Anoxia-induced increases in intracellular calcium concentration in primary cultures of rabbit thick ascending limb of Henle's loop

Anoxia-induced increases in intracellular calcium concentration in primary cultures of rabbit thick ascending limb of Henle's loop

Biock et Bk Biochimica et Biophysica Acta 122 t increases in intracel as( in primary cultures of rabbit thick thi U.M. Rose a, A . H a r t o g a,...

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Biock et Bk

Biochimica et Biophysica Acta 122

t increases in intracel as( in primary cultures of rabbit thick thi U.M.

Rose

a, A . H a r t o g

a, J . W . C . M .

JJ~a n s e n

b,

P. O. B a Department of Cell Physiology, University of' Nijmegen, Nijmt BV, P. b Department of Vascular Pharmacology, Solvay¢ Duphar Du~

99

tlcium concentr limb of Henle's 10s

a, R . J . M .

Bindel

HB Nijmegen, The Netherlands 80 DA Weesp, The Netherlands

Received 11 January

Iract

cultures of medullary (mTA ?he effect of anoxia on intracellular Ca 2+ concentration ([Ca2+]i) to monitor [Ca 2 me kL) thick ascending limb of Henle's loop was investigated. Previously, we reported a method 'tic substrates [1]. Corn lbation in the absence of glycolyti~ altured proximal tubule cells during 1 h of anoxic incubati of substrate-free es was 0 2 w a s realised by inclusion of a mixture of oxygenases in an anoxic chamber. As a r result levels within 60 min a and reached maximal le monol startLed to rise in individual cells of mTAL and cTAL monolayers from 76 + 1 (n = 176) to 469_+18 nM (n= dc incubation. Anoxia induced significant increases in [Ca2+]i [( anoxlc 106) in cTAL monolayers ( P < 0.( 0.05). At the re-introd tolayers and from 58 + 1 (n = 91) to 442 + 27 nM (n = 106 monola : 167) and 105_ 5 nM (n = 87) in mTAL and cT and glucose, elevated [Ca2+] i rapidly declined to 110 + 4 (n = evented anoxia-induced incJ ( P <." 0.05). Removal of extracellular Ca 2÷ and addition of 0.1 mM La 3+ partially prevente~ ~oth cell types. The L-type Ca 2÷ channel blocker D600 D60( (1 /.tM) was as effective as Ca z+ removal and in both evention of Ca 2+ influx by e observed. Preventio ce Comaparing mTAL and cTAL cells, only one difference was consistently h~nocl with ~vith Ca U'n2+ removal romovnl oor nr addition nrlclltic~n of 1/zM 1 nlk,t l)fiflO hnrl an greater inhibitory effect on al anoxic [Ca2+] i values i D600 had combined cTAL monolayers, indicative of a larger role of Ca 2÷ influx through L-type Ca 2+ channels im anoxia-induced inc aclusion, substrate-free anoxia reversibly increases [Ca2+]i in primary p cultures of cq in the former cell type. In conclusion ensitive Ca 2+ channels. ase from stores as well as from Ca 2÷ influx via D600-sensitive which results from Ca 2÷ release aannel blocker; Methoxyverapamil Key words: Ischemia; Ca 2+ ch~

1. I n t r o d u c t i o n

R e n a l i s c h e m i a results in a v a r i e t y o f p a t h o p h y s i o logical a l t e r a t i o n s at t h e level o f t h e t u b u l a r e p i t h e lium, i n c l u d i n g i m p a i r m e natt o f i n t r a c e l l u l a r Ca 2÷ homeostasis, p e r t u r b a t i o n o f c y t o s k e l e t a l o r g a n i s a t i o n a n d activation o f d e g r a d a t i v e e n z y m e s [2]. T h e c o m p l e x c o m p o s i t i o n of t h e kidney, in t e r m s o f m o r p h o l o g y a n d p h y s i o l o g i c a l a n d b i o c h e mnical i c a l functions, n e c e s s i t a t e s d e t a i l e d investigations o fF r e s p o n s e s o f t h e various emia. D i s t i n c t sensitivities to n e p h r o n s e g m e n t s to ischemia. anoxia a n d hypoxia w e r e o b s e r v e d in the e p i t h e l i a f r o m s e p a r a t e s e g m e n t s o f t h e n e p h r o n [3-5]. T h e s e different s u s c e p t i b i l i t i e s to a n o x i a can be a t t r i b u t e d to

