The Structure and Nephroprotective Activity of Oligo-Porphyran on Glycerol-Induced Acute Renal Failure in Rats

marine drugs

Article The Structure and Nephroprotective Activity of Oligo-Porphyran on Glycerol-Induced Acute Renal Failure in Rats

Jing Wang 1,2, Yun Hou 1,3, Delin Duan 1,2,4 and Quanbin Zhang 1,2,* 1 Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, Shandong, China; [email protected] (J.W.); [email protected] (Y.H.); [email protected] (D.D.) 2 Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China 3 Graduate University of the Chinese Academy of Sciences, Beijing 100049, China 4 State Key Lab of Seaweed Bioactive Substances, Qingdao 266500, China * Correspondence: [email protected]; Tel.: +86-532-8289-8708

Academic Editor: Keith B. Glaser Received: 1 February 2017; Accepted: 4 May 2017; Published: 9 May 2017

Abstract: Porphyran is a sulfate galactan in the cell wall of Porphyra. Its acid hydrolysis product, oligo-porphyran (OP), was prepared and the structure studied by electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). This oligosaccharide was mainly composed of monosulfate-oligo-galactan, disufate-oligo-galactan, trisulfate-oligo-galactan, trisulfate oligo-methyl-galactan, and 3,6-anhydrogalactose with the degree of polymerization ranging from 1 to 8. The effects of OP were investigated in the glycerol-induced acute renal failure (ARF) model. Compared with the normal group, rats from the glycerol-induced group exhibited collecting duct and medullary ascending limb dilation and casts. The OP-treated group exerted a protective effect against glycerol-induced changes. The results showed that the administration of OP markedly decreased mortality in female ARF rats. For male ARF rats, all of which survived, OP significantly decreased the blood urea nitrogen and serum creatinine levels. Ion levels in plasma and urine were significantly changed in ARF rats, whereas OP treatment almost recovered ion levels back to normal. This study showed a noticeable renal morphologic and functional protection by OP in glycerol-induced ARF rats.

Keywords: sulfated galactan; oligo-porphyran; acute renal failure; gender difference

1. Introduction Acute renal failure (ARF) is a syndrome characterized by a rapid loss of kidney function [1]. During ARF, tubular fluid flow rate and glomerular filtration rate decrease rapidly. Subsequently, the kidney is unable to produce sufficient amounts of urine, and nitrogen-containing waste substances accumulate in the blood [2–4]. ARF is a contributor to many other diseases, including cardiovascular diseases, hypertension, and diabetes mellitus [5]. This syndrome has a major impact on the life quality of patients, and it is even life-threatening in some conditions [6]. Porphyran, with a linear backbone of alternating 3-linked β-D-galactose and 4-linked α-L-galactose-6-sulfate or 3,6-anhydro-α-L-galactose (3,6-AG) units [7], is the main polysaccharide in the cell wall of Porphyra. Porphyran has various biological activities, which are mainly attributed to its sulfated group [8,9]. In our previous study, we found that the sulfated polysaccharides from Laminaria and its hydrolysis products had protective effect on the rats with renal impairment [10,11]. One of an important factor for glycerol-induced renal injury is oxidative stress [12–14]. Porphyran has been proven to have strong antioxidant activity in vitro, however, few people studied its inhibition

Mar. Drugs 2017, 15, 135; doi:10.3390/md15050135 www.mdpi.com/journal/marinedrugs Mar. Drugs 2017, 15, 135 2 of 13 has been proven to have strong antioxidant activity in vitro, however, few people studied its Mar. Drugs 2017, 15, 135 2 of 13 inhibition effect on ARF. Glycerol was a common agent for the induction of ARF in vivo and can lead to renal oxidative stress, as previously reported [15]. Therefore, the protective effect on glycerol‐ inducedeffect on ARF ARF. of Glycerol porphyan was is obvious. a common agent for the induction of ARF in vivo and can lead to renal oxidativeResearchers stress, asfound previously many factors reported of [sulfated15]. Therefore, polysaccharides, the protective such effect as molecular on glycerol-induced weight, degree ARF ofof sulfation, porphyan and is obvious. type of sugar, that influence their antioxidant activity. The earlier studies in our laboratoryResearchers showed found that manydifferent factors molecular of sulfated weights polysaccharides, of porphyan suchcould as influence molecular the weight, antioxidant degree activityof sulfation, [16]. Lower and type molecular of sugar, weight that influence porphyan their exhibited antioxidant stronger activity. antioxidant The earlier activity studies compared in our withlaboratory native porphyan. showed that In this different paper, molecular we prepared weights oligo of‐porphyran porphyan by could acid hydrolysis influence the and antioxidant evaluated theactivity structure [16]. and Lower effect molecular of oligo weight‐porphyran porphyan on the exhibited glycerol stronger‐induced antioxidant ARF in rats. activity Dexamethasone compared was with usednative in porphyan.the positive In control this paper, group we due prepared to its anti oligo-porphyran‐inflammatory and by acid antioxidant hydrolysis property and evaluated in ARF rats the [6].structure The levels and effectof blood of oligo-porphyran urea nitrogen (BUN), on the serum glycerol-induced creatinine (SCR), ARF in and rats. ions Dexamethasone in the rats’ blood was usedand urinein the were positive investigated. control group The dueaim toof itsthis anti-inflammatory study was to clarify and antioxidantthe structure property and nephroprotective in ARF rats [6]. activityThe levels of oligo of blood‐porphyran urea nitrogen on glycerol (BUN),‐induced serum creatinineacute renal (SCR), failure and in ionsrats. in the rats’ blood and urine were investigated. The aim of this study was to clarify the structure and nephroprotective activity of 2.oligo-porphyran Results on glycerol-induced acute renal failure in rats.

2.1.2. Results The Structure Study of Oligo‐Porphran

2.1.1.2.1. The Preparation, Structure StudyFraction, of Oligo-Porphran and Analysis of Oligo‐Porphyran (OP)

2.1.1.The Preparation, chemical composition Fraction, and of Analysis the oligo of‐porphyran Oligo-Porphyran (OP) used (OP) in this study was shown in Table 1. It contains 71.39% galactose, 7.05% 3,6‐anhydrogalactose, and 18.32% sulfate groups. Its average The chemical composition of the oligo-porphyran (OP) used in this study was shown in Table1. molecular weight was 1431 Da. This oligosaccharide was mainly composed of sulfated galactan with It contains 71.39% galactose, 7.05% 3,6-anhydrogalactose, and 18.32% sulfate groups. Its average the degree of polymerization ranging from 1 to 8. molecular weight was 1431 Da. This oligosaccharide was mainly composed of sulfated galactan with the degree of polymerization ranging from 1 to 8. Table 1. The chemical analysis of OP.

