FEMS Microbiology Letters 41 (1987) 295-298 295 Published by Elsevier

FEM 02747

Effect of iron on early changes in Escherichia coli-induced ascending pyelonephritis in rats

S. Sharma a, U.C. Garg b, N.K. Ganguly b and B.K. Sharma c

a Department of Microbiology, Panjab University, and Departments of b Experimental Medicine and ~ Internal Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Received 19 December 1986 Accepted 29 December 1986

Key words: Iron; Escher ich ia coli; Pyelonephritis; Virulence factor; (Rat)

1. SUMMARY

Uptake of D-glucose, L-lysine and L-proline and the kinetic parameters of alkaline phosphatase and ~,-glutamyl-transpeptidase were evaluated in renal brush border membrane (BBM) vesicles pre- pared from control, pyelonephritic and iron-ad- ministered pyelonephritic rats. The uptake of D- glucose and amino acids and Vm~ of both the enzymes studied were found to be altered in pyelonephritic and iron-administered pyeloneph- ritic rats, but changes appeared earlier and more severely in iron-administered infected animals than in other infected animals. These early physiologi- cal alterations were accompanied by higher bacterial colonisation in iron-administered rats.

2. I N T R O D U C T I O N

Urinary tract infection (UTI) is one of the common diseases leading to renal damage. Escher i ch ia coli is the commonest organism caus- ing uncomplicated UTI [1]. The exact role played

by host factors versus the virulence of the organism are still not well understood. In experimental hematogenous pyelonephritis, iron administration is known to enhance inflammatory lesions in the kidney [2,3]. Iron administration has also been shown to enhance other experimental infections [4-6]. E. coli possesses more than one mechanism, including haemolysin production, which make iron available to the organism [7].

Guze et al. [3] have shown that only early acute inflammatory changes are affected, while the course of chronic disease remains unaltered by iron administration. However, how iron exerts its effect is not understood. Recently, we have shown that pyelonephritis alters the reabsorption of nutrients and BBM enzymes of rat kidney as early as 3 or 4 days post-infection in rat ascending pyelonephritis [8,9]. The present study has been undertaken to see the effect of iron administration on these physiological parameters in the rat model ascending of pyelonephritis.

3. MATERIALS A N D METHODS

Correspondence to: N.K. Ganguly, Dept. of Experimental Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, 160 012, India.

3.1. Bac t e r ia l s t ra in

E. coli serotype O6K13, a wild-type strain iso- lated from a patient with acute t,TTI, was used to

0378-1097/87/$03.50 ,v, 1987 Federation of European Microbiological Societies

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induce infection. The strain was non-haemolytic, expressed mannose-resistant pili, and was resistant to the bacteriolytic action of normal human serum [10].

3.2. Iron treatment One week before infection, rats were injected

intramuscularly with iron-sorbitol (10 mg /kg body wt.).

3.3. Pyelonephritis Wistar female rats (180+ 20 g) were used.

Animals were deprived of water for 18 h, and ascending pyelonephritis was produced and as- sessed as described earlier [8]. In brief, after par- tial ligation of the left ureter, 0.4 ml of inoculum c o n t a i n i n g 10 6 bacteria was injected into the bladder lumen. The sham-operated animals were injected with 0.4 ml of tryptic soy broth into the bladder. The animals were killed 2 or 3 days post-infection. Left obstructed and right un- obstructed kidneys were removed separately. For colonisation, bacterial counts /g of kidney was made as described earlier [11].

3.4. Preparation of BBM vesicles The BBM vesicles from renal cortex were pre-

pared and their quality was checked as described by Turner and Moran [12]~ The final preparation of vesicles was suspended in the uptake buffer (300 mM mannitol, 1 mM Tris-Hepes, pH 7.5) to a protein concentration of 10-15 mg/ml.

3.4. Transport studies Uptake of D-glucose, L-lysine and L-proline was

determined by Millipore filtration technique of Hopfer et al [13]. In brief, 10 #1 of membrane vesicles (60 /~g protein) were incubated at 20°C into the incubation medium having 50/~m D-glu- cose or L-lysine or L-proline and 0.2 #Ci of [U- 14C]-labelled respective substrate. At 0.15 s, 30 s, 1 rain, 5 rain and 15 rain, the reaction was stopped by the addition of 5 ml stopping buffer (150 mM NaC1, 1 mM Tris-Hepes, pH 7.5). The mixture was filtered through a 0.45-/~m Millipore filter, and the radioactivity on the filter paper was counted in a LKB 1215 Rackbeta liquid scintilla- tion counter.