h e m o d y n a m i c factors as well as to biochc tional h e t e r o g e n e i t y o f t h e n e p h r o n [6] t h e d i f f e r e n c e s in bbloq l o o d flow distributio~ tex a n d m e d u l l a c o u l d result in r e g k d a m a g e in case o f llim i m i t e d oxygen supply for e x a m p l e , has b e e rn shown to be espe for t h e d e v e l o p m e n t of c injury d u r i n g an [5,7]. Also, cell swelling swelli or o b s t r u c t i o n causing v a s c u l a r cong;estion, can l e a d te f e r e n c e s in b l o o d supp p l y to t h e various [8]. I n d e p e n d e n t o f ttlh e s e h e m o d y n a m i c distinct intrinsic cchal haracteristics of the ithelial cell types m ma~. a y u n d e r l i e t h e vari sensitivity to ischemi s c h e m i c injury a l o n g th gh d e g r e e o f ischemic d a •. S 3 p o r t i o n o f t h e proxil ry thick a s c e n d i n g limb c tred to t h e o t h e r segmenl

U . M Rose et al. / Biochimica et Biophysica

y has severely h a m p e r e d injury in the various rompted several investind cultured tubular cells ed studies in which cell can be analyzed [6]. An in L,itro models is that in the absence of hemoestablished a method to racellular Ca 2+ levels est~gate Increases in intracellular a2+]~) in proximal tubules in primary culture in:ed by anoxic periods up to 60 min [1]. In that study aplete absence of oxygen was achieved by inclusion Dxyrase ®, a mixture of oxygenases, to preclude any Ltribution of reactive oxygen species to cellular ini which may occur in model studies using chemical ~xia or hypoxia [6]. In the present study, we applied ilar conditions to determine and compare the ~xia-induced increases in [Ca 2÷]i in primary cultures t o T A L and c T A L to delineate biochemical and ,siological mechanisms which may explain cell type cific sensitivities to anoxia.

2. Materials and methods 2.1. Isolation of kidney epithelial cells Rabbit Rabbit m T A L and c T A L cells were isolated by immunodissection nunodissection as described previously for proximal tubule ule cells [1]. Briefly, kidneys were excised from New Zealand white rabbits ( = 0.5 ).5 kg). A cell suspension was obtained by enzymatic dig estion of dissected medullary or cortical tissue to obtain fin m T A L and c T A L cells, ~ension was incubated for 60 respectively. The cell suspension Lal antibody C 1 0 9 E l l , recograin on ice with monoclonal nizing T A L cell surface srpecific antigens. After three don was added to goat antiwashings, the cell suspension mouse IgG-coated petri dishes. shes. After 15 min of incuba•e, the dishes were washed tion at room temperature, scraped off the dishes, carefully and adherent cellss were re scra ~, The isolated m T A L or c T"AL A L ceils were collected and seeded at a density of 5 x< 105 c e l l s / c m 2 on collagencoated round coverslips ( 0 22 mm; Menzel, Germany) or on collagen-coated 24-- or 96-well plates (Costar, ~rlands). Cells were grown to Badhoevedorp, The Netherlands ire of Dulbecco's Modified subconfluency in a mixture Eagles medium (Imperial #1-466-14, Hampshire, UK): H a m ' s F12 medium (Gibco), #041-01765M, Paisley, UK) (1 : 1), supplemented withI 5% ( v / v ) fetal calf serum ; g / m l ) , N a H C O 3 (25 mM), (FCS), gentamycin (10 /~g [in (5 # g / m l ) , transferrin (5 glutamine (14 mM), insulin izg/ml), hydrocortisone (50 nM) sential amino acids (Gibco, #043prostaglandin E a (70 n g / m l ) , trii Na~SeO~ (50 nM). o H 7.4: h

mec cT~ gas, useq

rred to as K t medium. "I ~e incubated in a humidi CO 2 in air, at 370( `. r seeding.