Mw SulfateTable 1. GroupThe chemical Galactose analysis of OP.3,6‐anhydrogalactose Sample (Kda) (%) (%) (%) SampleOP Mw (Kda)1.43 16.90 Sulfate ± 0.51 Group (%)59.93 ± Galactose 1.44 (%)6.45 3,6-anhydrogalactose ± 0.17 (%) OP 1.43 16.90 ± 0.51 59.93 ± 1.44 6.45 ± 0.17 The eluting profile of chromatography of OP was shown in Figure 1. Three fractions were obtained:The F1, eluting F2, and proﬁle F3 from of chromatography 0 to 0.3 M NaCl of elutions OP was from shown column in Figure chromatography,1. Three fractions and the were F4 fractionobtained: was F1, obtained F2, and from F3 from a 0.5 0 toM 0.3NaCl M elution. NaCl elutions The sulfate from group column of chromatography, F1–F4 was 16.54%, and 16.06%, the F4 18.98%,fraction and was 23.18%, obtained respectively. from a 0.5 M NaCl elution. The sulfate group of F1–F4 was 16.54%, 16.06%, 18.98%, and 23.18%, respectively.

2 0.6 Water 0.5 1.5 0.4 F1 F4 1 F2 F3 0.3 0.2 0.5

0.1 NaCl（mol/L） Absorbance 0 0 0 50 100 150 200 250 Elution volume(mL)

Figure 1. Chromatography of eluted OP on DEAE-sepharose CL-6B chromatography. Figure 1. Chromatography of eluted OP on DEAE‐sepharose CL‐6B chromatography.

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2.1.2. Structure of Oligo-PorphyranOligo‐Porphyran (OP) The degree of polymerization (DP) of OP and its fractions fractions F1–F4 F1–F4 was was tested tested by by HPLC HPLC (Figure (Figure 22).). The number of sulfate groups and DPs of OP increased from F1 to F4. The sulfate group numbers of F1~F4 were were 1, 2, 3, and >3, respectively, respectively, while the DPs of their main components were were 2–4, 4–6, 6–8, and 7–13, respectively.

Figure 2. Cont.

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FigureFigure 2. 2. TheThe degreedegree ofof polymerizationpolymerization of of OP OP and and its its fractions fractions tested tested by by HPLC. HPLC.

WeWe studied the the molecular molecular weight weight distribution distribution and and structure structure of F1–F4 of F1–F4 using ESI using‐TOF ESI-TOF-MS‐MS (Figure (Figure3). From3). the From MS‐spectrum the MS-spectrum of F1, we found of F1, a wedistribution found a of distribution singly‐charged of singly-chargedions corresponding ions to − − corresponding[Galn‐SO3H‐H] to (n [Gal = 2–6).n-SO There3H-H] were(n =less 2–6). intensive There weresignals, less which intensive were signals, found to which be [3,6 were‐AG found‐Galn‐ − − toSO be3H [3,6-AG-Gal‐H] (n = 3–4).n-SO Analysis3H-H] of(n the= 3–4). spectrum Analysis of F2 of found the spectrum a distribution of F2 foundof doubly a distribution‐charged ions of 2− 2− doubly-chargedcorresponding to ions [Gal correspondingn‐(SO3H)2‐2H] to (n [Gal = 3–7),n-(SO which3H)2 -2H]means (F2n =was 3–7), mainly which made means of disulfate F2 was mainly‐oligo‐ madegalactan. of disulfate-oligo-galactan.A distribution of trivalently A‐charged distribution ions ofcorresponding trivalently-charged to [Galn‐ ions(SO3H) corresponding3‐3H]3− (n = 5–9) to 3− [Galweren-(SO found3H) in3-3H] the MS( nspectrum= 5–9) were of F3. found Three in ions the MSat m spectrum/z 467.75 of(−3), F3. 565.11 Three (− ions3) and at m 625.08/z 467.75 (−3) (were−3), 565.11also detected (−3) and in 625.08 ESI‐TOF (−3)‐MS were spectrum also detected of F3, in suggesting ESI-TOF-MS the spectrum presence of of F3, [CH suggesting3‐Gal7‐(SO the3H) presence3‐3H]3−, 3− 3− 2−3− 2− of[3,6 [CH‐AG3‐-GalGal87‐(SO-(SO3H)3H)3‐33H]-3H], and, [3,6-AG-Gal[NaSO3‐Gal68‐-(SO(SO33H)H)2‐32H]-3H]. The, and molecular [NaSO weight3-Gal6 -(SOdistribution3H)2-2H] and. Thestructure molecular of F4 was weight complicated; distribution the andmain structure ions at m/z of 355.04 F4 was (−3), complicated; 367.04 (−4), 374.25 the main (−5), ions 409.06 at (m/z−6), 355.04433.07 ((−−7),3), 439.07 367.04 (−5),4), 448.07 374.25 ( (−−4),5), 463.08 409.06 (− (3),−6), 471.48 433.07 (− (5),− 7),and 439.07 488.58 (− (5),−4) 448.07 were (identified−4), 463.08 as m− m− ([Gal−3),n(SO 471.483H)m (‐−mH]5), and(n = 488.58 5–13, m (− = 4)3–7), were while identiﬁed the less‐ asintensive [Galn(SO ions3H) detectedm-mH] at m(n/z= 331.03 5–13, (−m2),= 412.06 3–7), while(−5), 491.87 the less-intensive (−5), 514.07 ions(−4), detected517.09 (−3), at m554.58/z 331.03 (−4), (517.11−2), 412.06 (−3), and (−5), 605.09 491.87 (−3) (− corresponded5), 514.07 (− 4),to 2− 5− 5− 2− 517.09[Gal3(SO (−3H)3),2‐2H] 554.58, [(CH (−4),3)2‐ 517.11Gal10‐(SO (−33),H)5‐ and5H] 605.09, [NaSO (−3‐3)Gal corresponded12‐(SO3H)5‐5H] to, [NaSO [Gal3(SO3‐Gal3H)10‐2(SO-2H]3H)4,‐ 4− 3− 5− 4− 5− 3− 4− [(CH4H] 3,) 2[Gal-Gal8‐10(SO-(SO3H)3H)3‐3H]5-5H], [NaSO,3‐ [NaSOGal11‐(SO3-Gal3H)124‐-(SO4H]3H), and5-5H] [NaSO,3‐Gal [NaSO8‐(SO33-GalH)3‐3H]10-(SO, respectively.3H)4-4H] , 3− 4− 3− [Gal8-(SO3H)3-3H] , [NaSO3-Gal11-(SO3H)4-4H] , and [NaSO3-Gal8-(SO3H)3-3H] , respectively.

