Path. Res. Pract. 178, 280-288 (1984)

The Renal Lysosomes in Acute Experimental Pancreatitis inDogs Treated with Prostacyclin (PGh)

Andrzej T. Triebling, J. Dlugosz, J. Brzozowski, A. Andrzejewska,U. Wereszczynska, and A. GabryelewiczGastroenterology Clinic, Medical School, Bialystok, POLAND

SUMMARY

The inflammatory process in pancreas affects the function and structure of hulneys bothby enzymatic toxemia and impairment of the renalcirculation.In this study the stabiuty of renallysosomes in AEP in dogs treated with cytoprotectiveagent PGI2 was Investigated. AEP was inducedby injection of the bileand trypsin into thepancreatic duct; experiments were terminated after 12 hours. In lysosomal enrichedsub­fraction of the kidney cortex (sedimenting In 15000 x g) in untreated group (N = 5)relative free activity (rIa.) of cathepsins (Cs), acid phosphatase (APh) and beta -glucuro­nidase (BG) increased to 51,67 and 62% respectiuely, whereas in healthy dogs (N = 6)these activities were 20,38 and 25%. In dogs (N = 6) treated with PGI2 at the dose of20 ng/kg/min. during 12 hrs, the r.j.a. of Cs, APh and BG was 18,40 and 49%, whereasindogs (N = 5) additionally pretreated during 1 hr before induction of AEP with the samedose of PGh Its values achieved 19,40 and 47% respectively.Our results suggest the stabilizing effect of PGI2 on kidney lysosomes damaged in acuteexperimental pancreatitis in dog. As possible mechanisms of prostacyclin action are dis­cussed:

1) limitation of necrotic process in the pancreas2) improvement of renal haemodynamics3) direct cytoprotective effect on the kidney.

Introduction

Numerous studies have demonstrated the frequent asso­ciation of disturbances in renal function and acute pan­creatitis (AP) !-5. Acute renal failure as a complication ofAP is estimated to account for about 10% of all cases':".The mortality in this group of patients amounts as high as90%4,6.

Despite many observations, the etiopathogenesis ofthis complication is obscure. Several factors are taken intoconsideration: oligovolemic shock':", enzymatic toxe­mia8

-10 disturbances in the clotting and fibrinolytic

system!', activation of kinins'? and complement':' sy­stems.

The consequences of the impaired lipids metabolismwere also stressed".

0344-0338/84/0178-0280$3.50/0

The lposomes play an important role in physiology ofkidneys 5; their significance in renal necrosis" and insevere pathological states i.e. in shock", intoxication" orendotoxemia'" is emphasized. To our knowledge thereare no reports causally relating damage of the kidneys inthe course of AP with disturbances on the lysosomal level.Musiatowicz et aI,2° found significant increase of nonspe­cific phosphatase activity in kidney of dogs with acuteexperimental pancreatitis (AEP). In our previous work"we have reported the labilization of renal lysosomes inAEP in dogs terminated after 24 hrs.

Since Robert's et al.22 observations of cytoprotectiveeffect exerted by prostaglandins on gastric mucosa, a greatdeal of interest has been shown in this phenomenon.Manabe and Steer23 found the protective effect of PGE2 oncholinedeficient diet-induced acute pancreatitis in mice.

© 1984 by Gustav Fischer Verlag, Stuttgart

Another prostaglandin analog-prostacyclin (PGI2) hasbeen shown to limit the tissue damage observed in endoto­xin shock2~. Prostac yclin seems to ~lay an essential role inrenal physiology and pathology' < 7. Recently the protec­tive effect of this drug on renal allograft in dogs has beenshown".

The purpose of present stud y was to evaluate the pot­entially protective effect of prostacycline on renal lysoso­mes damaged during the course of AEP in dogs.

Material and Methods

The study was performed m 26 mongrel dogs of both sexes,weighing 12-16 kg, fed with a standard diet. The animals weregiven no food except water for 12 hours before the expenment.They were subdivided into 5 groups as follows:

I Control group - healthy dogs (N = 6)II Dogs with AEP (N =5) untreated with any drug (only supp­

lementary i.v. saline drop infusion at a constant rate 2 ml/kg/hrfor 12 hrs was applied)

III Dogs with AEP (N = 6) treated with prostacyclin* in thedose of 20 nglkg/min. I. V. dur ing 12 hrs, starting immedratelyafter the induction of AEP. Volume of saline infusion as in groupI.