2.2. cult

ration of mTAL and c7;

t (cA prir 96-~ buff Ca( alal sulI (HE /xl/ 1-3 E2 met

duced cyclic adenosine n tion was measured to et es. m T A L and c T A L cel vere washed twice with K :omposition in mM: 128 1 cose, 10 Na-acetate, 4 4-(2-hydroxyethyl)- 1-pipe [d / tris(hydroxymethyl)a p H 7.4) and incubated al VI bovine parathyroid ho in, vasopressin (AVP) or K H B containing 1 ml~ (IBMX). After 10 min w of the buffer waas~ ~ t U I J l ~ U u y aspiration a~pna 50/zl 0.2 N HCI to each ea well. The cAMF was determined accor )rding to Brown et The Na +-K+-2C1 - co-transport acti~ mined by measuring bumetanide-sensit take in the presence of ouabain to inhib pump. All incubatk cubations were perforr Medium from the 24-well 2, plates was ceils were preincuba incubated for 5 min in containing 1 mM ouabain ol and in th~ absence of 10 txM bumetanide t (Leo I Products, Ballerup, Denmark). Subs medium was replace placed by 0.2 ml of tl medium to which 0.5 t t C i / m l 86RbC1 w 86Rb+ uptake was stopped st after 3 min KHB supple times with 0.4 ml ice-cold ice mM ouabain and 10 1( /zM bumetanid( allowed to air-dry and ant cells were subseq adding 0.5 ml 0.05% ( v / v ) sodium dode each well. Radioacti~ tioactivity in the cell lys mined by liquid scintillation scinti counting. P tration was determim Iermined with the Coom; tein assay (Biorad, Richmond, R CA), usi globulin as a standar~ ~rd. In a separate s( ermined that 86Rb+ upt ments was determine up to at least 5 min (d a t a not shown).

2.3. Fura-2 loading m T A L and c T A L cells were loaded incubating coverslips with monolayers f containing 5 lzM fura-2 MM; Molecular Probes, ~luronic F127 (Molecula I 3 mM probenecid. Aft~

U.M. Rose et al. / Biochirnica et Biophysica

e experimental medium experiments were pernM probenecid in order ganic anion transporters,

con fluc Rm sity the abs coil (E( ma: sig~ fur~

tuantitative fluorescence e Photonic System (NPS: wcastle, UK). This system contains a photomulti~r tube which is connected to a Nikon Diaphot erred microscope with a 40 × quartz oil immersion ective. The p h o t o m e t e r contains a pin-hole di~ragm to determine the viewing field. In this studyy data represent fluorescence m e a s u r e m e n t s from 2 4 cells, captured at 400 x magnification. The fura-2 ded m T A L or c T A L cells were alternatingly excited 340 and 380 nm and emitted light was collected at intervals at 510 nm. iCa2+]i was calculated according to the formula •ived by Grynkiewicz et al. [10]: [Ca2+] i = K d × Rb~ bf (R-Rmin)/(Rma x - R)], where g d is the dissociation

con

be me3 ion cell det lel~ pro

[Ca OUS t~llllCld tair

?