3.5. Enzyme assay Alkaline phosphatase was assayed as described

by Bergmeyer [14]. Method of Naftalin et al. [15] was used to determine the activities of y-gluta- myltranspeptidase. To calculate the K m and Vma ~ of the enzymes, various substrate concentrations were used. One enzyme unit is defined as 1/~mol of substrate hydrolysed per rain per g protein. Protein was estimated by the method of Lowry et al. [16].

4. RESULTS

The kidneys were examined for bacterial col- onisation, uptake of D-glucose, L-lysine and L-pro- line and kinetic parameters of alkaline phos- phatase and ~,-glutamyltranspeptidase were stud- ied in renal BBM vesicles prepared from control, infected and iron-administered infected rats. The uptake of all nutrients and the activities of both the enzymes studied were found to be different in experimental obstructed and unobstructed kid- neys, while no difference could be observed in the 2 kidneys of control group. Both the experimental kidneys were affected, but the changes were more pronounced in partially obstructed kidney than in unobstructed kidney.

Table 1 shows the effect of iron on the bacterial multiplication. The bacterial count was noted to be significantly higher (P < 0.001) in iron-admin- istered infected animals than in animals to which

Table 1

Effect of iron administration on the bacterial multiplication in the kidney

Days post-infection Log no. of bacteria/g tissue a

Iron No iron

2 Days Unobstructed kidney 4.37_+0.31 b 3.19___0.29 Obstructed kidney 5.99_+0.48 bx 4.03 -+0.58 c

3 Days Unobstructed kidney 4.89_+0.53 b 3.51 +0.26 Obstructed kidney 6.32-+0.79 b,c 4.29_+0.46 ~

a Each value is mean_+ S.D. of 6 animals. b p < 0.001 as compared to when no iron was administered

but infection was given. c p < 0.05 as compared to unobstructed kidney.

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Table 2 Uptake of D-glucose, L-lysine and L-proline in the renal BBM vesicles prepared from control, infected and iron-administered infected

animals killed 2/3 days post-infection

Nutrient Control Infection only (2) Iron + Infection (2) Infection only (3) Iron + Infection (3)

RK d LK d RK LK RK LK RK LK

D-Glucose 664- 3.1 62_+3.0 61_+ 3.2 60_+4.5 52+3.9 a'bx 60+3.0 a 41+_2.2 a,~ 59-+2.8 a 36+_1.8 a'bx L-lysine 109_+11.0 103_+9.8 97_+10.8 94_+6.9 87_+6.9 a 101_+9.5 81_+3.9 a'¢ 90_+7.3 a 70_+ 5.0a,b,c L-proline 110_+ 9.4 102_+8.8 95+11.1 93-+6.0 84+_8.7 ~ 99-+6.2 85_+6.1 ~,c 87_+5.6 a 71±5.9~.b,c

a p < 0.05 as compared to control. b p < 0.05 as compared to when iron was not administered but infection was given. c p < 0.05 as compared to RK, in the same group. d RK, right unobstructed kidney;

mean_+ S.D. of 4 determinations.

LK, left obstructed kidney. Days post-infection in parentheses. Results are expressed as

i r o n was n o t a d m i n i s t e r e d bu t wh ich had b e e n

in fec ted . T a b l e 2 shows the u p t a k e of D-glucose, L-lysine

a n d L-pro l ine in to the B B M vesicles p r e p a r e d

f r o m con t ro l , i n f ec t ed a n d i r o n - a d m i n i s t e r e d in-

f ec ted rats. T h e u p t a k e o f all nu t r i en t s s h o w e d the

s o d i u m g r a d i e n t o v e r s h o o t p h e n o m e n o n . T h e

ear l ies t s ign i f i can t changes ( P < 0 . 0 5 ) d e t e c t e d

w e r e 2 days pos t - i n f ec t i on , on ly in the o b s t r u c t e d

k i d n e y of i r o n - a d m i n i s t e r e d in fec t ed g roup as

c o m p a r e d to con t ro l , whi le no s ign i f i can t c h a n g e

in the u p t a k e of these nu t r i en t s cou ld be o b s e r v e d

in e i the r k i d n e y of the in fec t ed g roup as c o m p a r e d

to con t ro l . 3 days pos t - i n f ec t i on , b o t h the k idneys

o f the i r o n - a d m i n i s t e r e d g roup s h o w e d s ign i f i can t

c h a n g e s ( P < 0.05) as c o m p a r e d to con t ro l , whi le

the in fec t ed g r o u p s h o w e d s ign i f i can t changes ( P

< 0.05) o n l y in the o b s t r u c t e d k idney . O n l y in the

case of D-glucose c o u l d a s ign i f i can t dec rease ( P

< 0.05) also be o b s e r v e d in u n o b s t r u c t e d k idney .