IV Dogs with AEP (N =5) with analogical treatment as mgroup III and additional pretreatment with the same rate of PGI1infusion 1 hour before the induction of AEP. Volume of salineinfusion as in group I.

V "Sham operated" dogs (N = 4): laparotomy, mcisron of duo­denum, sacrification after 12 hrs.

The dose and the way of treatment with the prosracychn waschosen according to study in merr' ".

Induction of AEP. The anesthesia was induced with l. V. injec­tion of hexobarbital. The femoral artery and vem were cannula­ted and the urine bladder was catheterised. After a stenle laparo­tomy and the incision of duodenum the main pancreatic duct wascannulated WIth a metal cannula. AEP was produced accordingto the method of Elliott et a1. 30, by injection into the pancreaticduct of a mixture of dog bile and trypsin under a pressure excee­ding 30 em of a water column , manometrically controlled. Thebile was collected from several healthy dogs, pooled, and kept insterile, cooled conditions. The diluted (1 : 1) with saline solutionsamples of bile (10 ml) were incubated WIth 12 mg of trypsin(Ferak, Berlin) at 37° during 24 hrs before the experiment. For10 kg of animal body weight 7 ml of the mixture was administe­red and Its volume was increased by an extra 0.2 ml for everysubsequent kg. All portions of the mixture after mcubanon werecontrolled microbiologically m agar cultures with negative re­sults.

During experiment at the fixed intervals (0 ' , 30', 4 h, and 12hrs) the peripheral blood was taken from femoral vern to deter­mme the activity of amylase and lipase, and the concentration ofurea and creatinine in serum. The activity of amylase was deter­mined by method of Caraway!' and lipase by the method ofCherry and Crandall, as referred by Kingll. The urea level wastested according to urease merhod' :' and the level of creatmine bythe method of Rehberg-Fohn, modified by Kokor' ",

In both groups treated with PGI1 and in the animals With AEPwithout any treatment the permanent control of arterial bloodpressure (estimated in femoral artery) and collection of unne intwo-hours intervals were carr ied on.

• Prostacyclin was a gift trom Upjohn Company, Kalamazoo,Michigan, USA

PGI1 and Renal Lysosomes . 281

Preparations of fractions. After 12 hrs the dogs were killed bycardiac embolisation with air to avoid farmacological effects onsubcellular structures. As soon as possible the autopsy was madeand the kidneys and pancreas were taken for biochemical andultrastructural examinations. Immediately thereafter the strips,taken out of renal cortex were homogenised in 0.25 M sucrose,using a Porter-Elvejhem homogeniser with Teflon pestle at 1500rpm. 3 down and up strokes (the whole procedure in ice bath).

The part of the homogenate was then centrifuged at 800 x gfor 10 min in ]anetzki's centrifuge with cooling to 4 °C. Thenuclear and debris pellet was discarded and supernatant was cen­trifuged at 15 000 x g for 20 min. The resulting lysosomal enri­ched subfraction was washed with 0.25 M sucrose and centrifu­ged in the same conditions. The supernatants were mixed anddesignated as a final supernatant. Enzymes assay: In wholehomogenates of the cortical parts of the left and right kidney, inlysosomal enriched subfraction and in supernatant of the corticalpart of the right kidney the "free" and "total" activities ofcathepsins (Cs), acid phosphatase (APh) and beta-glucuronidase(BG) were determined using as substrats hemoglobin (Sigma Co),natnum-bera-glycerophosphare (BDH) and phenolphtaleinmono-beta-glucuronic acid (sodium salt, Sigma Co) respectivelyaccording to the method of Gianetto and de Duve'".

The "total" activity was determined in the presence of 0.1 %v/v concentration of Triton X-lOa (Rohm and Haas Co) in theincubation mixture" .