a-2 for Ca 2+ of 224 nM; I f the cell at 340 and 380 : the ratios of fura-2 fluo 380 nm excitation obtai3 5 /~M ionomycin in the ated by addition of 2 m ethyl ether)-N.N.N'N'-t~ tracellular Ca 2÷, respecti' m signal divided by the n f the results, however, ar ence ratio values instea~ , since a calibration proce in every preparation bec ~ms. Firstly, addition )mycin led to abrupt rout ndly, addition of EGT,a the cells from the coversl encountered in studies e cells [1]. However, in or e d in m T A L and c T A L c d data, Ca 2+ concentral 111.9111 O_11 i:lV~lii] average calibration curv,

i

:r: : i } }

i}[ i~N

N Fig. 1. I m m u n o p e r o x i d a s e s t a i n i n g w i t h r r a b b i t kidney. In b o t h sections, only t h i c k and qD n M fnr flonra~ R and I~

d cortical (A, B) or m e d u l l a r y The b a r r e p r e s e n t s 2 0 0 / z m fo

U.M. Rose et al. /Biochimica et Biophysica

, :,~. . . .

;~/£•

,,•

~ ,•,~,

I/,~::.

(continued)•

2.5. Anoxic chamber experiment T o e s t i m a t e [Ca2+]i d u rring i n g anoxia, an anoxic c h a m b e r was u s e d as d e s c r i b e d p r e v i o u s l y [1]. Briefly, fura-2 l o a d e d t o T A L or c T A L monola' 9nolayers on a glass coverslip w e r e m o u n t e d in an anoxxc 3xic c h a m b e r at 37°C. T h e c h a m b e r was filled with 100% N 2 gassed, m o d i f i e d K H B ( c o m p o s i t i o n in m M : : 138 NaCI, 5 KC1, 5 l-lactate, lanine, 20 H E P E S / T r i s a n d 1 MgSO4, 2 CaCI2, 1 1-alanine.

Table 1 Hormone-induced cAMP formation tion in primary cultures of mTAL and cTAL Hormone

cAMP (nmol. m protein- i. 10 min 1) amol.mg mTAL

Control bPTH (1-34) Calcitonin AVP PGE 2

~i~nifienntlv

52+_ 13 186+_ 53" 8214- 104 * 290+- 75 * 155 + 29 *

differe.nl

2.6. Materials

cTAL 31_+ 7 78+-

frnrn

22 *

552_4- 85 * 284+_ 89 *

Values are mean+-S.E, with n_> 5. In were applied at a concentration of 10 7 1 • P < [/fig

360 m U / m l of t h e oxygenases coml ( O x y r a s e Inc., A s h l a n d , O H ) , which rec oxygen using l a c t a t e as a s u b s t r a t e , m o u n t i n g t h e c h a m b e r on t h e stage of t [Ca2+]i was m e;asured a s u r e c using the NPS. ,a was s t a r t e d by p e r f u anoxia, r e p e r f u s i o n w b e r with o x y g e n a t e d K H B c o n t a i n i n g It In a d d i t i o n a l e x p)eerni m n e n t s cell viability after 1 h of s u b s t r a t e - f r e e anoxia in the by m e a n s of l a c t a t e d e h y d r o g e n a s e (LD] the i n c u b a t i o n m e d i u m . L D H concentr~ d e s c r i b e d pre~ s u r e d using a L D H assay a~

cot

C o l l a g e n a s e A aann d h y a l u r o n i d a s e ' from Boehringer M M~a n n h e i m ( G e r m a n c h a n n e l b l o c k e r D600 D60 was kindly pro~ , Germany). All other c ljugated antibodies were tis, M O ) unless otherwis~ of t h e p u r e s t g r a d e avai

U.M. Rose et a l. /Biochimica et Biophysica ~

n / 380 nm)

F

0

]

anoxia

0

2'0 3'0

anoxia

I rep

I

/

40

50

60

l'0

70

I

;

I

30

40

5'o T

Time (min)

lture in response to substrate-fi • After 60 min of anoxia, oxyge itation. Three typical experimer

2. Time-dependency of increase in [Ca2+]i in mTAL (A) and cTAL cT~ (B) cell: - 10 min the anoxic chamber is closed and [Ca2+]i measurements measuremen start at I ltroduced (reperfusion: rep). [Ca2+] i is presented as the 340 and 380 i nm rati are shown.

