Hg UAE, mg/24 hours Serum creatinine, mmol/L eGFRMDRD, mL/min ACEi/ARB therapy, n (%)

21 62 13 28 28 7.6 7.1 54 4.3 1.1 2.4 119 72 0 75 86 1

54 64 13 31 39 9.6 7.6 66 4.0 0.9 2.0 135 78 755 101 65 60

8 (44) 52 (41e64) d 23 (21e27) d 4.8 (3.8e6.6) 5.4 (5.0e5.8) d 5.3 (4.2e6.6) 1.4 (0.8e1.8) 3.1 (2.3e3.8) 120 (100e135) 78 (60e85) 0 (0e13) 76 (60e93) 100 (78e134) d

<0.001 0.04 0.40 0.03 0.30 0.03 0.40 0.10 0.20 0.007 0.10 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

(36) (32e81) (2e30) (21e54) (47%) (4.8e18.3) (4.1e13.2) (92%) (2.5e7.9) (0.4e2.7) (0.9e4.5) (88e154) (55e86) (0e12) (40e112) (55e162) (2%)

(81) (38e89) (3e34) (19e45) (58%) (4.8e18.0) (5.7e10.3) (99%) (2.7e7.7) (0.6e3.1) (0.6e5.6) (105e186) (58e99) (305e8540) (56e272) (20e111) (90%)

Data are expressed as median (range) unless otherwise indicated. *UAE <30 mg/24 hours versus UAE >300 mg/24 hours. ACEi, angiotensin-converting enzyme inhibitor; ARB, angiotensin II receptor blocker; DBP, diastolic blood pressure; eGFRMDRD, estimated glomerular filtration rate determined by the Modification of Diet in Renal Disease; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; SBP, systolic blood pressure UAE, urinary albumin excretion.

4

ajp.amjpathol.org

-

The American Journal of Pathology

FLA 5.4.0 DTD  AJPA2648_proof  28 June 2017  10:49 pm  EO: 2017_39

Q15

435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496

497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558

sTAM Receptors in Diabetic Nephropathy rate (eGFR), suggesting a significant decline in renal function when compared with controls with diabetes and normoalbuminuria. At the same time, the severity of diabetes was comparable in both study groups, as indicated by the lack of significant differences in HbA1c, duration of diabetes, and proportion of patients receiving insulin treatment. Still, patients with macroalbuminuria had increased body mass index (BMI), reduced high-density lipoprotein cholesterol levels, and elevated blood pressure when compared with controls with diabetes. Finally, patients with DN were slightly older, and the proportion of males in this group was also higher than in controls with diabetes. In line with the inclusion criteria, the healthy volunteers in the control group used to define normal values of sTAM receptors had normal glucose, HbA1c, blood pressure, cholesterol, and serum creatinine values.

Plasma Levels of Total and Free Protein S in DN ½F1

No significant differences were found between the study groups in plasma levels of total (Figure 1A), free (Figure 1B), or C4b binding proteinebound (data not shown) protein S, all approaching the levels measured in healthy individuals. Nevertheless, protein S levels correlated positively with the level of albuminuria (rs Z 0.19, P < 0.05 and rs Z 0.24, P < 0.01 for total and free protein S, respectively) and negatively to eGFR (rs Z 0.22, P < 0.01 and rs Z 0.18, P < 0.05) in the overall cohort.

Plasma Levels of sTAM Receptor Subtypes in DN Soluble forms of all three subtypes of TAM receptors were detected in plasma of both healthy controls and patients with diabetes, indicating a nonnormal skewed distribution (Figure 1, CeE). No significant difference was found between the study groups with diabetes in sTyro3 levels [mean (range), 4.40 (1.29 to 31.20) ng/mL versus 5.81 (0.97 to 32.2) ng/mL] (Figure 1C) or sAxl levels [mean (range), 17.1 (4.93 to 22.3) ng/mL versus 18.3 (10.6 to 24.1) ng/mL, for patients without and with DN, respectively] (Figure 1D), although in both cases the levels were significantly higher than the values measured in healthy controls [mean (range), 1.93 (0.79 to 25.0) ng/mL and 12.4 (8.10 to 18.7) ng/mL for sTyro3 and sAxl, respectively]. On the other hand, sMer plasma levels were elevated [mean (range), 14.4 (6.31 to 27.6) ng/mL versus 19.3 (9.29 to 47.4) ng/mL, P < 0.005] (Figure 1E) in patients with DN when compared with controls with diabetes, whereas no significant difference was found between the levels in patients without macroalbuminuria and healthy controls [mean (range), 13.2 (6.59 to 21.8) ng/mL]. Moreover, sMer systemic levels were significantly correlated with sTyro3 and sAxl levels in the whole cohort (sMer versus Tyro3 rs Z 0.45, P < 0.001; sMer versus sAxl rs Z 0.37, P < 0.001). Furthermore, plasma levels of sTyro3 were significantly related to the levels of HbA1c and