Units of enzymatic activity

1 Uof cathepsins = 1 !J.Eq of thyrosine liberated from substrate(hemoglobin) within 10 min of incubation in pH 5.0 at 37 °C.

1 U of phosphatase = 1 mg of inorganic P liberated from thesubstrate under similar incubation conditions.

1 U ofbeta-glucuronidase = 1 mg of phenolphtalein, liberatedfrom substrate under identical conditions.

The activities of enzymes were expressed in units per gram ofprotein estimated according to the method of Lowry et ap 7. Theratio of free activity to the total one (%) called relative freeactivity (r.f.a.) was accepted as an index of lysosomal stability.

The statistical analysis of the results was performed accord ingto the Student's t test and differences with the probability lessthan 0.05 were accepted as statistically significant.

Ultrastructural examinations.The specimens of the kidney tis­sue were fixed in 3,6% glutaraldehyde at pH 7,4 and postfixed inMilloning 's phosphate buffered 1% osmium tetraoxide. Afterdehydratarion they were embedded in Epon 812. Ultrathin sec­tions were contrasted with lead citrate, and uranyl acetate , andexamined with a Tesla BS-613 electron microscope.

Results

Twelve hours after the induction of AEP poor conditionof the animals from all groups was observed . Autopsyrevealed in all dogs severe haemorrhagic and necrotic pan­creatitis confirmed by histological examination.

During the experiments considerable and statisticallysignificant increase of amylase (p < 0,001) from 216 ± 56to 794 ± 175,382 ± 119 to 1149 ± 415, 624 ± 47 to1400 ± 264 rc in groups II, III and IV respectively andlipase (p < 0,001) from 91 ± 31 to 582 ± 171,79 ± 9 to387 ± 127 IV and from 78 ± 14 to 700 ± 112 after 12 hrs

282 . Andrzej T. Triebling et al.

Table 1. Arterial blood pressure measured III femoral artery (mmHg). Mean values and SD are shown.

Time after induction of AEP ,hr.,

Group befol"llDper. AEP 1 2 4 & a 1D 12

tian

II 170 160 120 120 125 125 125 120 115- 10 - 8 --10 - 10 - 12 - 10 - L; - Lf - 4+ + + + + + + + +AEP

PGI2 infusion

IIIAEP+PG~ 155 135 90 115 120 120 115 110 110

treatment - 7 - 14 - L, - 8 - 8 - Lf - 4 - 4 - 4+ + + + + + + + +

PGI2 infusion

IVAEP+PGI; 160 130 120 90 105 110 100 100 100 95

- 8 - 0 - 7 - 11 - 9 - 7 - 7 - 4 - 7 - 7pretreat- + + + + + + + + + +ment

Table 2. Diuresis (m1l2 hrs). Mean values and SD are shown.

Time after induction of AEP 'hrs,

Group2 4 & a 1D 12

II12 28 25 24 21 20

AEP - 1,9 - 5,3 - 5,9 - 5,7 - 2,2 - 2,5+ + + + + +

III~EP+PGIe 12 31 29 33 21 20

- 2,5 - 7,8 - 6,8 - 9,4 - 4,0 - 4,2+ + + + + +

IVAEP"-=, 10 18 19 23 25 19

- 2,1 - 5,7 : 1,2 - 2,5 - 1,1 - 0,'+pretreat- + + + + ...ment

was noted. At the same time neither urea nor creatininewere changed in any of experimental groups.

One hour after the induction of AEP the drop in arterialblood pressure was observed. Pretreatment with PGIz ingroup III produced decrease in arterial blood pressure.Subsequently its mean values were kept approximately onthe same level (90-125 mmHg) in the all experimentalgroups (Table 1).

During experiment no significant changes in diuresiswere observed (Table 2). In none of the animals anuriawas noted.

As illustrates Fig. 1, in the lysosomal enriched subfrac­tion the relative free activity (r.f.a.) of cathepsins increased

PGlz and Renal Lysosomes . 283

significantly (p < 0,001) from 20% in control group to51% in dogs with AEP without any treatment (group II).In the case of beta - glucuronidase (Fig. 2) in dogs withAEP without any treatment the increase of r.f.a. to 62%from initial 25% in group I was observed (p < 0,001).