Statistical

analysis

~dl reported data are expressed as means + S.E. tistical analysis was performed on fura-2 ratio valu e s using analysis of variance ( P < 0.05 is significant). )sequently, statistical significant differences between Subse( experimental ~erimental groups were estimated by means of contrast~t analysis according to Fisher [11].

3. Results 3.1. Characterization o f prlmary •imary cultures o f m T A L and c TAL

Conventional immunoperoxida .eroxidase Jdase staining with monoclonal antibody C109Ell, confirmed that this anti-

[ C a 2+]

Ratio (340 nm / 380 nm)

(nM)

ly recognizes m T A L (Fi bod t, C). This antibody did cT! ]:/IUMIIIill I. L J U U l I ~es. b~ ~IU pro glomeruli, connecting convoluted tubules or collecting ducts. T sected m T A L and cq cTAL cells were sul tured on glass coversl :overslips. The cultured c to 100% confluency vwithin 48 h after sc and exhibited an epitl 9ithelial-like appeara] ments were performe rformed 2 days after see( cellular de-differenti~ation which is a c culturing. Table 1 shows sh the hormone. cellular cAMP accumulation accur in cultun cultures, bPTH(1-34), c cTAL. In both cultur and P G E 2 stimulate( nulated cAMP productic Subsequently, the pr, )resence of Na+-K +port as indicated by bumetanide-sensit sensitive S6Rb+ uptake was investigat

Ratio (340 nm / 380 nm) O o

°

6-

o

o

o?

+8

,

~5

o

o

O O

°

q3

-> 2/~M

o

1764

O

°o

°o cP

o 0

_~

0 o

c~ c ~ °°° ~

337

A

0

I

I

I

I

I

I

I

I

I

0

50

100

150

200

0

50

100

150

Jp number Cell grout Fig. 3. Summary of heterogeneous respon basal [Ca2+]i (mTALH: n = 174; cTALH: min after filling the anoxic chamber); o , n = 154; cTALH: n = 98) reached 10 min

Cell group number cTAL (B) cells to substrate-fre anoxic chamber on the stage of fter 60 rain of anoxia; z~, mean g buffer. [Ca2÷] i is presented a

U.M. Rose et al. / Biochimica et Biophysica

ence of 1 mM ouabain, ~-0 _+ 2 nmol • min - 1. mg anide inhibited 69 + 1%

Tabl creases in [Ca -,+ ]i in primary.

Ano and

[Ca2+ ]i (nM)

Bas~ Max

ly, oxygen was totally :hamber within 10 min 1 " medium ',dium contain' ~,r filling the chamber with hypoxic the enzyme complex Oxyrase ® [1]. As a result of substrate-free anoxia, [Ca2+] i started to rise in ividual cells of m T A L and c T A L monolayers and ched maximal levels within 60 min after starting the asurements (Fig. 2). At the introduction of oxygen 1 glucose, i.e., reperfusion, the elevated fura-2 ratio ',lined rapidly towards pre-anoxic levels. As shown in . 3, the maximal levels attained varied strongly ween cells on one coverslip and between subse,'nt preparations. On average, the fura-2 fluoresce ratio increased significantly from 1.14 + 0.01 (n ~76) to 2.33 + 0.09 (n = 203) in m T A L and from 7 _+ 0.02 (n = 91) to 2.27 + 0.14 (n = 106) in c T A L ( P < 0.05). After the introduction of oxygen and gluc o s ee the elevated fura-2 ratio declined within 10 min to 1.277 + 0.04 (n = 167) and 1.25 + 0.06 (n = 87) in t o T A L and1 cTAL, respectively. These reperfusion values were n o t significantly different from the basal values ( P > 0.1),), indicating that no irreversible damage occurred of :ing the anoxic period. In addition, after 1 h of durin a n o x)xia l a L D H release did not increase above 5% in both m T A L and c T A L cells. Moreover, no abrupt fura-2 loss occurred during 60 rain of anoxia in both cell ell m e m b r a n e barrier propertypes, an indication that cell ties remained intact. Corn!aparing the effects of anoxia o n [ C a 2 + ] i in m T A L and cTAL, no significant differences in maximal anoxic nor reperfusion levels were