The American Journal of Pathology

-

559 560 561 562 563 564 Urinary Excretion of sTAM Receptor Subtypes in DN 565 566 Intriguingly, all three sTAM receptor subtypes were 567 excreted in the urine. Western blot analysis revealed that all 568 569 three sTAM receptors were present in plasma and urine in 570 full-length form (Axl, 120 kDa; Tyro3, 105 kDa; Mer, 200 571 kDa) (Supplemental Figure S1). Absolute and creatinine corrected urinary levels are given in Table 3. Urinary con- ½T3 572 573 centrations of sTyro3 had skewed distribution and large 574 variability (Table 3). Importantly, sTyro3 daily urinary 575 excretion was increased more than twofold in patients with 576 DN compared with patients with normoalbuminuric diabetes 577 [mean (range), 0.94 (0.20 to 4.07) mg/24 hours versus 2.10 578 (0.24 to 7.40) mg/24 hours, P < 0.001, for patients without 579 and with DN, respectively] (Figure 1F). Control sTyro3 580 581 excretion did not differ from the excretion in normoalbu582 minuric patients [mean (range), 1.06 (0.74 to 1.43) mg/24 583 hours] (Figure 1F). To estimate the role of systemic versus 584 renal contribution to urinary excretion of sTAM receptors, 585 renal clearance was calculated for each individual TAM 586 receptors subtype, as described in Materials and Methods. 587 sTyro3 renal clearance was significantly higher in patients 588 with DN when compared with controls with diabetes [mean 589 (range), 0.17 (0.01 to 0.66) mL/minutes versus 0.25 (0.05 to 590 2.10) mL/minutes, P < 0.001] (Figure 1I), suggesting 591 changes in renal sTyro3 handling (filtration/reabsorption) or 592 593 active production of sTyro3 in the kidney on DN. 594 Although no changes were observed in urinary excretion 595 of sAxl [mean (range), 46.0 (20.0 to 62.0) mg/24 hours 596 versus 44.5 (12.2 to 171) mg/24 hours versus 47.5 (14.8 to 597 150) mg/24 hours] (Table 3 and Figure 1G) or renal clear598 ance [mean (range), 2.26 (1.25 to 3.20) mL/minute versus 599 1.82 (0.54 to 5.79) mL/minute versus 1.81 (0.54 to 6.52) 600 mL/minute for healthy controls, patients with diabetes 601 without DN, and patients with diabetes and DN, respec602 tively] (Figure 1J), daily excretion of sMer was approxi603 mately doubled in patients with DN when compared with 604 605 controls with diabetes or healthy controls [mean (range), 606 1.23 (0.28 to 2.440) mg/24 hours versus 0.91 (0.14 to 2.33) 607 mg/24 hours versus 2.35 (0.49 to 7.72) mg/24 hours, 608 P < 0.001] (Table 3 and Figure 1H). Furthermore, renal 609 clearance of sMer was markedly increased as well [mean 610 (range), 0.046 (0.004 to 0.137) mL/minute versus 0.087 611 (0.008 to 0.323) mL/minute, P < 0.001] (Figure 1K), 612 indicating that in addition to elevated systemic sMer, 613 increased urinary sMer excretion is due to altered renal 614 handling and/or production of sMer in the diabetic kidneys. 615 In logistic regression, we observed that independent of the 616 617 eGFR, sTyro3 and sMer but not sAxl (Supplemental Table 618 S1) was associated with the prevalence of DN. A similar 619 pattern was observed when we investigated the fractional 620

UAE; plasma levels of sAxl were significantly related to BMI, HbA1c, UAE, and eGFR; and sMer was positively correlated with BMI, HbA1c, systolic blood pressure, eGFR, and UAE in the complete study population (Table 2). ½T2

ajp.amjpathol.org

FLA 5.4.0 DTD  AJPA2648_proof  28 June 2017  10:49 pm  EO: 2017_39

5

621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682

Ochodnicky et al

Figure 1 Comparison of plasma protein S levels (A and B) and concentrations of soluble TAM receptors (CeE) in healthy controls and patients with type 2 diabetes with [urinary albumin excretion (UAE) >300 mg/day] and without (UAE <30 mg/day) diabetic nephropathy (DN). Urinary excretion (FeH), renal clearance (IeK), and fractional excretion (LeN) of soluble TAM receptors in healthy controls and patients with type 2 diabetes with (UAE >300 mg/day) and without (UAE <30 mg/day) DN. Data are expressed as means  SEM (IeN). *P < 0.05, ***P < 0.005.

excretion of the sTAM receptors (Figure 1, LeN). In the theoretical situation of free sTAM receptor filtration and no renal sTAM receptor excretion, the maximum fractional excretion is 100% (taking into account that no creatinine is excreted by the renal tubules). In healthy controls, but also in patients with diabetes (with or without macroalbuminuria), the fractional excretion exceeded 100% by a factor two to four for sTyro3 and a factor 18 to 26 for sAxl,

6

indicating that potentially renal excretion (at least of these two sTAM receptors) takes place. In the complete cohort, urinary levels of sTyro3 and sMer correlated positively with BMI, blood pressure, and UAE, whereas urinary sAxl was only related to BMI and eGFR levels (Table 3). There was a significant positive relation between systemic and urinary levels for sMer (r Z 0.26, P < 0.01) but not sTyro3 or sAxl. In the correlation matrix,

ajp.amjpathol.org

-

The American Journal of Pathology

FLA 5.4.0 DTD  AJPA2648_proof  28 June 2017  10:49 pm  EO: 2017_39

683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744

745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806

sTAM Receptors in Diabetic Nephropathy Table 2

Associations between the Levels of sTAM Receptors and Clinical Parameters Per Group Circulating sTAM

Parameter BMI DM r p DMDN r p HC r p HbA1C, % DM r p DMDN r p HC r p SBP, mm Hg DM

Urinary sTAM

sTyro, ng/mL

sAxl, ng/mL

sMer, ng/mL

sTyro, pg/mL

sAxl, ng/mL

sMer, mg/mL

0.26 0.07

0.01 >0.99

0.08 0.60

0.22 0.12

0.31 0.03

0.30 0.03

0.14 0.30

0.11 0.42

0.16 0.24

0.14 0.28

0.25 0.06

0.19 0.16

0.26 0.35

0.28 0.31

0.04 0.88

0.06 0.83

0.15 0.62

0.39 0.17

0.03 0.82

0.02 0.90

0.06 0.68

0.07 0.64

0.12 0.38

0.21 0.14

0.08 0.56

0.09 0.49

0.12 0.38

0.20 0.12

0.21 0.10

0.11 0.41

0.29 0.28

0.55 0.03

0.01 0.98

0.11 0.71

0.14 0.62

0.41 0.13 Q16

0.09 p 0.56 DMDN r 0.34 P <0.01 HC r 0.01 p 0.98 eGFRMDRD, mL/min DM r 0.03 p 0.82 DMDN rS 0.04 p 0.74 HC r 0.22 p 0.41 UAE, mg/24 hours DM r 0.16 p 0.25 DMDN rS 0.07 p 0.58 HCs r 0.02 p 0.94 S