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Figure 3. ESIESI-TOF-MS‐TOF‐MS spectra spectra of of the the fractions F1–F4 in a negative mode. All All of of the main peaks n− correspondFigure to 3. [MESI −‐TOFnnH]H]‐nMS− of ofspectraeach each individual individualof the fractions sulfated sulfated F1–F4 galactan galactan in a negative oligomer. oligomer. mode. All of the main peaks correspond to [M−nH]n− of each individual sulfated galactan oligomer. 2.2. The The Nephroprotective Nephroprotective Activity of Oligo Oligo-Porphyran‐Porphyran on Glycerol Glycerol-Induced‐Induced Acute Renal Failure in Rats 2.2. The Nephroprotective Activity of Oligo‐Porphyran on Glycerol‐Induced Acute Renal Failure in Rats 2.2.1. The The Mortality Mortality Effect of Oligo Oligo-Porphyran‐Porphyran (OP) in ARF Rats 2.2.1. The Mortality Effect of Oligo‐Porphyran (OP) in ARF Rats After the OP treated for seven days in ARF rats, all male rats in each group and female rats in After the OP treated for seven days in ARF rats, all male rats in each group and female rats in thethe normal group survived, whereas deaths of female rats occurred in all ARF groups. The The mortality mortality the normal group survived, whereas deaths of female rats occurred in all ARF groups. The mortality of the negative control (NC) group was 85.71%. Dexamethasone reduced the mortality slightly to of theof negative the negative control control (NC) (NC) group group was was 85.71%. 85.71%. Dexamethasone reduced reduced the the mortality mortality slightly slightly to to 71.43%.71.43%. The The TheARF ARF ARF rats rats rats treated treated treated with with with 30 30 30 mg mg mg oligo oligo-porphyran oligo‐‐porphyranporphyran per per per body body body weight weight weight (BW) (BW) (BW) per per perday day had hadthe the lowestlowestlowest mortality mortality (28.57%), (28.57%), while while the the mortality mortality of of thethethe OP10OP10 group groupgroup (ARF (ARF(ARF + +oligo+ oligo-porphyranoligo‐porphyran‐porphyran 10 mg/kg 1010 mg/kgmg/kg BW per BW day) per day) was was 57.14% 57.14% (Figure (Figure4 4).). 4).

100100 80 80 60 60 40 40 20 20 0 0 Normal NC PC OP10 OP30 Mortality of female rats/% Normal NCExperimental PC group OP10 OP30

Mortality of female rats/% Experimental group FigureFigure 4. Mortalities 4. Mortalities of femaleof female rats rats in in experimental experimental groups. Normal: Normal: normal normal rats rats without without acute acute renal renal failurefailure (ARF); (ARF); NC: negative NC: negative control control group (ARFgroup rats); (ARF PC: rats); positive PC: controlpositive group control (ARF group + dexamethasone (ARF + Figure 4. Mortalities of female rats in experimental groups. Normal: normal rats without acute renal 0.1 mg/kgdexamethasone BW per 0.1 day); mg/kg OP10: BW ARFper day); rats OP10: treated ARF with rats oligo-porphyrantreated with oligo‐porphyran (10 mg/kg (10 body mg/kg weight body per failureweight (ARF); per day);NC: OP30:negative ARF ratscontrol treated group with oligo (ARF‐porphyran rats); PC:(30 mg/kg positive body controlweight pergroup day). (ARF + day); OP30: ARF rats treated with oligo-porphyran (30 mg/kg body weight per day). dexamethasone 0.1 mg/kg BW per day); OP10: ARF rats treated with oligo‐porphyran (10 mg/kg body weight per day); OP30: ARF rats treated with oligo‐porphyran (30 mg/kg body weight per day).

Mar. Drugs 2017, 15, 135 6 of 13 Mar. Drugs 2017, 15, 135 6 of 13 2.2.2. The BUN and SCR Level in ARF Rats

2.2.2.Before The BUN glycerol and SCR injection, Level inwe ARF tested Rats the BUN and SCR levels in all male and female rats, the average levels of BUN and SCR were 7.64 ± 0.71 nmol/L and 14.82 ± 0.84 μmol/L, respectively. Then the ratsBefore were glycerol randomly injection, divided we into tested five the groups BUN and(seven SCR male levels rats in and all maleseven and female female rats rats, in each the averagegroup). levelsThe rats of BUNwere and deprived SCR were of water 7.64 ± for0.71 24 nmol/L h and received and 14.82 an± 0.84intramuscularµmol/L, respectively. injection of Then 50% theglycerol rats were in NS randomly into both divided hind limbs into ﬁve at a groups total dose (seven of male 10 mL/kg rats and BW, seven except female for the rats normal in each group).group, Thewhich rats was were injected deprived with of NS. water Then for the 24 hanimals and received were allowed an intramuscular access to a injection standard of diet. 50% glycerolBefore the in OP NS intoinjection both began hind limbs at 48 ath aafter total glycerol dose of injection, 10 mL/kg we BW, tested except the for BUN the and normal SCR group, levels whichto see waswhether injected the withARF NS.models Then were the successful animals were (Table allowed 2). From access the data to a standardshowed in diet. Table Before 2, we the could OP see injection the levers began of atBUN 48 hand after SCR glycerol in ARF injection, group were we much tested higher the BUN than and in SCRthe Normal levels to group. see whether Thus, we the concluded ARF models the wereARF models successful were (Table successful.2). From the data showed in Table2, we could see the levers of BUN and SCR in ARF group were much higher than in the Normal group. Thus, we concluded the ARF models wereTable successful. 2. Levels of BUN and SCR in the blood of experimental groups after injection 50% Glycerol for 48 h.

GenderTable 2. LevelsIndex of BUNNormal and SCR in the bloodNC of experimentalPC groups after injectionOP10 50% GlycerolOP30 for BUN 48 h. 7.73 ± 0.27 11.48 ± 1.23 ** 12.27 ± 0.74 ** 11.96 ± 1.08 ** 12.32 ± 2.11 ** (nmol/L) Male SCR Gender Index15.32 Normal ± 0.74 26.72 ± NC 3.18 ** 24.48 ± PC1.64 ** 27.11 ± OP10 2.54 ** 26.22 OP30± 1.71 ** (μmol/L) BUN (nmol/L) 7.73 ± 0.27 11.48 ± 1.23 ** 12.27 ± 0.74 ** 11.96 ± 1.08 ** 12.32 ± 2.11 ** Male BUN 6.91 ± 0.54 13.44 ± 0.88 ** 13.18 ± 0.68 ** 12.64 ± 0.67 ** 14.97 ± 1.29 ** SCR (µmol/L) 15.32 ± 0.74 26.72 ± 3.18 ** 24.48 ± 1.64 ** 27.11 ± 2.54 ** 26.22 ± 1.71 ** (nmol/L) (n = 7) (n = 4) (n = 4) (n = 5) (n = 5) Female SCR 13.946.91 ±± 1.050.54 25.6413.44 ± ±1.940.88 ** ** 24.9913.18 ± ±0.960.68 ** ** 26.7012.64 ± ±1.270.67 ** ** 26.0214.97 ±± 1.131.29 ** ** BUN (nmol/L) Female (μmol/L) (n( n= =7) 7) (n( n= =4)4) (n( =n 4)=4) (n( n= =5) 5) (n(n == 5) 5) 13.94 ± 1.05 25.64 ± 1.94 ** 24.99 ± 0.96 ** 26.70 ± 1.27 ** 26.02 ± 1.13 ** Normal:SCR normal (µmol/L) rats without acute renal failure (ARF); NC: negative control group (ARF rats); PC: (n = 7) (n =4) (n =4) (n = 5) (n = 5) positive control group (ARF + dexamethasone 0.1 mg/kg BW per day); OP10: ARF rats treated with Normal: normal rats without acute renal failure (ARF); NC: negative control group (ARF rats); PC: positive control groupoligo‐porphyran (ARF + dexamethasone (10 mg/kg 0.1 body mg/kg weight BW perper day); day); OP10: OP30: ARF ARF rats rats treated treated with with oligo-porphyran oligo‐porphyran (10 mg/kg (30 bodymg/kg weight body per weight day); OP30:per day); ARF ** rats p treated< 0.01 withcompared oligo-porphyran with the (30Normal mg/kg group. body weight Statistical per day); analysis ** p < was 0.01 compared with the Normal group. Statistical analysis was performed using ANOVA. Data are presented as meanperformed± S.D. using (n = 7 ANOVA. in male rats, Datan = are 4–7 presented in female rats). as mean ± S.D. (n = 7 in male rats, n = 4–7 in female rats).