Rep

A

cT

76_+ 1 469 _+ 18 * 109_+ 4 393 _+ 15 *

5 44 10 38

of m T A L and cTAL in prim; ad reperfusion [Ca2+]i, and L ]i) are presented as calculated 12 ~ean+_S.E. of at least 38 cell lrations (* P < 0.05: compared versus cTAL),

Ano maxi mira All sepa #p,

0.1). The maximal [Ca2+i as the basal [Ca2+] i was si 9 _+ 0.10 (n = 203) and 1. ~ly ( P > 0.1). Only the ba uer in c T A L than in m T 1I1n1 ' I I 1 U l k , /., tllk, lllk, all mean values are given both preparations. values observed in bo )endence of anoxia-ind Next, the depend( xtracellular C a 2 + ( C a 2 + ) in [Ca2+] i on extracel preventing Ca 2+ infl influx and the results ~a~2+ from the mediur Fig. 4. Omitting Ca [Ca2+]o of ~ 20 ~ M and therefore 0.1 added to block any residual Ca 2+ ir C a 2+ influx slightly reduced the basa albeit significantly ( P t < 0.05), and part increases in fluorescenc anoxia-induced incre~ indicates that the anox cell types. This indic )endent on Ca 2+ inflm in [Ca2+]i is depend~ intracellular Ca 2+ store~ release from intrace prevention of Ca 2+ influx had a gre anoxic and reperfusi~ effect on maximal an obs

ing and 103 sigr

380 nm) Ratio (340 nm / 38

Ratio (340 nm 380 nm)

A __7___

T 2-

2

l

basal

anoxia

repeffusion

Fig. 4. T h e effect of nominally CaE+-free and on Zl ratio values (i.e., maximal ano: Ca 2 +-free situation (dashed columns) are /~nan

mTAL

o~||amnc~

P~I11mnc

r~nrocPnt

m~r

0

Aratio

I

basal

I

anoxia

I

reperfusion

aaximal anoxic and repeffusion , (A) and cTAL (B). Fura-2 rat moxic incubations in the preser - C a / + La versus control ratk

U.M. Rose et al. /Biochimica et Biophysica

nm / 380 nm)

A T

I

basal

anoxia

reperfusion

1

Aratio

,effusion fura5. The effect of 1 /xM D600 on basal, maximal anoxic and reperl tl ratios) in primary cultures of mTAL (A) and cTAL (B). Fura-2 ratios obse ~s observed in the absence of D600 (open columns). Columns rep,resent mea :rol ratio values).

1 differences between basal and anoxic ratios in 'AL than in cTAL ( P < 0.05). This points to a larger ", of Ca 2+ in anoxia-induced increases i n [Ca2+]i in m T A L cell type. Since ~ince L-type Ca 2+ channel blockers such as verapamil nil have been shown to reduce in vivo ischemic renal al injury [12-14], and since D600 also reduced anoxia-induced ,xia-induced increases in [Ca 2+]i in primary cultures of proximal tubules [1], the effect of D600 on anoxiainduced uced increases in [Ca2+] i in m T A L and cTAL was investi estigated. Fig. 5 demonstrates that in both cell types 1 /zM ;M D600 significantly reduced the maximal [Ca2+] i reached during anoxia. A• reduction in the difference between basal and anoxicz fura-2 ratio was observed, while no effect was seen on ,n basal [Ca2+] i. After reperfusion, [Ca2+] i rapidly declined ,~clined and stabilised in the presence of D600 at basal1 levels. When the effects of D600 on m T A L and c T A LL are compared, it is evident that 1 /~M D600 has a larger inhibitory effect on reperfusion [Ca2+]i and on the difference between basal and anoxic fura-2 ratio atio in m T A L than in cTAL ( P < 0.05), which confirmss that anoxia-mediated Ca 2+ influx is more substantial in m T A L than in cTAL.