T a b l e 3 shows the k ine t i c p a r a m e t e r s of al-

ka l ine p h o s p h a t a s e and y - g l u t a m y l t r a n s p e p t i d a s e .

N o s ign i f i can t c h a n g e in the K m of e i the r g r o u p

c o u l d be f o u n d t h r o u g h o u t the s tudy, whi le Vm~ ,

was f o u n d to be s ign i f i can t ly a l t e red ( P < 0.05) 3

days pos t - in fec t ion , on ly in the i r o n - a d m i n i s t e r e d

i n f ec t ed g r o u p as c o m p a r e d to con t ro l . Ea r l i e r

t h a n 3 days, n o s ign i f i can t c h a n g e in Vm~ x c o u l d be

o b s e r v e d (da t a n o t shown) . T h e Vma x of a lka l ine

p h o s p h a t a s e and y - g l u t a m y l t r a n s p e p t i d a s e de-

c r eased in the o b s t r u c t e d k idney , b u t n o t in the

u n o b s t r u c t e d k idney . T h e Vm~ , o f a lka l ine phos -

Table 3

Kinetic parameters of alkaline phosphatase and y-glutamyltranspeptidase in renal BBM of control, infected and iron-administered infected animals killed 3 days post-infection.

Results are expressed as mean + S.D. of 4 determinations. Vm~ is given in gmol of substrate hydrolysed, min-1 (g protein) l, Km is given in mM. RK, right unobstructed kidney; LK, left obstructed kidney.

Enzyme/Group Alkaline phosphatase y-Glutamyl transpeptidase

Vm~x Km Vmax Km

Control 395 _+ 52 6.2 _+ 0.7 1475 + 60 0.43 + 0.08 RK (Only infected) 423 _+ 18 6.1 _+ 0.7 1462 + 108 0.38 _+ 0.04 LK (Only infected) 365 _+ 15 ¢ 6.0 _+ 0.5 1 368 + 132 0.42 + 0.05 RK (Iron + infection) 440 _+ 28 6.3 + 0.8 1316 + 78 a 0.41 +_ 0.07 LK (Iron + infection) 320 + 17 a'bx 6.1 + 0.7 1214 _+ 36 a.b 0.42 + 0.09

a p < 0.05 as compared to control. b p < 0.05 as compared to when no iron was administered but infection was given. c p < 0.05 as compared to right kidney, in the same group.

298

phatase was found to be increased, while the Vm~ of y-glutamyltranspeptidase decreased in the un- obstructed kidney.

5. DISCUSSION

We have earlier shown that in rat model of ascending pyelonephritis, the uptake of nutrients and BBM enzymes are altered prior to histopatho- logical and serological changes [8,9]. In the present investigation, bacterial colonisation and these early physiological changes were evaluated after admin- istering iron.

Bacterial multiplication and the uptake of D- glucose, L-lysine and L-proline were different when iron administration preceded infection. The unal- tered g m of alkaline phosphatase and ~,-gluta- myltranspeptidase ruled out the major molecular modifications of these enzymes by iron adminis- tration [17]. No significant change in the Vma x of these enzymes could be observed in the infected group as compared to control throughout the study period. However, iron administration did cause significant changes ( P < 0.05) in the Vma x of these enzymes in comparison to control in both kidneys 3 days post-infection. This again demonstrates that iron administration enhances the changes. The increase in the Vma x of alkaline phosphatase in right kidney could be attributed to a compensa- tory phenomenon. Such changes are often seen during various stresses on BBM [8,9,18].

The virulence-enhancing effect of iron in urinary tract as well as other infections is known [2-6], but the underlying mechanism is ill-under- stood. Increased bacterial colonisation in the kid- neys of iron-administered infected group could be responsible for early and severer changes in renal physiology, but the virulence-enhancing effect of iron is not restricted to facilitating the bacterial growth alone [19]. The explanation for the dis- crepancy between bacterial number and pathology is still not forthcoming [20]. Guze et al. [3] found the effect of iron to be strain-related. E. coli isolates from pyelonephritic cases have a greater ability to synthesise siderophores, which help in iron uptake from the iron-limiting surrounding milieu [21]. It is also known that iron-regulated outer membrane proteins are expressed as bacteria recovered without subculture from infected hu-

man urine [22]. We thus feel that further work using the study of these physiological parameters could help us to understand the exact role of iron in the pathogenesis of the disease in relation to other uropathogenic properties of the organism.

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