As described in Figure 4, after the experiment, the mortality of the female ARF rats ranged from As described in Figure4, after the experiment, the mortality of the female ARF rats ranged from 28.57% to 85.71%, which means most of the female ARF rats died in NC, PC, and OP10 groups. From 28.57% to 85.71%, which means most of the female ARF rats died in NC, PC, and OP10 groups. From these results we suggest that the female ARF rats may not be suitable for studying the these results we suggest that the female ARF rats may not be suitable for studying the nephroprotective nephroprotective activity of OP with such high mortality. Thus, in the following study, we use just activity of OP with such high mortality. Thus, in the following study, we use just the data from male the data from male ARF rats to analyze the nephroprotective activity of the OP. Figure 5A shows that ARF rats to analyze the nephroprotective activity of the OP. Figure5A shows that the levels of BUN in the levels of BUN in the normal, dexamethasone‐treated ARF and two oligo‐porphyran‐treated ARF the normal, dexamethasone-treated ARF and two oligo-porphyran-treated ARF groups are signiﬁcantly groups are significantly lower than that in the NC group (p < 0.05). The concentrations of SCR in the lower than that in the NC group (p < 0.05). The concentrations of SCR in the oligo-porphyran-treated oligo‐porphyran‐treated groups are also significantly lower than that in the NC group in Figure 5B groups are also signiﬁcantly lower than that in the NC group in Figure5B( p < 0.01). Comparing the (p < 0.01). Comparing the rats in OP10 and OP30 groups, both OP10 and OP30 injections could lower rats in OP10 and OP30 groups, both OP10 and OP30 injections could lower the BUN and SCR levels the BUN and SCR levels (p < 0.01, respectively). (p < 0.01, respectively).

15 12 A * * 9 * * 6 BUN /nmol/L 3 0 Normal NC PC OP10 OP30 Experimental group

Figure 5. Cont.

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35 B 30

25 * 20 ** ** ** 15 10

SCR/µmol/L 5 0 Normal NC PC OP10 OP30

Experimental group

Figure 5.5. Blood urea nitrogen (BUN, Figure 55A)A) andand serumserum creatininecreatinine (SCR,(SCR, FigureFigure5 B)5B) levels levels of of experimental groups. Normal: normal normal rats rats without acute renal failure (ARF); NC: negative control group (ARF rats); PC:PC: positivepositive controlcontrol groupgroup (ARF(ARF ++ dexamethasonedexamethasone 0.10.1 mg/kgmg/kg BW BW per day); OP10: ARF ratsrats treatedtreated withwith oligo-porphyranoligo‐porphyran (10(10 mg/kgmg/kg body weight per day); OP30: ARF rats treated with oligo-porphyranoligo‐porphyran (30 mg/kgmg/kg body body weight weight per day); * p < 0.05, ** p < 0.01 compared with NC. Data are presented asas meanmean ±± S.D.S.D. ( (nn == 7). 7).

2.2.3. The Ions and Protein Levels in the Urine and PlasmaPlasma ofof ARFARF RatsRats The ions and protein levels in the urine and plasmaplasma of eacheach experimentalexperimental group were analyzed − (Table(Table3 3).). InIn blood, OP OP treatment treatment reduced reduced only only the the concentration concentration of of Cl Cl− significantlysignificantly (p < (0.05).p < 0.05). The Thelevels levels of albumin of albumin and and other other ions, ions, such such as as Ca Ca2+,2+ Na, Na+, +and, and K+ K, +did, did not not change change significantly significantly by by OP, OP, although the normal, OP10, and OP30 groups hadhad lowerlower concentrationconcentration ofof KK++ than that of the NC − group. In urine,urine, itit waswas notednoted that that K K++,, NaNa++,, and Cl − levelslevels in in the the normal, normal, OP10, OP10, and and OP30 OP30 groups were significantlysignificantly higherhigher thanthan thosethose inin thethe NCNC groupgroup ((pp << 0.01).

Table 3.3. LevelsLevels ofof protein,protein, potassiumpotassium ions,ions, sodiumsodium ions,ions, chlorinechlorine ions,ions, andand calcium ions in the blood ((A)) andand urineurine ((BB)) ofof experimentalexperimental groups.groups.