4. Discussion

The present study demonstrat tonstrates that substrate-free anoxia increases [Ca2+]i in in m T A L and cTAL cells in primary culture. In addition on, the L-type Ca 2÷ channel blocker D600 and excluskion of Ca 2+ only partly reduced elevated levels of [Ca [Ca 2÷] 2+]~ in these monolayers, This suggests that in bothh cell types Ca 2÷ influx via L-type Ca 2+ channels as well a intracellular stores contribute t, creases in [Ca2+] i. The.

nrlmnrv

ennltnro~ n r i o i n n

I

anoxia

I

repeffusion

and on A ratio values (i.e., ma~ ~sence of D600 (dashed column calues + S.E. with N > 50 (* P <

and cTAL cells using ant recognized plasma mer ant i:l.llUggll~. 1 1 1 U l I ~is way, w a y , only m T A L and c" isolated from rabbit kidney [ medullary a suspensions, respecti, •.spectively. Recently, we this technique yieldec ridded monolayers of px or cortical collecting system that retaine acteristics of the ori~ginal nephron seg~ the present study, m T A L and cTAL c culture accumulated cAMP when treat, AVP, P G E 2 or calcitonin. calci This patter responses is similar to t those reported 1 freshly-isolated m mTA] T A L and cTAL segm~ P T H receptors which were only presenl lated cTAL [16,17]. The T cTAL cultures K+-2CI - co-transporl ,ort as indicated by bl nsensitive 86Rb + uptake. sitive ouabain-insensil demonstrate that the primary cultures tions which are typicl fical for the thick as¢ Henle's loop. Previously, we established est~ an in vitrc to study changes in [ C a 2 + ] i i n single cells during prolonge, ged periods of anoxi~ anoxia, i.e., the parti 9artial 0 2 pressure is established by adding addin an oxygenase r gassed medium. However, How anoxia-induc [Ca2+] i were only apparent ap in the abse and acetate. When these t conditions, i oxygen and substrate bstrates for glycolysis, w m T A L and cTAL cells ct in primary cu levels of [Ca2+]i were wer attained which neous with respect to the onset as steady-state level reached. real Nevertheless ~yers similar maximal 1 60 min of anoxia, with a ~42 + 27 nM, respectively nained stable until oxyge secl

U.3/L Rose et al. /Biochimica et Biophysica

electrochemical gradient acreases in [Ca2+]i are ervations are in line with duced cellular injury in dney cells where similar reported [1]. Additionn [Ca2+] i after metabolic tubule cells [18,19]. The 2+] i in m T A L and c T A L enation since " ~erfus were reversible on re-oxygenation repertua rapidly restored [Ca2+]i to pre-anoxic levels. This ling indicates that the cultured cells remain viable ing 60 min of anoxia, which is confirmed by the ence of cellular L D H or sudden fura-2 loss as a sign •ell death. Apparently, the rise in [Ca2+]i is an early nt in anoxia-mediated disturbances which leads ntually to cell injury or, alternatively, Ca 2 + is not a nary mediator of cellular damage associated with lemia. Increased [Ca2+] i can be involved in a number of cesses that are detrimental to the cell, as several '+-dependent compounds of cellular injury have .,n appreciated including disintegration of brushders, mitochondria and cytoskeletal organisation [2]. borders Of interest is the recent observation by Doctor et al. [3] thatt renal ischemia induces c T A L cells to specifically and1 extensively degrade ankyrin, a pivotal protein in the spectrin-connected cytoskeleton. These authors hyhesised that anoxia-mediated increases in [Ca2+]i pothe~ ivate CaZ+-dependent proteases, such as calpain activate whiich in turn are capable of degrading proteolytically a ~s including cytoskeletal provariety of different proteins ml cells, activation of calpain teins. Also in cultured renal has been correlated withI molecular mechanisms inry [2,5]. Future studies are volved in renal cell injury :letrimental mental role of ischemiaawaited to delineate the detrime oskeleton-related proteins in induced proteolysis of cytoskek TAL. ant role for increased Ca 2+ In general, an important ae pathogenesis of ischemic influx is implicated in the tance, lowering [Ca 2+]o prerenal injury [5,7]. For instance ,'velopment of hypoxic T A L vented effectively the develo rfused rat kidney [20]. Therenecrosis in the isolated perfused signed to prevent Ca 2+ entry fore, manoeuvres were desi zh into the cell, all of which reduced anoxia-mediated as as effective as removal of [Ca2+] i increase. D600 was 3+, which identifies L-type Ca2o+ and addition of Laa 3+ -mediated Ca 2+ entry pathCa 2+ channels as anoxia-medi~ resence of such channels in way. Evidence for the pres ¢ious studies in cultures of a T A L originates from previous mixture of cells from T A L and distal convoluted tubules [21]. Using these cultures, Bacskai and Friedman demonstrated that PTH-induced were significantly inhibited by C while pre-stimulated Ca 2+ level study, were not influenced. Und •