0.22 0.14

0.06 0.69

0.29 0.04

0.05 0.75

0.17 0.24

0.00 >0.99

0.13 0.33

0.14 0.31

0.06 0.65

0.18 0.18

0.04 0.88

0.24 0.41

0.41 0.16

0.49 0.09

0.07 0.82

0.25 0.07

0.08 0.57

0.04 0.77

0.06 0.66

0.08 0.56

0.13 0.33

0.16 0.24

0.30 0.02

0.52 <0.01

0.18 0.18

0.02 0.96

0.44 0.09

0.49 0.06

0.50 0.06

0.06 0.83

0.40 <0.01

0.13 0.37

0.33 0.02

0.25 0.08

0.29 0.04

0.08 0.52

0.06 0.66

0.22 0.09

0.07 0.61

0.33 0.01

0.15 0.58

0.28 0.29

0.60 0.02

0.69 <0.01

0.62 0.01

Numbers in bold indicate statistically significant associations. BMI, body mass index; DBP, diastolic blood pressure; DM, diabetes mellitus without nephropathy; DMDN, diabetes mellitus with diabetic nephropathy; eGFR, estimated glomerular filtration rate determined by the Modification of Diet in Renal Disease; HCs, healthy controls; SBP, systolic blood pressure; sTAM, soluble TAM; UAE, urinary albumin excretion.

The American Journal of Pathology

-

ajp.amjpathol.org

FLA 5.4.0 DTD  AJPA2648_proof  28 June 2017  10:49 pm  EO: 2017_39

7

807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868

Ochodnicky et al

print & web 4C=FPO

869 Table 3 Absolute and Creatinine-Corrected Urinary Levels of sTAM Receptors in Healthy Controls and Patients with Diabetes with and 870 without DN 871 Urinary concentration 872 Patients without DN Patients with DN 873 sTAM receptor Healthy controls (UAE <30 mg/24 hours) (UAE >300 mg/24 hours) 874 875 sTyro, pg/mL 484 (357e905) 495 (180e3443) 1152*y (180e3646) 876 sTyro, ng/mmolCr 96 (67e149) 87 (16e329) 150*y (12e632) 877 sAxl, ng/mL 22 (9e39) 24 (5e56) 23 (6e54) 878 sAxl, mg/mmolCr 4 (2e6) 4 (1e9) 3 (1e8) 879 sMer, pg/mL 649 (120e1254) 505 (120e2586) 1203*y (120e3086) 880 sMer, ng/mmolCr 121 (23e298) 93 (13e737) 173* (41e434) 881 Data are expressed as median (range). 882 *P < 0.05 versus patients with diabetes without DN. 883 y P < 0.05 versus healthy controls. 884 Cr, creatinine; DN, diabetic nephropathy; sTAM, soluble TAM. 885 886 887 we observed that particularly in healthy individuals, the conditions. Indeed, control renal biopsy specimens were 888 fractional excretion indices of the sTAM receptors were largely protein S negative, with only minor positivity in 889 strongly correlated with the corresponding serum concentubuli (Figure 2A), possibly reflecting reabsorption of the ½F2 890 Q10 trations (Supplemental Figure S2). This pattern was lost in filtered protein. In contrast, DN is characterized by promi891 nent glomerular deposition of protein S (Figure 2, B and C), patients with diabetes with or without nephropathy 892 whereas no significant difference was noted in tubular (Supplemental Figure S2, B and C). 893 protein S signal (Figure 2D). 894 895 Follow-Up and Predictive Value of sTAM Receptors 896 Changes in Renal Expression of TAM Receptor Subtypes 897 On follow-up, 39% of the patients with diabetes mellitus 898 had a doubling in UACR or a doubling of their serum in the Kidney after DN 899 creatinine level compared with baseline. The sTAM re900 All three subtypes of TAM receptors were found to be ceptors in the serum, the urine, or their fractional excretion 901 expressed in control and diabetic human kidneys by were not better at predicting progressive DN than albu902 immunohistochemistry. Tyro3 was abundantly expressed in minuria or the Modification of Diet in Renal Disease eGFR 903 proximal tubular epithelium (Figure 3A), and mild positivity ½F3 ½T4 (Table 4). 904 was observed in glomeruli (podocytes and endothelial cells). 905 Protein S Immunoreactivity in the Diabetic Kidneys 906 907 Being a typical systemic liverederived circulating antico908 909 agulant protein, protein S immunoreactivity would not be 910 readily expected in organs, such as kidneys, under normal 911 912 Table 4 AUROC Analysis for the Prediction of Doubling of Urinary 913 Albumin/Creatinine Ratio or Serum Creatinine on Follow-Up 914 0.621 (0.526e0.716) 915 Q17 eGFRMDRD Serum sTyro3 0.482 (0.367e0.597) 916 Urine sTyro3 0.567 (0.462e0.672) 917 FE sTyro3 0.515 (0.396e0.634) 918 Serum sAxl 0.552 (0.445e0.659) 919 Urine sAxl 0.517 (0.409e0.626) 920 FE sAxl 0.463 (0.349e0.578) 921 Serum sMer 0.468 (0.358e0.578) 922 Urine sMer 0.527 (0.416e0.639) 923 Figure 2 Representative immunohistochemical staining of protein S in Q11 FE sMER 0.505 (0.385e0.625) 924 the control kidneys (A; arrow indicate a positive staining of tubular 925 Q18 Data are expressed as median (range). epithelial cells) and in the kidneys of patients with diabetic nephropathy 926 AUROC, area under the receiver operating characteristic curve; eGFR, (DN) (B; arrows indicate extensive glomerular depositions of protein S). 927 estimated glomerular filtration rate determined by the Modification of Diet Quantitative comparison of glomerular (C) and tubular (D) score of protein 928 in Renal Disease; FE, fractional excretion index; s, soluble; UACR, urinary S staining in control and diabetic kidneys. Data are expressed as 929 Q19 albuminecreatinine ratio. Q12 means  SEM. *P < 0.05 versus control. 930