A A Group Albumin (g/L) K+ (mmol/L) Na+ (mmol/L) Cl− (mmol/L) Ca2+ (mmol/L) Group Albumin (g/L) K+ (mmol/L) Na+ (mmol/L) Cl− (mmol/L) Ca2+ (mmol/L) Normal 32.95 ± 2.27 5.48 ± 0.63 148.27 ± 0.81 ** 106.43 ± 2.00 ** 2.52 ± 0.13 NormalNC 32.9533.19± 2.27 ± 1.34 5.486.27± 0.63± 2.22 148.27144.53± ±0.81 2.04 ** 106.4399.41 ±± 3.842.00 **2.54 2.52 ± 0.23± 0.13 NCPC 33.1936.96± 1.34 ± 1.79 6.277.03± 2.22± 3.04 144.53145.68± ± 2.041.98 97.04 99.41 ± ±3.663.84 2.66 2.54 ± 0.19± 0.23 PCOP10 36.9632.84± 1.79 ± 1.55 7.035.46± 3.04± 0.49 145.68144.24± ± 1.981.66 103.70 97.04 ± ±1.993.66 * 2.49 2.66 ± 0.10± 0.19 OP10 32.84 ± 1.55 5.46 ± 0.49 144.24 ± 1.66 103.70 ± 1.99 * 2.49 ± 0.10 OP30 32.82 ± 1.53 5.12 ± 0.24 143.63 ± 1.35 103.55 ± 0.54 * 2.56 ± 0.07 OP30 32.82 ± 1.53 5.12 ± 0.24 143.63 ± 1.35 103.55 ± 0.54 * 2.56 ± 0.07 B BGroup Albumin (g/L) K+ (mmol/L) Na+ (mmol/L) Cl− (mmol/L) Ca2+ (mmol/L) GroupNormal Albumin 0.41 ± (g/L) 0.19 K+64.56(mmol/L) ± 10.27 * 228.48 Na+ (mmol/L) ± 27.75 ** 338.58Cl− ±(mmol/L) 68.46 ** 3.67Ca2+ ± 2.06(mmol/L) NormalNC 0.410.29± 0.19 ± 0.22 64.5647.13± 10.27 ± 10.60 * 228.4874.07± ±27.75 33.67 ** 113.93 338.58 ±± 54.3168.46 **5.00 3.67 ± 2.58± 2.06 NCPC 0.290.62± 0.22 ± 0.57 47.1342.88± 10.60± 17.50 74.0743.33 ±± 31.8933.67 * 71.49 113.93 ± 66.48± 54.31 2.16 5.00 ± 2.10± 2.58* PCOP10 0.620.25± 0.57 ± 0.12 42.8862.19± ±17.50 5.22 ** 43.33185.21± ± 31.8929.06 *** 278.41 71.49 ± 60.09± 66.48 ** 3.78 2.16 ± 2.40± 2.10 * OP10OP30 0.250.29± 0.12 ± 0.12 62.1967.45± 5.22± 4.19 ** ** 185.21201.38± ± 29.0661.62 **** 282.80 278.41 ±± 05460.09 ** **4.09 3.78 ± 1.01± 2.40 OP30Normal: normal 0.29 ± 0.12rats without 67.45 acute± 4.19 renal ** failure 201.38 (ARF);± 61.62 NC: ** negative 282.80 control± 054group ** (ARF 4.09rats);± PC:1.01 Normal:positive normal control rats group without (ARF acute + dexamethasone renal failure (ARF); 0.1 NC: mg/kg negative BW control per day); group OP10: (ARF rats);ARF PC: rats positive treated control with groupoligo‐ (ARFporphyran + dexamethasone (10 mg/kg 0.1 body mg/kg weight BW perper day); day); OP10: OP30: ARF ARF rats rats treated treated with with oligo-porphyran oligo‐porphyran (10 mg/kg (30 body weight per day); OP30: ARF rats treated with oligo-porphyran (30 mg/kg body weight per day); * p < 0.05, **mg/kgp < 0.01 body compared weight with per NC. day); Statistical * p analysis< 0.05, was** p performed < 0.01 compared using ANOVA. with Data NC. are Statistical presented asanalysis mean ± wasS.D. (performedn = 7). using ANOVA. Data are presented as mean ± S.D. (n = 7).

Mar. Drugs 2017, 15, 135 8 of 13 Mar. Drugs 2017, 15, 135 8 of 13 2.2.4. Histopathological Studies on Renal Tissues of ARF Rats 2.2.4.Renal Histopathological tissues stained Studies with hematoxylin on Renal Tissues and eosin of ARF were Rats examined under light microscopy for the histopathologicalRenal tissues stainedassay (Figure with hematoxylin 6). The rats and in eosinthe normal were examinedgroup were under found light to microscopy be in normal for thephysiological histopathological condition. assay These (Figure animals6). The had rats normal in the normalglomeruli group with were tubules found (data to be not in normalshown). physiologicalHowever, the condition. NC group These rats had animals marked had normaldilations glomeruli in the blood with capillaries tubules (data of glomeruli not shown). and However, diffuse themononuclear NC group infiltrations rats had marked in the dilations partial lymphocytes in the blood capillaries of glomeruli. of glomeruli Severe expansion and diffuse and mononuclear cast‐shaped inﬁltrationsaccumulations in the of dense partial bodies lymphocytes of proteinosis of glomeruli. also appeared Severe expansion in the tubules and (Figure cast-shaped 6A,B). accumulations With respect ofto densethe PC, bodies OP10, of and proteinosis OP30 groups, also appeared no marked in the alteration tubules was (Figure detected6A,B). in With the glomerulus, respect to the and PC, normal OP10, andcorticomedullary OP30 groups, nodemarcations marked alteration were observed, was detected although in the there glomerulus, were slight and normal dilations corticomedullary in the tubules demarcations(Figure 6C,D). were observed, although there were slight dilations in the tubules (Figure6C,D).

FigureFigure 6.6. Histopathological studies on renal tissues of rats in the NC group (A,,B) and OP10 ((C,,D)) group.group. ((AA,,CC):): cortex;cortex; ((BB,,DD):): medulla. NC group: negative control group (acute(acute renalrenal failurefailure rats);rats); OP10OP10 group:group: acuteacute renalrenal failurefailure ratsrats treatedtreated with with oligo-porphyran oligo‐porphyran (10 (10 mg/kg mg/kg body weight per day).

3.3. DiscussionDiscussion WeWe gotgot anan oligo-porphyranoligo‐porphyran (OP)(OP) usingusing acidacid hydrolysishydrolysis methodmethod fromfrom porphyran,porphyran, itsits averageaverage molecularmolecular weight was was 1431 1431 Da. Da. The The main main composition composition of the of theOP OPwere were galactose galactose (71.39%), (71.39%), 3,6‐ 3,6-anhydrogalactoseanhydrogalactose (7.05%), (7.05%), and andsulfate sulfate group group (18.32%), (18.32%), respectively. respectively. OP can OP be can further be further classified classified using usinga DEAE a DEAE Sepharose Sepharose Fast Flow Fast Flowanion anion-exchange‐exchange column column and obtained and obtained four fourfractions fractions (Figure (Figure 1). The1). Thedegree degree of polymerization of polymerization (DP) (DP),, molecular molecular weight weight distribution, distribution, and and structure structure of of F1–F4 F1–F4 were studied usingusing HPLC and ESI ESI-TOF-MS‐TOF‐MS (Figures (Figures 2 and3 3).). The DPs of F1–F4 F1–F4 was was increased increased from from two two to to 13, 13, in inaccordance accordance with with the the ESI ESI-TOF-MS‐TOF‐MS results. results. Analysis Analysis with with the theESI‐ ESI-TOF-MSTOF‐MS spectrums, spectrums, we found we found F1 was F1 wasmainly mainly made made of monosulfate of monosulfate-oligo-galactan,‐oligo‐galactan, F2 was F2 mainly was mainly made of made disulfate of disulfate-oligo-galactan,‐oligo‐galactan, F3 was F3mainly was mainlymade of made trisulfate of trisulfate-oligo-galactan,‐oligo‐galactan, trisulfate trisulfate oligo‐ oligo-methyl-galactan,methyl‐galactan, and andtrisulfate trisulfate 3,6‐ 3,6-anhydrogalactose.anhydrogalactose. The Themolecular molecular weight weight distribution distribution and andstructure structure of F4 of was F4 wascomplicated; complicated; it was it wasmainly mainly made made of sulfate of sulfate oligo oligo-galactan,‐galactan, and and the the sulfate sulfate groups groups number number from from three three to seven. TheThe fractionsfractions F1–F4 were all mainly mainly made made of of sulfate sulfate oligo oligo-galactan,‐galactan, the the differences differences were were in in the the number number of ofsulfate sulfate groups. groups. From From our our previous previous knowledge knowledge and and experiments, experiments, we we know know the the sulfate sulfate group is anan importantimportant factorfactor which which could could influence influence the antioxidant the antioxidant activity activity of sulfated of polysaccharides.sulfated polysaccharides. However, thisHowever, is not this to say is not that to higher say that is better. higher Thus,is better. in thisThus, study in this we study chose we OP chose for the OP tested for the samples. tested Wesamples. should We confirm should if confirm OP had if nephroprotective OP had nephroprotective activity in activity vivo, firstly. in vivo, If OPfirstly. exhibited If OP exhibited excellent nephroprotectiveexcellent nephroprotective activity, we activity, will using we fractions will using to studyfractions the relationshipto study the between relationship the structure between and the thestructure nephroprotective and the nephroprotective activity furthermore activity in furthermore the future. in the future.