r ~

tion absl beo ( onb. La 3 infl~ ano Hei was The fres ano

-~ channels are apparen~ ing anoxia or hormonal sl ed. ~tion that the increase i ?lished by removing Ca~ ing D600, suggests that tracellular Ca 2+ stores increases in [Ca2+]i. Wit tribution of the Ca 2 + inf ounced in toTAL comp~ are in contrast with c and cultured proximal increases in [Ca2+] i com on However, this functional ~xia between proximal tul rest o different susceptibiliti~ did both nephron segments stre Las Its role in anoxia-indu( the~ ains unclear• In addition, d also become apparen sen ons than 1 h. ano t esent study it cannot be intrinsic properties oof nephron segmen )tibilities to hypoxia or i different susceptibilit since primary renal c served in vivo [5,7], sil oxidative stress in tel equally sensitive to ox attained during anoxia, levels of [Ca 2+]i attai accumulated Ca 2+ and an absence of cellul namic factors might b sequently, hemodyna~ renal damage. D~ in ischemic related pply, for example, val levels of blood suppl during ischemia [ hypoxia are obtained ported that the bl~ spect, it has been rel the medullary than creased more in , resulting in a hig region after ischemia [7]. Unfortunately, injury in the medulla ~reater capability to col cells express a great( cells tive stress than freshly-isolated fres excluded that the d possibility cannot b e ( cultured cells masks s tivity to anoxia of cul aspects of nephron heterogeneity• h~ In conclusion, ano~~xia induced increas primary cultures of TAL, which resu release from stores as well as from ( D600-sensitive Ca2+cchannels• However cellular funct of anoxia these altered alte ,totoxic effects resultir accompanied by cytot

Acknowledgements

")4-~

was financed by Solva 4etherlands). A.H. was su Foundation (Grant 91•11

[20]

U.M. Rose et aL / Biochimica et Biophysica

[I11 [12] A., Jansen, J.W.C.M. and Van 3, 378-386. d. 44, 27-37. Mandel, L.J. (1993) Am. J.

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0) Am. J. Physiol. 259, F338(1986) Kidney Int. 29, 11721179. Weinberg, J.M. (1991) Kidney Int. 39, 476-500. Bonventre, J.V. (1993) Kidney Int. 43, 1160-1178. Mason, J., Joeris, B., Welsch, J. and Kriz, W. (1989) Miner. Electrolyte. Metab. 15, 114-124. Brown, B.L., Albano, J.D.M., Ekins, R.P. and Sgherzi, A.M. (1971) Biochem. J. 121,561-562. Grynkiewicz, G., Poenie, M. and Tsien, R.Y. (1985) J. Biol. Chem. 260, 3440-3450.

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