8

ajp.amjpathol.org

-

The American Journal of Pathology

FLA 5.4.0 DTD  AJPA2648_proof  28 June 2017  10:49 pm  EO: 2017_39

931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992

sTAM Receptors in Diabetic Nephropathy

The American Journal of Pathology

-

1055 Finally, Axl receptor was prominently expressed in 1056 glomeruli and renal vasculature, reflecting endothelial localization of this subtype (Figure 5A). In addition, clear ½F5 1057 1058 membrane staining of Axl was noticed in proximal tubuli 1059 (Figure 5A). Although hardly any changes were observed in 1060 Axl glomerular (Figure 5, B and C) or tubular (Figure 5, B 1061 and D) staining after DN, some CD68-positive interstitial 1062 macrophages had expression as well (Figure 5, E and F). 1063

Differential Expression of TAM Receptor Subtypes by Tubular Epithelial Cells in Vitro To further gain insight in the regulation of TAM receptor expression by tubular epithelial cells, we cultured human kidney HK-2 cells under diabetic conditions, using an established in vitro model of diabetic milieu.35 Incubation of cells with high glucose and transforming growth factor-b levels for 48 hours resulted in significant down-regulation of Tyro 3 and Mer mRNA, although Tyro3 mRNA levels were not different between the high-glucose and osmotic control circumstances (Figure 6, A and C). Axl mRNA levels remained unchanged among the different experimental conditions (Figure 6B). Interestingly, sTAM receptor protein concentrations in the culture supernatants did not reflect

1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 ½F6 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116

print & web 4C=FPO

print & web 4C=FPO

993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 Figure 3 A and B: Immunohistochemical staining of Tyro3 receptor in 1018 the control kidneys (A) and in the kidneys of patients with diabetic 1019 nephropathy (DN) (B). Arrows indicate the positive interstitial cells; as1020 terisks, the positive tubular epithelium. CeE: Quantitative comparison of 1021 glomerular (C), tubular (D), and interstitial (E) score of Tyro3 receptor 1022 staining in control and diabetic kidneys. F: Double staining of macrophage 1023 marker CD68 (red/brown) and Tyro3 (blue) shows co-localization (arrows) in the renal interstitium of patients with DN. Data are expressed as 1024 means  SEM. *P < 0.05 versus control. 1025 1026 1027 The latter was not altered in diabetic kidneys (Figure 3C); 1028 however, epithelial Tyro3 expression was markedly reduced 1029 in DN (Figure 3, B and D). Interestingly, abundant inter1030 stitial influx of Tyro3-positive infiltrating cells (Figure 3B), 1031 presumably macrophages, was found in diabetic kidneys, 1032 although such cells were also observed in normal kidneys, 1033 albeit in markedly lower number (Figure 3E). Indeed, using 1034 macrophage marker CD68 and Tyro3 staining, we 1035 confirmed massive interstitial influx of macrophages in 1036 diabetic kidneys with numerous cells double positive for 1037 both the macrophage marker and Tyro3 receptor 1038 1039 (Figure 3F). 1040 In contrast to ubiquitous renal expression of Tyro3, Mer 1041 receptor had a more restricted expression pattern, including 1042 specific subset of tubular epithelial cells, presumably in 1043 proximal portions, often strongly localized in the cell mem1044 ½F4 brane (Figure 4A). Little glomerular and weak interstitial 1045 expression of Mer was noticed in control kidneys. No changes 1046 in glomerular Mer expression were found in diabetic kidneys 1047 (Figure 4C). On the other hand, in diabetic kidneys, loss of 1048 tubular Mer staining (Figure 4, B and D) was associated with 1049 increased presence of Mer-positive interstitial cells (Figure 4, 1050 1051 B and E). Comparable to Tyro3 interstitial expression pattern, 1052 macrophage marker CD58 co-localized with Mer receptor in 1053 the renal interstitium (Figure 4F). 1054

Figure 4 A and B: Immunohistochemical staining of Mer receptor in the control kidneys (A; asterisk indicates the positive tubular epithelium) and in the kidneys of patients with diabetic retinopathy (DN) (B; arrow indicates a positive interstitial cell). CeE: Quantitative comparison of glomerular (C), tubular (D), and interstitial (E) score of Mer receptor staining in control and diabetic kidneys. F: Double staining of macrophage marker CD68 (red/brown) and Mer (blue) shows co-localization (arrow) in the renal interstitium of patients with DN. Data are expressed as means  SEM. *P < 0.05 versus control.

ajp.amjpathol.org

FLA 5.4.0 DTD  AJPA2648_proof  28 June 2017  10:49 pm  EO: 2017_39

9

with respect to the sAxl levels in the culture supernatants (Figure 6E). Taken together, these data suggest that diabetic circumstances result in down-regulation of Tyro3 and Mer on the mRNA level but at the same time cause increased shedding of the Tyro3 and Mer receptor proteins.

Discussion

print & web 4C=FPO

1117 1118 1119 1120 1121 1122 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178

Ochodnicky et al

Figure 5

A and B: Immunohistochemical staining of Axl receptor in the control kidneys (A) and in the kidneys of patients with diabetic retinopathy (DN; B). Asterisk indicates the positive tubular epithelium; arrow, interstitial vascular staining. CeE: Quantitative comparison of glomerular (C), tubular (D), and interstitial (E) score of Axl receptor staining in control and diabetic kidneys. F: Double staining of macrophage marker CD68 (red/ brown) and Axl (blue) shows co-localization (arrows) in the renal interstitium of patients with DN. Arrowheads point to the vascular Axl staining of vascular endothelium. Data are expressed as means  SEM.

the cellular mRNA levels. sTyro3 and Mer levels were significantly increased after 48 hours of incubation with high glucose and transforming growth factor-b levels (Figure 6, D and F). Again, no differences were measured