Mar. Drugs 2017, 15, 135 9 of 13

Glycerol-induced renal injury in rats, which closely resembles the lesions in the human kidney with ARF caused by transfusion accidents or crush injuries [2], is the most widely used model for studying acute renal failure. Therefore, glycerol injection was employed in the present study to develop ARF rat models. The results described above indicated that glycerol-induced rats lost their renal function efficiently. Compared with the normal group, the negative control group had significantly higher BUN and SCR levels (Figure5), and its renal tissues were in much worse physiological condition. As mentioned above, our previous study revealed that the sulfated polysaccharides from Laminaria and its hydrolysis products had an inhibitory effect on renal impairment [10,11]. We hypothesized that low molecular weight porphyran, a sulfated galactan, could also inhibit the development of renal failure in rats, and this speculation was verified by the present study. In general, the syndrome of ARF was characterized by a rapid reduction in the ability of the kidney to eliminate waste products, such as BUN and SCR. BUN is a nitrogen-containing waste that cannot be filtrated into the urine in renal failure diseases. SCR is the product of creatine high-energy phosphate consumption [17]. They are important indicators of ARF. The reduction of SCR and BUN underscores the improvement of renal filtration function. In our experiment, OP reduced the levels of BUN and SCR in ARF rats, the activity were similar, or better, than the PC group. This means OP treatment could be notably ameliorated by the impairment of renal filtration function. We also observed significant changes of electrolyte levels in serum and urine for the rats with ARF. However, the concentrations of K+, Na+, and Cl− in the urine of the OP-treated groups were statistically higher than that in the NC group. According to this result, we can also conclude that the renal failure could be ameliorated by OP and the low concentration dose group exhibited excellent protection of renal function. Finally, we analyzed the renal histopathology of all of the rats, and the results demonstrated that OP reduced the lesion of kidneys compared with the NC group. Collectively, these results suggest that OP has a protective effect against glycerol-induced ARF. However, in this experiment, we only analyzed the renal function index at the sixth experimental day. From our results we obtained that the OP treatment has a positive effect on ARF rats, however, we still did not know on which day OP treatment had exhibited the renal protective effect because of a lack of data regarding the day-by-day observation. As for the findings in the histopathological study, we need to provide additional data from further study, which could indicate that the OP treatment cures the damaged tissues day by day. The fact that OP relieves glycerol-induced ARF might be due to the antioxidant activities of this sulfated saccharide. Many studies revealed that free radicals were critical mediators of heme-induced renal tubular damage and that antioxidants could be used to protect against renal failure [17,18]. Our previous report demonstrated that OP derived from Porphyra was an antioxidant, which had an inhibitory effect on superoxide, hydroxyl radicals, and hydrogen peroxide [16]. Therefore, the antioxidant activities of oligo-porphyran might contribute to its protective effect against ARF. In this study, we also obtained an interesting result: the gender of rats affected their tolerance to the glycerol-induced ARF. Differing mortalities suggest that female rats were more sensitive to glycerol than male rats. The gender differences had been documented in the studies of other ARF models. For instance, female rats were more refractory to ischemic ARF than male rats [19], whereas female rats appeared more sensitive to cisplatin-induced ARF [20]. According to these studies, the sex hormone was considered an important reason for this difference. Our results indicated that OP reduced the mortality of female rats effectively (Figure4). The mechanism of the differential mortality of the different sexes is still not clear, which will be the focus of future studies.

4. Materials and Methods

4.1. Preparation and Analysis of Oligo-Porphyran Porphyran used for hydrolysis was extracted from Porphyra yezoensis, which was collected from Lianyungang (35◦010 N, 119◦150 E), China in 2015 [21]. For the preparation of oligo-porphyran, Mar. Drugs 2017, 15, 135 10 of 13

◦ porphyran was dissolved in 0.5 mol/L H2SO4 and incubated for 2 h at 80 C. After the acid hydrolysis, BaCl2 was added to neutralize the solution to pH 7.0. The solution was centrifuged, and then the supernatant was concentrated and lyophilized to obtain oligo-porphyran [22]. Oligo-porphyran was dissolved in normal saline (NS) for animal treatment. The chemical characteristics of oligo-porphyran were investigated. Its total sugar content was analyzed with the phenol-sulphuric acid method [23]. The monosaccharide composition [24] and the molecular weight [22] of oligo-porphyran were determined by high-performance liquid chromatography. 3,6-anhydrogalactose content was analyzed according to the method of Yaphe [25]. Sulfate group content was determined using barium chloride-gelatin method [26]. The MWs of OP was determined by high-performance gel permeation chromatography with a TSK G3000PWxl column (TOSOH Co. Ltd., Tokyo, Japan). The mobile phase was 0.2 M Na2SO4 aqueous solution, and the ﬂow rate was 0.5 mL min−1. The column temperature was maintained at 40 ◦C, and the samples were detected with a refractive index detector (Shimadzu RID-10A). We used dextran standards with molecular weights of 2.5 KDa, 7.8 KDa, 12.2 KDa, 21.4 KDa, 41.1 KDa, 84.4 KDa, 133.8 KDa, and 2000 KDa as standards. The dextran standards dissolved in 0.2 M Na2SO4 aqueous solution to form a 1% solution and added 20 µL each time. We used the molecular weight and the retention time to make the standard curve. The OP samples were analyzed using this standard curve. All data were recorded and processed using Shimadzu LC-Solution software [22].