The relative free activity of acid phosphatase increasedfrom 38% in healthy dogs to 67% in group II (0,001 < P< 0,005) - Fig. 3.

In the groups treated, as well as pretreated with prosta­cyclin the r.f.a. of lysosomal hydrolases was significantlylower than that noted in dogs with AEP without any pro­tection. In the case of Cs the r.f.a. approximated thatnoted in healthy animals. In the dogs with AEP (group II),

CATHEPSINS

o Tat:. ,

Lysosomal enrichedsub'ractlon

201. 517. 18'/. 19'/.

~F"'.

Supernatant

66"1. 78 '1. 78 '1. 61'1.

Fig. 1. Total, free and relative freeactivity of cathepsins in lysosomalenriched subfracnon and in super­natant from cortex of right kidneyin experimental groups. Verticalbars - S.E.M.

320Ie

II. 2400~

Q.

- 1600

ClISO...

~

0

Group II III IV II III IV

p- GLUCURONIDASE

o Tat:.1 ~ Fr••

Lysosomal enriched Superna'an'sub'raction

251. 62". 49.,. 477- SO". 79.,. 797. 69.,.

Fig. 2. Total, free and relative free acnvi­ty of beta-glucuronidase in lysosomal enri­ched subfraction and in supernatant fromcortex of right kidney in experimentalgroups. Designations as in Fig. 1.

10

.s 8:0.. 6Go

'04

Dl.....:::I 2

0

Group II III IV II III IV

284 . Andrzej T. Triebling et al.

ACID PHOSPHATASE

Lysosomal enriched Supernatant

subfraction

18

16c

"-o 12..~

38", 67", 401, 40",

r

r94", 100'/,100'/, 93'/.

o 8

Group II III IV II III IV

Fig. 3. Total , free and relative free activi­ty of acid phosphatase in lysosomal enri­ched subfraction and supernatant fromcortex of right kidney in experimentalgroups. Designations as in Fig. 1.

Fig. 4. Portion of the prox imal tubule cell of kidney from groupII (AEP without any treatment) with numerous dilated cisternaeof endoplasmatic reticulum. TEM x 4750.

Fig. 5. Portion of the proximal tubule cell of kidney from groupII (AEP without any treatment) Numerous lysosomes (Ly) areseen in the cytoplasm. TEM x 4750.

PGlz and Renal Lysosomes . 285

Fig. 6. Mitochondria in the proximal tubule cell from group IIare swollen and severely damaged. TEM x 14400.

the drop of the total activity of three estimated lysosomalhydrolases was observed. Pretreatment with PGI2 partiallyprotected from this decrease. In the case of Cs in the groupIV statistically significant (0,01 < p < 0,05) increase ofthe total activity was noted.

In supernatant, the evident decrease of total activity ofall hydrolases in group II was observed. It is noteworthythat r.f.a. of lysosomal hydrolases in group with PGI2 pre­treatment (group IV) is lowered in comparison with con­trol group, suggesting some regenerative process.

The "sham" operation (group V - not shown) did notaffect significantly the stability of kidney lysosomes andthis group was omitted.

No differences in the total, free and relative free activi­ties of the lysosomal hydrolases from the cortex betweenthe right and left kidneys were noted.

The results obtained from analysis of the whole homo­genates were not presented here because directions of thechanges were the same as in lysosomal enriched subfrac­tion. Ultrastructural examinations revealed high degreedeterioration in most of cells from proximal convoluted

Fig. 7. Epithelium of the proximal tubule of kidney from groupIII (AEP + PGlz). Some lysosomes (Ly),slightly dilatedendoplas­matic reticulum (SER) and almost unchangedmitochondria (Mi)are seen. Bb- brush border, Bm- basementmembrane. TEM x4750.

tubules in untreated group. The significant feature was anappearance of numerous vacuoles (probably dilatatedendoplasmic reticulum) in the cells' cytoplasm (Fig. 4).