In the present study, we provide evidence that type 2 diabetes is associated with excessive circulating levels of sTAM receptors sTyro3 and sAxl, whereas sMer levels are increased only in patients with diabetes and DN. Surprisingly, all sTAM receptors are excreted in the urine, and urinary excretion of sTyro3 is enhanced in patients with diabetes and DN. This is associated with the loss of tubular Tyro3 and Mer expression in diabetic kidneys. In vitro, tubular epithelial cells respond to diabetic circumstances with a down-regulation of Tyro3 and Mer mRNA production, and at the same time the shedding of sTyro3 and sMer is increased. Furthermore, DN is characterized by excessive infiltration of Mer-, Tyro3-, and Axl-positive macrophages and glomerular deposition of TAM ligand protein S. So far, elevations in circulating levels of TAM receptors sMer and sTyro3 have been reported in patients with autoimmune disease, including systemic lupus erythematosus and rheumatoid arthritis.30,37,38 It has been proposed that excessive sMer levels might reflect impaired clearance of apoptotic debris and excessive cytokine production by macrophages and dendritic cells, both key pathologic characteristics of autoimmune disease. The current study suggests that a primarily nonimmune disease, such as type 2 diabetes, might be associated with systemic elevations of sTAM receptors as well. Diabetes mellitus is characterized by chronic low-grade inflammation, activation of innate immune system, and elevated systemic levels of cytokines,

Figure 6 TAM receptor production and shedding in human tubular epithelial cells. A and C: Incubation of cells with high glucose (30 mmol/L) and 10 ng/mL of transforming growth factor (TGF)b for 48 hours results in significant downregulation of Tyro 3 (A) and Mer mRNA (C), although Tyro33 mRNA levels are not different between the high-glucose and osmotic (mannitol) control circumstances. B: Axl mRNA levels remain unchanged among the different experimental conditions. Soluble TAM (sTAM) receptor protein concentrations in the supernatant were determined by enzyme-linked immunosorbent assay. D and F: Soluble Tyro3 (sTyro3) (D) and soluble Mer (sMer) (F) levels are significantly increased after 48 hours incubation with high glucose (30 mmol/ L) and 10 ng/mL of TGF-b. E: sAxl levels remain unchanged. Data are expressed as means  SEM. n Z 3 independent experiments. *P < 0.05, **P < 0.01. AU, arbitrary units; Glu, glucose; Man, mannitol.

10

ajp.amjpathol.org

-

The American Journal of Pathology

FLA 5.4.0 DTD  AJPA2648_proof  28 June 2017  10:49 pm  EO: 2017_39

Q13

Q14

1179 1180 1181 1182 1183 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240

1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 1253 1254 1255 1256 1257 1258 1259 1260 1261 1262 1263 1264 1265 1266 1267 1268 1269 1270 1271 1272 1273 1274 1275 1276 1277 1278 1279 1280 1281 1282 1283 1284 1285 1286 1287 1288 1289 1290 1291 1292 1293 1294 1295 1296 1297 1298 1299 1300 1301 1302

sTAM Receptors in Diabetic Nephropathy such as IL-1b, IL-6, or tumor necrosis factor-a,39 potentially negatively regulated by TAM receptors. Direct correlations between circulating levels of sTyro3, sAxl, and sMer on the one hand and clinical parameters, such as BMI or the level of glycemic control (HbA1c), on the other hand may suggest that elevations of TAM receptors in patients with diabetes are the reflection of such systemic metabolically induced inflammation. Possibly, systemic or resident immune cells, such as monocytes or macrophages, might represent the primary source for circulating TAM receptor elevations, although TAM receptors are expressed by many other systemic cell types, including vascular endothelium and platelets.40,41 For instance, shedding of the sMer domain in macrophages is probably initiated by metalloproteinase ADAM17.29 Increased ADAM17 expression along with reduced Mer expression have been recently described in circulating monocytes isolated from patients undergoing dialysis.32 On the other hand, in our study, systemic levels of sMer were increased only in patients with DN when compared with patients with diabetes and normoalbuminuria. This finding is in line with a previous finding that renal function loss is associated with increase in sMer circulating levels in a mixed population of patients with chronic kidney disease.32 Increase of systemic sMer levels on renal injury might reflect more severe cardiovascular and metabolic dysfunction associated with impaired renal function or alternatively indicate contribution of kidneyderived sMer to circulating levels. Indeed, in the current study, DN was characterized by excessive renal TAM receptorepositive macrophage infiltration and reductions in Tyro3 and Mer receptor expression in renal cell types, such as tubular epithelium. Notably, we recently found an excessive renal expression of Mer-shedding protease ADAM17 in patients with DN.35 Collectively, in contrast to sTyro3 and sAxl levels affected only by metabolic and/or cardiovascular disease, systemic sMer concentration is directly or indirectly elevated by diabetic renal dysfunction. It remains unclear whether renal shedding of Mer receptor might contribute to the systemic changes. Intriguingly, we have found all three soluble receptors being excreted in urine in a range of micrograms per day. This is the first study to find urinary excretion of sTAM receptors in healthy individuals and patients with diabetes. It is a rather surprising finding because the soluble forms of TAM receptors are high-molecular-weight proteins (105 to 200 kDa)31 that are not likely to be filtered in intact glomeruli. All three subtypes are expressed in tubular epithelium of normal as well as diabetic kidneys and could be shed in tubular lumen, leading to urinary excretion of sTAM. In addition, urinary excretion of sMer and sTyro3 is increased on diabetic renal injury when compared with patients with dibetes and normoalbuminuria. Interestingly, the fractional excretion of two of the sTAMs exceeds 100%, which means that there is significant intrarenal production and shedding of TAMs into the urine. The observed reduced tubular expression of sTyro3 and sMer in diabetic kidney