4.2. Fraction of Oligo-Porphyran OP 0.5 g OP was dissolved in 2 mL water and then filtered through a microporous membrane (0.45 µm) before injecting into a DEAE Sepharose Fast Flow anion-exchange column (2.8 × 40 cm). The glass column (2.8 × 40 cm) was bought from Huideyi Company (Beijing, China) and DEAE Sepharose Fast Flow gel from GE Company (Boston, MA, USA), and then the column was filled with DEAE Sepharose Fast Flow by ourselves. Following loading of the sample, the column was pre-equilibrated with distilled water and then eluted by NaCl solution, gradiently, from 0 to 0.3 M at a flow rate of 1.5 mL min−1. Then eluted by 0.5 M NaCl solution. The elution was monitored by the concentration of total sugar by the phenol-sulfuric acid method at 490 nm [23], and pooled into four fractions (F1–F4) as illustrated in Figure1. A Sephadex G10 column (GE Company, Boston, MA, USA) was then used to desalinize the four fractions.

4.3. The Structural Analysis of Oligo-Porphyran The degree of polymerization of OP, and its fractions F1–F4, was tested by HPLC [27]. A Mal-CF column was bought from the Dalian Institute of Chemical Physics (CAS, DaLian, China), and a silica-maltose and evaporative light-scattering detector were used. Chromatographic conditions were as follows: oven temperature, 30 ◦C; sample concentration, 10 mg/mL; sample volume, 10 µL; mobile phase buffer, 100 mmol/L ammonium formate-formic acid (pH 3.1); ﬂow rate, 1 mL/min; and the elution conditions were as shown in Table4.

Table 4. Mobile phase gradient.

Time (min) Acetonitrile (%) Buffer (%) 0 80 20 30 40 60 40 10 90

MW distributions of F1–F4 were determined by MS analysis with an LTQ Orbitrap XL mass spectrometer (Thermo Scientiﬁc, Sparta, NJ, USA). Mar. Drugs 2017, 15, 135 11 of 13

4.4. Experimental Animals and Treatments SD rats were reared in standard conditions of humidity (50 ± 5%), temperature (22 ± 1 ◦C), and lighting (12 h light/day). The animals were allowed access to a standard diet containing 20% protein and water ad libitum during the experiments. In this study, all drugs were injected into the abdominal cavity at a dose of 5 mL/kg body weight (BW) per day. These experiments were conducted in accordance with the National Guide for the Care and Use of Laboratory Animals of China. The rats were deprived of water for 24 h and received an intramuscular injection of 50% glycerol in NS into both hind limbs at a total dose of 10 mL/kg BW. Then the animals were allowed access to a standard diet. The following experiments began at 48 h after glycerol injection, when the ARF models were stable. The rats were randomly divided into five groups (seven male rats and seven female rats in each group): a normal group, an ARF group as a negative control (NC), a positive control group (PC, ARF + dexamethasone 0.1 mg/kg BW per day), an OP10 group (ARF + oligo-porphyran 10 mg/kg BW per day), and an OP30 group (ARF + oligo-porphyran 30 mg/kg BW per day). All of these groups were administered by intraperitoneal injection with the samples at the dosages described above for six days, while the rats in the NC and normal groups were treated with the same volume of saline. Urine specimens were collected on the fifth and sixth experimental days. After urine sampling, the rats were anaesthetized with chloral hydrate at the dose of 10 mg/kg and killed after blood samples were taken from their hearts. The blood was centrifuged at 2250× g for 20 min at 4 ◦C to obtain plasma for the biochemical analysis. The kidneys were taken and fixed in 10% formalin.

4.5. Biological Measurement The urinary and plasma protein was measured by using a Pierce Coomassie Protein Assay Kit (Thermo Fisher Scientiﬁc, Petaluma, CA, USA) [28]. The principle is that, when mixed with a protein solution, the acidic coomassie-dye reagent changes color from brown to blue in proportion the amount of protein present in the sample. We used 20 µL urine and blood, each extracted from rats, to test levels of ions, SCR, and BUN with an automated biochemical analyzer (CX5, Beckman Instruments, Brea, CA, USA). The ion selective electrodes (Beckman, Brea, CA, USA) was used to measure ions, such as Na+, + 2+ – K , Ca , and Cl . We used the 10–300 mM NaCl, 10–100 mM KCl, and 1–10 mM CaCl2 as standard sfor Na+,K+, and Ca2+, respectively. We used 10–400 mM NaCl as the standard for Cl−. An ISE is a sensor that determines the concentration of ions in a solution by measuring the current ﬂow through an ion-selective membrane. The renal histopathology was investigated using the previous method [5].

4.6. Statistical Analysis The Student’s t-test was used to determine the level of signiﬁcance of differences between the NC group and the other experimental groups. A signiﬁcant difference was accepted with p < 0.05.

5. Conclusions This study revealed the fractions F1–F4 were all mainly made of sulfate oligo-galactan with the degree of polymerization ranging from 1 to 8 and the sulfated groups numbering 1–3. We also found the protective effect of OP against glycerol-induced ARF. OP improved the renal functions of ARF rats markedly, as shown by renal histology and lower levels of SCR and BUN. A higher survival rate of male rats suggests a gender-related dependency of the sensitivity of rats to glycerol-induced ARF. This initial study of the OP-treated ARF did not clarify the mechanism of oligo-porphyran’s protective effect and the gender differences in this ARF model, which will be the subject of future work.

Acknowledgments: This study was supported by the Science and Technology Project of Shandong province (no. 2016GSF115031, 2014GHY115017), Jiangsu Science and Techoogy Program (BE2015335), the Jiangsu Innovation Entrepreneurs Talent Program, Youth Innovation Promotion Association of CAS, under grant no. 2016190, the Natural Science Foundation of China (NSFC) (41376166), and the Special Fund for Marine Scientiﬁc Research in the Public Interest (201405040). Mar. Drugs 2017, 15, 135 12 of 13

Author Contributions: Jing Wang and Yun Hou conceived and designed the experiments, designed the study, provided most of the data, and wrote the article; Delin Duan helped revise the English language aspect; and Quanbin Zhang co-designed the study. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