Additionally the numerous lysosomes, containing theheterogenous, electron dense material, were observed.(Fig. 5). In many cells the mitochondria showed the diffe­rent degree of alterations from swelling, dilatation of thecristal spaces to homogenization of their matrix and therupture of their membranes (Fig. 6). The ultrastructure ofproximal convoluted tubules cells from the dogs treatedand pretreated with PGIz was much better preserved thanin the untreated animals (Fig. 7).

Conclusions

1. During the course of acute experimental pancreatitisin dogs, the renallysosomes are damaged.

2. Treatment with prostacyclin (PGIz) protects the renallysosomes against their damage during the course of acutepancreatitis.

286 . Andrzej T. Triebhng et at.

3. Additional pretreatment with PGI1 does not improvethe effect of treatment alone.

Discussion

Our results indicate that after 12 hrs of AEP the lysoso­mes of dog kidney are labilized. Relative free activity ofCs, APh and B-G in homogenates and in lysosomal enri­ched subfraction from cortical part of canine kidney isenhanced. These changes were even more rronouncedthan after 24 hrs found in our previous work I.

The ultrastructural appearance of kidney in our modelof AEP is not characteristic and rowly correspond withrenal morphology during endotoxemia'", shock'? andintoxication". By analogy we assume that observed chan­ges are simple manifestation of sequela of enzymatic tox­emia and circulatory - ischemic disturbance observed inacute pancreatitis.

It is not certain if the mild labilization of renal lysoso­mes could be responsible for reversible symptoms of kid­ney dysfunction i.e. proteinuria, which is frequently obser­ved in AP3

.

Release of lysosomal hydrolases as an aspect of irrever­sibility of changes could reflect the cellular necrosis inkidney followed by irreversible renal failure. Participationof kidney lysosomal hydrolases in irreversibility of shockwas suggested by Lefer et a1.38

• Therefore the systemiceffects of renal acid hydrolases liberated in AP should alsobe taken into consideration.

In both groups of dogs treated with prostacyclin ther.f.a. of renal hydrolases was lower in comparison withuntreated group.

In the case of cathepsins its values approximated thosein control group of healthy animals. Treatment with PGI2prevented also against decrease of total hydrolases acti­vity. These data indicate a stabilizing effect of PGI2 onrenal lysosomes damaged in the course of acute experi­mental pancreatitis in dogs.

The mechanism of stabilizing effect of prostacyclinremains not solved. It appears likely that at least threegroups of factors come into play with respect to protectiveaction exerted by PGI2 on kidney Iysosomes damagedduring AEP.

A) The limitation of pathologic process in pancreas

Konturek et al.39 have reported the inhibiting effect ofPGI2 on pancreatic exocrine secretion. Similarly Kauff­mann et al.40 found inhibition of gastric secretion by PGI2•

Thus prostacyclin could depress the action of aggressivefactors in acute pancreatitis. It is noteworthy that otherprostaglandins (PGE2) protect the pancreatic ductal sys­tem against bile salts" . Moreover Manabe and Steer23

have shown that PGE2 stabilizes the pancreatic lysosomesduring cholino-deficient diet-induced pancreatitis in mice.In our observationf PGI2 exerted the protective effect onpancreatic lysosomes during experimental pancreatitis indogs. Therefore one can suspect that the limitation of

pathologic process in pancreas could reduce the quantityof potentially toxic substances to gain access to systemiccirculation and on this way protect kidneys against theirdamage.

B) Improvement of renal haemodynamic

Albrecht and Nieth" stated the decrease of effectiverenal plasma flow in patients with acute pancreatitis. Bal­sov et al." observed the impairment of endogenous creati­nine clearance in AP patients with fatal course.

According to Oates and co-workers'" prostacyclin pro­duces an increase in renal blood flow at very low doses,redistributes this flow to the inner cortical zone. Baer etal.43 found that PGI2 increases urine flow and is about tentimes more potent than PGE2 with regard to natriuresis.Therefore despite its depressing action on systemic circula­tion in higher dose" prostacyclin at the moderate dosagecould counteract against majority of hemodynamic distur­bances in the course of AP.