The American Journal of Pathology

-

biopsy specimens supports this finding. Western blot analysis revealed that sTAM receptors are excreted in full-length form, which suggests active production of TAM receptors in the kidney under healthy and disease conditions. In addition, our in vitro data indicate decreased Tyro3 and Mer mRNA and increased Tyro3 and Mer shedding under diabetic circumstances. On the other hand, urinary sMer levels correlate with sMer circulating levels and in addition are directly proportional to the severity of albuminuria in patients with DN, indicating at least a partial contribution of excessive filtration through damaged glomeruli to urinary sMer excretion. Because the exact role of TAM receptors in the development of kidney injury has not been defined so far, we can only speculate about the functional consequences of excessive sTAM shedding. TAM receptors are required for limitation of cytokine production in macrophages16,42; therefore, reduced expression and/or excessive shedding might contribute to the activation of circulating or renal interstitial macrophages on inflammatory injury. Furthermore, protein Seinduced activation of Tyro3 receptors protects microvascular endothelial cells against apoptosis and preserves endothelial barrier function.25,40 Because Tyro3 is expressed in glomeruli, it might contribute to the regulation of glomerular permeability. Interestingly, although circulating levels of protein S were not different in patients with diabetes, we observed marked glomerular deposits of protein S in diabetic kidneys, suggesting that the TAM ligand local renal levels are increased on injury. Furthermore, systemic protein S levels were positively correlated with albuminuria in patients with diabetes. This is in line with a recent study that found elevated protein S levels in patients with chronic kidney disease.43 The is similarly true for circulating levels of Gas-6,43 an additional ligand of Axl and other TAM subtypes. Glomerular Gas-6 has been proposed to stimulate mesangial Axl, inducing glomerular hypertrophy in DN.26,44 We, however, did not detect any changes in Axl glomerular staining in our study. Furthermore, Gas-6 circulating levels could be related to systemic vascular pathology because Gas-6 and Axl expressed on vascular smooth muscle cells seem to be involved in prevention of vascular calcifications associated with chronic kidney disease.43,45 This was, however, not reflected by any changes in circulating sAxl levels in DN in the current study. In conclusion, renal injury in type 2 diabetes is associated with elevated systemic levels of sMer and increased urinary excretion of sMer and sTyro3. In the current study, we found a measurable excretion of sTAM in the urine, attributable to filtration and local tubular production or shedding, thus identifying the kidney as a potential source of sTAM shedding. We could not establish an additional role for sTAM receptors as predictive biomarkers of renal functional decline besides albuminuria. Further studies will be required to gain better understanding of the mechanisms of TAM shedding in renal epithelial cells and the role of

ajp.amjpathol.org

FLA 5.4.0 DTD  AJPA2648_proof  28 June 2017  10:49 pm  EO: 2017_39

11

1303 1304 1305 1306 1307 1308 1309 1310 1311 1312 1313 1314 1315 1316 1317 1318 1319 1320 1321 1322 1323 1324 1325 1326 1327 1328 1329 1330 1331 1332 1333 1334 1335 1336 1337 1338 1339 1340 1341 1342 1343 1344 1345 1346 1347 1348 1349 1350 1351 1352 1353 1354 1355 1356 1357 1358 1359 1360 1361 1362 1363 1364

1365 1366 1367 1368 1369 1370 1371 1372 1373 1374 1375 1376 1377 1378 1379 1380 1381 1382 1383 1384 1385 1386 1387 1388 1389 1390 1391 1392 1393 1394 1395 1396 1397 1398 1399 1400 1401 1402 1403 1404 1405 1406 1407 1408 1409 1410 1411 1412 1413 1414 1415 1416 1417 1418 1419 1420 1421 1422 1423 1424 1425 1426

Ochodnicky et al TAM receptors and their ligands in the development of renal injury. 21.

Q20

Supplemental Data

22.

Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.ajpath.2017.05.004.

23.

References 24. 1. Collins AJ, Foley RN, Chavers B, Gilbertson D, Herzog C, Ishani A, et al: US Renal Data System 2013 Annual Data Report. Am J Kidney Dis 2014, 63(Suppl 1):A7 2. Gross JL, de Azevedo MJ, Silveiro SP, Canani LH, Caramori ML, Zelmanovitz T: Diabetic nephropathy: diagnosis, prevention, and treatment. Diabetes Care 2005, 28:164e176 3. Diez-Sampedro A, Lenz O, Fornoni A: Podocytopathy in diabetes: a metabolic and endocrine disorder. Am J Kidney Dis 2011, 58: 637e646 4. Jefferson JA, Shankland SJ, Pichler RH: Proteinuria in diabetic kidney disease: a mechanistic viewpoint. Kidney Int 2008, 74:22e36 5. Satchell SC, Tooke JE: What is the mechanism of microalbuminuria in diabetes: a role for the glomerular endothelium? Diabetologia 2008, 51:714e725 6. Forbes JM, Cooper ME: Mechanisms of diabetic complications. Physiol Rev 2013, 93:137e188 7. Qian Y, Feldman E, Pennathur S, Kretzler M, Brosius FC III: From fibrosis to sclerosis: mechanisms of glomerulosclerosis in diabetic nephropathy. Diabetes 2008, 57:1439e1445 8. Galkina E, Ley K: Leukocyte recruitment and vascular injury in diabetic nephropathy. J Am Soc Nephrol 2006, 17:368e377 9. Navarro-Gonzalez JF, Mora-Fernandez C, Muros de Fuentes M, Garcia-Perez J: Inflammatory molecules and pathways in the pathogenesis of diabetic nephropathy. Nat Rev Nephrol 2011, 7:327e340 10. Nguyen D, Ping F, Mu W, Hill P, Atkins RC, Chadban SJ: Macrophage accumulation in human progressive diabetic nephropathy. Nephrology (Carlton) 2006, 11:226e231 11. Tesch GH: Macrophages and diabetic nephropathy. Semin Nephrol 2010, 30:290e301 12. Rothlin CV, Ghosh S, Zuniga EI, Oldstone MB, Lemke G: TAM receptors are pleiotropic inhibitors of the innate immune response. Cell 2007, 131:1124e1136 13. Linger RM, Keating AK, Earp HS, Graham DK: TAM receptor tyrosine kinases: biologic functions, signaling, and potential therapeutic targeting in human cancer. Adv Cancer Res 2008, 100:35e83 14. Lemke G, Rothlin CV: Immunobiology of the TAM receptors. Nat Rev Immunol 2008, 8:327e336 15. Lemke G, Burstyn-Cohen T: TAM receptors and the clearance of apoptotic cells. Ann N Y Acad Sci 2010, 1209:23e29 16. Lemke G, Lu Q: Macrophage regulation by Tyro 3 family receptors. Curr Opin Immunol 2003, 15:31e36 17. Lu Q, Lemke G: Homeostatic regulation of the immune system by receptor tyrosine kinases of the Tyro 3 family. Science 2001, 293: 306e311 18. Rothlin CV, Lemke G: TAM receptor signaling and autoimmune disease. Curr Opin Immunol 2010, 22:740e746 19. Hafizi S, Dahlback B: Gas6 and protein S. Vitamin K-dependent ligands for the Axl receptor tyrosine kinase subfamily. FEBS J 2006, 273:5231e5244 20. Stitt TN, Conn G, Gore M, Lai C, Bruno J, Radziejewski C, Mattsson K, Fisher J, Gies DR, Jones PF, Masiakowski P, Ryan TE, Tobkes NJ, Chen DH, DiStefano PS, Long GL, Basilico C, Goldfarb MP, Lemke G, Glass DJ, Yancopoulos GD: The