1. Koza, Y. Acute kidney injury: Current concepts and new insights. J. Inj. Violence Res. 2016, 8, 58–62. [PubMed] 2. Jim, S.; Oken, D.E.; Arce, M.L.; Wilson, D.R. Glycerol-induced hemoglobinuric acute renal failure in the rat. I. Micropuncture study of the development of oliguria. J. Clin. Investig. 1966, 45, 724–735. 3. Thiel, G.; Wilson, D.R.; Arce, M.L.; Oken, D.E. Glycerol-induced hemoglobinuric acute renal failure in the rat. Nephron 1967, 4, 276–297. [CrossRef][PubMed] 4. Ferenbach, D.A.; Bonventre, J.V. Mechanisms of maladaptive repair after AKI leading to accelerated kidney ageing and CKD. Nat. Rev. Nephrol. 2015, 11, 264–276. [CrossRef][PubMed] 5. Basile, D.P.; Anderson, M.D.; Sutton, T.A. Pathophysiology of acute kidney injury. Compr. Physiol. 2012, 2, 1303–1353. [PubMed] 6. Shusterman, N.; Strom, B.L.; Murray, T.G.; Morrison, G.; West, S.L.; Maislin, G. Risk factors and outcome of hospital-acqusiology of acute renal failure. Am. J. Med. 1987, 83, 65–71. [CrossRef] 7. Bell, S.; Dekker, F.W.; Vadiveloo, T.; Marwick, C.; Deshmukh, H.; Donnan, P.T.; Diepen, M.V. Risk of postoperative acute kidney injury in patients undergoing orthopaedic surgery—Development and validation of a risk score and effect of acute kidney injury on survival: observational cohort study. BMJ 2015, 351, 5639–5642. [CrossRef][PubMed] 8. Zhang, Q.; Qi, H.; Zhao, T.; Deslandes, E.; Ismaeli, N.M.; Molloy, F.; Critchley, A.T. Chemical characteristics of a polysaccharide from Porphyra capensis (Rhodophyta). Carbohydr. Res. 2005, 340, 2447–2450. [CrossRef] [PubMed] 9. Bhatia, S.; Sharma, K.; Sharma, A.; Nagpal, K.; Bera, T. Anti-inﬂammatory, Analgesic and Antiulcer properties of Porphyra vietnamensis. Avicenna J. Phytomed. 2015, 5, 69–77. [PubMed] 10. Isaka, S.; Cho, K.; Nakazono, S.; Abu, R.; Ueno, M.; Kim, D.; Oda, T. Antioxidant and anti-inﬂammatory activities of porphyran isolated from discolored nori (Porphyra yezoensis). Int. J. Biol. Macromol. 2015, 74, 68–75. [CrossRef][PubMed] 11. Zhang, Q.; Li, N.; Zhao, T.; Qi, H.; Xu, Z.; Li, Z. Fucoidan inhibits the development of proteinuria in active Heymann nephritis. Phytother. Res. 2005, 19, 50–53. [CrossRef][PubMed] 12. Wang, J.; Zhang, Q.; Jin, W.; Niu, X.; Zhang, H. Effects and mechanism of low molecular weight fucoidan in mitigating the peroxidative and renal damage induced by adenine. Carbohydr. Polym. 2011, 84, 417–423. [CrossRef] 13. Ustundag, S.; Sen, S.; Yalcin, O.; Ciftci, S.; Demirkan, B.; Ture, M. L-Carnitine Ameliorates Glycerol-Induced Myoglobinuric Acute Renal Failure in Rats. Ren. Fail. 2009, 31, 124–133. [CrossRef][PubMed] 14. Singh, P.; Ricksten, S.E.; Bragadottir, G.; Redfors, B.; Nordquist, L. Renal oxygenation and hemodynamics in acute kidney injury and chronic kidney disease. Clin. Exp. Pharmacol. Physiol. 2013, 40, 138–147. [CrossRef] [PubMed] 15. Ratliff, B.B.; Abdulmahdi, W.; Pawar, R.; Wolin, M.S. Oxidant Mechanisms in Renal Injury and Disease. Antioxi. Redox Signal. 2016, 25, 119–146. [CrossRef][PubMed] 16. Aydogdu, N.; Atmaca, G.; Yalcin, O.; Taskiran, R.; Tastekin, E.; Kaymak, K. Protective effects of 1-caenitine on myoglobinuric acute renal failure in rats. Clin. Exp. Pharmacol. Physiol. 2006, 33, 119–124. [CrossRef] [PubMed] 17. Zhao, T.; Zhang, Q.B.; Qi, H.M.; Zhang, H.; Niu, X.; Xu, Z.; Li, Z. Degradation of porphyran from Porphyra haitanensis and the antioxidant activities of the degraded porphyrans with different molecular weight. Int. J. Biol. Macromol. 2006, 38, 45–50. [CrossRef][PubMed] 18. Zager, R.A. Rhabdomyolysis and myohemoglobinuric acute renal failure. Kidney Int. 1996, 49, 314–326. [CrossRef][PubMed] Mar. Drugs 2017, 15, 135 13 of 13

19. Park, C.H.; Tanaka, T.; Cho, E.J.; Park, J.C.; Shibahara, N.; Yokozawa, T. Glycerol-Induced Renal Damage Improved by 7-O-Galloyl-D-sedoheptulose Treatment through Attenuating Oxidative Stress. Biol. Pharm. Bull. 2012, 35, 34–41. [CrossRef][PubMed] 20. Müller, V.; Losonczy, G.; Heemann, U.; Vannay, Á.; Fekete, A.; Reusz, G.; Tulassay, T.; Szabó, A.J. Sexual dimorphism in renal ischemia-reperfusion injury in rats: Possible role of endothelin. Kidney Int. 2002, 62, 1364–1371. [CrossRef][PubMed] 21. Wei, Q.; Wang, M.H.; Dong, Z. Differential Gender Differences in Ischemic and Nephrotoxic Acute Renal Failure. J. Am. Soc. Nephrol. 2005, 25, 491–499. [CrossRef][PubMed] 22. Zhang, Q.; Yu, P.; Li, Z.; Zhang, H.; Xu, Z.; Li, P. Antioxidant activities of sulfated polysaccharide fractions from Porphyra haitanesis. J. Appl. Phycol. 2003, 15, 305–310. [CrossRef] 23. Hou, Y.; Wang, J.; Simerly, T.; Jin, W.; Zhang, H.; Zhang, Q. Hydrogen peroxide released from Pyropia yezoensis induced by oligo-porphyrans: Mechanisms and effect. J. Appl. Phycol. 2015, 27, 1639–1649. [CrossRef] 24. DuBois, M.; Gilles, K.A.; Hamilton, J.K.; Rebers, P.A.; Smith, F. Colorimetric Method for Determination of Sugars and Related Substances. Anal. Chem. 1956, 28, 350–356. [CrossRef] 25. Zhang, J.; Zhang, Q.B.; Wang, J.; Shi, X.L.; Zhang, Z. Analysis of the monosaccharide composition of fucoidan by precolumn derivation HPLC. Chin. J. Oceanol. Limnol. 2009, 27, 578–582. [CrossRef] 26. Yaphe, W.; Arsenault, G.P. Improved resorcinol reagent for the determination of fructose, and of 3,6-anhydrogalactose in polysaccharides. Anal. Biochem. 1965, 13, 143–148. [CrossRef] 27. Kawai, Y.; Seno, N.; Anno, K. A modiﬁed method for chondrosulfatase assay. Anal. Biochem. 1969, 32, 314–321. [CrossRef] 28. Guo, Z.; Lei, A.; Zhang, Y.; Xu, Q.; Xue, X.; Zhang, F.; Liang, X. “Click saccharides”: Novel separation materials for hydrophilic interaction liquid chromatography. Chem. Commun. 2007, 24, 2491–2493. [CrossRef] [PubMed]

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