Musiatowicz et a1.44, Kwaan et al." found changes inrenal vessels of patients with AP corresponded to dissemi­nated intravascular coagulation (DIC). Prostacyclin is themost potent known disaggregating factor against plateletthrombi". Its role in preventing DIC-syndrome wasstressed". Antiaggregatory effect of PGI2 in the mainte­nance of blood circulation in renal allo~raft preventing itsrejection was reported by Mundy et a1. 8. It seems resona­ble to assume that improvement of renal circulation inacute pancreatitis treated with PGI2 could be conditionedalso by prevention of blood hypercoagulability.

C) Direct cytoprotective effect on the kidney

Ignarro et al47 have shown that some of prostaglandins(PGE1, PGE2, and PGF2 alia at low doses) inhibit the releaseof acid hydrolases from lysosomal fraction of kidneycortex. Weissman et al." have observed the inhibition ofrelease of lysosomal hydrolases from granulocytes aftertreatment with PGE1 and PGE2•

They ascribed this effect to increase of intracellular levelof cAMP. PGE2 exerted the cytoprotective effect durinfcarbon tetrachloride-induced liver cell necrosis in the rat" .Similar effect was observed after prostacyclin treatmentduring perfusion of hypoxic cat liver". The high activityof PGI2 in stimulation of adenylate cyclase and accumula­tion of cAMP was supported by extensive studies in cellu­lar culture of fibroblasts" . To our knowledge the directeffect of prostacyclin on renal cells or cellular componentsof kidney was not evaluated but by analogy we can suspectthat such stabilizing effect exist and could be mediated byincrease of cellular cAMP or by interaction with lysosomalmembrane after incorporation of PGI2 into the cell.

The results of present study in connection with our pre­vious studies indicating on stabilizing effect of PGI2 onhepatic Iposomes52 and beneficial effect on mitochondrialfunction 3 of liver in AEP let us to conclude that PGI2 has abroad protective effect on different organs damagedduring the course of acute experimental pancreatitis.

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47 Ignarro L], Oronsky A'L, and Perper R] (1973) Effects ofprostaglandins on release of enzymes from lysosomes of pan­creas, spleen and kidney cortex. Lrf SCI 12: 193-201

48 Weissmin G, Goldstein I, and Hoffstem S (1976) Prosra­glandms and the modulation by cyclic nucleoudes of lysosomalenzyme release. In Adv. m Prostaglandin and ThromboxaneResearch. Eds. Samuelsson B, and Padotti R, vol. 2, 803- 814Raven Press, New York

Received ]uly 29, 1982 . Accepted March 15. 1983

49 Stachura], Tarnawski A, Ivey K] , Mach T, Bogdal], Szczu­drawa], and Klimczyk B (1981) Prostaglandin protection of car­bon tetrachloride - induced liver cell necrosis in the rat. Gastro­enterology 81: 211- 217

50 Araki H, and Lefer A M (1980) Cytoprotective actions ofprostacyclin during hypoxia in the isolated perfused cat liver.Amer ] Physiol238: 176-1 81

51 Gorman R R, Hamilton R D, and Hopkins N K (1979)Prostacyclin and Throboxane A2 biosynth esis and regulation ofadenylate cyclase in human diploid cell lines. In: ProstacyclinEds. Vane,]. R. and Bergstrom S, Chapter 8, 85- 98 Raven Press,New York

52 Dlugosz], GabryelewiczA, AndrzejewskaA, Triebling AT,Brzozowski ], and Wereszczynska U (1982) Prostacyclin (PGI2)

stabilizes hepatic Iysosomes during acute experimentalpancreati­tis in dogs. Z Exper Chir 15: 210-218

53 Pawhcka E, Dlugosz], AndrzejewskaA, and GabryelewiczA (1982) The functional and ultrastructural changes of hepaticmitochondria in acute experimental pancreantis treated WIth pro­stacyclin (PGI2) . Exp Path 22: 157-164

Key word: Acute pancreatitis - KIdney - Lysosomes - Cathepsins - Beta-glucuronidase - Acid phosphatase - Pro stacy­clin - Dogs

]. Dlugosz, Gastroenterology Clinic, Medical School BIalystok, M. C. Sklodowskiej str. 24 a, 15-276 Bialystok, POLAND