12

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

anticoagulation factor protein S and its relative, Gas6, are ligands for the Tyro 3/Axl family of receptor tyrosine kinases. Cell 1995, 80: 661e670 Castoldi E, Hackeng TM: Regulation of coagulation by protein S. Curr Opin Hematol 2008, 15:529e536 Rezende SM, Simmonds RE, Lane DA: Coagulation, inflammation, and apoptosis: different roles for protein S and the protein S-C4b binding protein complex. Blood 2004, 103:1192e1201 Takagi T, Taguchi O, Aoki S, Toda M, Yamaguchi A, Fujimoto H, Boveda-Ruiz D, Gil-Bernabe P, Ramirez AY, Naito M, Yano Y, D’Alessandro-Gabazza CN, Fujiwara A, Takei Y, Morser J, Gabazza EC: Direct effects of protein S in ameliorating acute lung injury. J Thromb Haemost 2009, 7:2053e2063 Zhong Z, Wang Y, Guo H, Sagare A, Fernandez JA, Bell RD, Barrett TM, Griffin JH, Freeman RS, Zlokovic BV: Protein S protects neurons from excitotoxic injury by activating the TAM receptor Tyro3-phosphatidylinositol 3-kinase-Akt pathway through its sex hormone-binding globulin-like region. J Neurosci 2010, 30: 15521e15534 Zhu D, Wang Y, Singh I, Bell RD, Deane R, Zhong Z, Sagare A, Winkler EA, Zlokovic BV: Protein S controls hypoxic/ischemic bloodbrain barrier disruption through the TAM receptor Tyro3 and sphingosine 1-phosphate receptor. Blood 2010, 115:4963e4972 Nagai K, Arai H, Yanagita M, Matsubara T, Kanamori H, Nakano T, Iehara N, Fukatsu A, Kita T, Doi T: Growth arrest-specific gene 6 is involved in glomerular hypertrophy in the early stage of diabetic nephropathy. J Biol Chem 2003, 278:18229e18234 Yanagita M, Arai H, Ishii K, Nakano T, Ohashi K, Mizuno K, Varnum B, Fukatsu A, Doi T, Kita T: Gas6 regulates mesangial cell proliferation through Axl in experimental glomerulonephritis. Am J Pathol 2001, 158:1423e1432 Budagian V, Bulanova E, Orinska Z, Duitman E, Brandt K, Ludwig A, Hartmann D, Lemke G, Saftig P, Bulfone-Paus S: Soluble Axl is generated by ADAM10-dependent cleavage and associates with Gas6 in mouse serum. Mol Cell Biol 2005, 25:9324e9339 Thorp E, Vaisar T, Subramanian M, Mautner L, Blobel C, Tabas I: Shedding of the Mer tyrosine kinase receptor is mediated by ADAM17 protein through a pathway involving reactive oxygen species, protein kinase Cdelta, and p38 mitogen-activated protein kinase (MAPK). J Biol Chem 2011, 286:33335e33344 Wu J, Ekman C, Jonsen A, Sturfelt G, Bengtsson AA, Gottsater A, Lindblad B, Lindqvist E, Saxne T, Dahlback B: Increased plasma levels of the soluble Mer tyrosine kinase receptor in systemic lupus erythematosus relate to disease activity and nephritis. Arthritis Res Ther 2011, 13:R62 Sather S, Kenyon KD, Lefkowitz JB, Liang X, Varnum BC, Henson PM, Graham DK: A soluble form of the Mer receptor tyrosine kinase inhibits macrophage clearance of apoptotic cells and platelet aggregation. Blood 2007, 109:1026e1033 Lee IJ, Hilliard BA, Ulas M, Yu D, Vangala C, Rao S, Lee J, Gadegbeku CA, Cohen PL: Monocyte and plasma expression of TAM ligand and receptor in renal failure: links to unregulated immunity and chronic inflammation. Clin Immunol 2015, 158: 231e241 Ahdi M, Gerdes VE, Graaff R, Kuipers S, Smit AJ, Meesters EW: Skin autofluorescence and complications of diabetes: does ethnic background or skin color matter? Diabetes Technol Ther 2015, 17: 88e95 Schnog JB, Mac Gillavry MR, van Zanten AP, Meijers JC, Rojer RA, Duits AJ, ten Cate H, Brandjes DP: Protein C and S and inflammation in sickle cell disease. Am J Hematol 2004, 76:26e32 Lattenist L, Ochodnický P, Ahdi M, Claessen N, Leemans JC, Satchell SC, Florquin S, Gerdes VE, Roelofs JJ: Renal endothelial protein C receptor expression and shedding during diabetic nephropathy. J Thromb Haemost 2016, 14:1171e1182 Strober W: Trypan blue exclusion test of cell viability. Curr Protoc Immunol 2001, Appendix 3. Appendix 3B

ajp.amjpathol.org

-

The American Journal of Pathology

FLA 5.4.0 DTD  AJPA2648_proof  28 June 2017  10:49 pm  EO: 2017_39

1427 1428 1429 1430 1431 1432 1433 1434 1435 1436 1437 1438 1439 1440 1441 1442 1443 1444 1445 1446 1447 1448 1449 1450 1451 1452 1453 1454 1455 1456 1457 1458 1459 1460 1461 1462 1463 1464 1465 1466 1467 1468 1469 1470 1471 1472 1473 1474 1475 1476 1477 1478 1479 1480 1481 1482 1483 1484 1485 1486 1487 1488

1489 1490 1491 1492 1493 1494 1495 1496 1497 1498 1499 1500 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 1511 1512 1513 1514 1515 1516 1517 1518 1519 1520 1521 1522 1523 1524 1525 1526 1527 1528 1529 1530 1531 1532 1533 1534 1535 1536 1537 1538 1539 1540 1541 1542 1543 1544 1545 1546 1547 1548 1549 1550

sTAM Receptors in Diabetic Nephropathy 37. Recarte-Pelz P, Tassies D, Espinosa G, Hurtado B, Sala N, Cervera R, Reverter JC, de Frutos PG: Vitamin K-dependent proteins GAS6 and Protein S and TAM receptors in patients of systemic lupus erythematosus: correlation with common genetic variants and disease activity. Arthritis Res Ther 2013, 15:R41 38. Zizzo G, Guerrieri J, Dittman LM, Merrill JT, Cohen PL: Circulating levels of soluble MER in lupus reflect M2c activation of monocytes/macrophages, autoantibody specificities and disease activity. Arthritis Res Ther 2013, 15:R212 39. Pickup JC: Inflammation and activated innate immunity in the pathogenesis of type 2 diabetes. Diabetes Care 2004, 27:813e823 40. Fraineau S, Monvoisin A, Clarhaut J, Talbot J, Simonneau C, Kanthou C, Kanse SM, Philippe M, Benzakour O: The vitamin Kdependent anticoagulant factor, protein S, inhibits multiple VEGF-Ainduced angiogenesis events in a Mer- and SHP2-dependent manner. Blood 2012, 120:5073e5083 41. Gould WR, Baxi SM, Schroeder R, Peng YW, Leadley RJ, Peterson JT, Perrin LA: Gas6 receptors Axl, Sky and Mer enhance

The American Journal of Pathology

-

42.

43.

44.

45.

platelet activation and regulate thrombotic responses. J Thromb Haemost 2005, 3:733e741 Deng T, Zhang Y, Chen Q, Yan K, Han D: Toll-like receptor-mediated inhibition of Gas6 and ProS expression facilitates inflammatory cytokine production in mouse macrophages. Immunology 2012, 135: 40e50 Lee IJ, Hilliard B, Swami A, Madara JC, Rao S, Patel T, Gaughan JP, Lee J, Gadegbeku CA, Choi ET, Cohen PL: Growth arrest-specific gene 6 (Gas6) levels are elevated in patients with chronic renal failure. Nephrol Dial Transplant 2012, 27:4166e4172 Nagai K, Matsubara T, Mima A, Sumi E, Kanamori H, Iehara N, Fukatsu A, Yanagita M, Nakano T, Ishimoto Y, Kita T, Doi T, Arai H: Gas6 induces Akt/mTOR-mediated mesangial hypertrophy in diabetic nephropathy. Kidney Int 2005, 68:552e561 Collett GD, Sage AP, Kirton JP, Alexander MY, Gilmore AP, Canfield AE: Axl/phosphatidylinositol 3-kinase signaling inhibits mineral deposition by vascular smooth muscle cells. Circ Res 2007, 100:502e509

ajp.amjpathol.org

FLA 5.4.0 DTD  AJPA2648_proof  28 June 2017  10:49 pm  EO: 2017_39

13

1551 1552 1553 1554 1555 1556 1557 1558 1559 1560 1561 1562 1563 1564 1565 1566 1567 1568 1569 1570 1571 1572 1573 1574 1575 1576 1577 1578 1579 1580 1581 1582 1583 1584 1585 1586 1587 1588 1589 1590 1591 1592 1593 1594 1595 1596 1597 1598 1599 1600 1601 1602 1603 1604 1605 1606 1607 1608 1609 1610 1611 1612

1613 1614 1615 1616 1617 1618 1619 1620 1621 1622 1623 1624 1625 1626 1627 1628 1629 1630 1631 1632 1633 1634 1635 1636 1637 1638 1639 1640 1641 1642 1643 1644 1645 1646 1647 1648 1649 1650 1651 1652 1653 1654 1655 1656 1657 1658 1659 1660 1661 1662 1663 1664 1665 1666 1667 1668 1669 1670 1671 1672 1673 1674 1675 1676 1677 1678 1679 1680

Supplemental Figure S1 Western blot analysis of soluble Tyro3, Axl, and Mer in urine (A) and matched plasma (B) samples of nine randomly chosen individuals; three healthy (ctrl), three with diabetic nephropathy (DN) and normoalbuminuria (DN normo), and three with DN and macroalbuminuria (DN macro). Supplemental Figure S2

Correlation matrices with unsupervised hierarchical clustering in healthy individuals (A), patients with diabetes mellitus without nephropathy (B), and patients with diabetes mellitus with nephropathy (C). eGFR, estimated glomerular filtration rate; FE, fractional excretion; UAE, urinary albumin excretion.

FLA 5.4.0 DTD  AJPA2648_proof  28 June 2017  10:49 pm  EO: 2017_39

1681 1682 1683 1684 1685 1686 1687 1688 1689 1690 1691 1692 1693 1694 1695 1696 1697 1698 1699 1700 1701 1702 1703 1704 1705 1706 1707 1708 1709 1710 1711 1712 1713 1714 1715 1716 1717 1718 1719 1720 1721 1722 1723 1724 1725 1726 1727 1728 1729 1730 1731 1732 1733 1734 1735 1736 1737 1738 1739 1740 1741 1742 1743 1744 1745 1746 1747 1748