Urinary Concentrating Defect in Experimental Hemochromatosis1

X.J. Zhou, N.D. Vaziri,2 D. Pandian, Z.Q. Wang, M. Mazowiecki, S.Y. Liao, and F. Oveisi

ulation carry the gene for and 1% actually suffer from X.J. Zhou. ND. Vaziri, D. Pandian, Z.Q. Wang, M. iron overload (1). Tissue iron overload without tissue Mazowiecki, S.Y. Liao, F. Oveisi Division of Nephrology, damage, usually referred to as hemosiderosis or sid- Department of Medicine, University of California, Ir- erosis, may occur as a result of redistribution of iron vine, CA in such organs as the lungs, kidneys, or liver with little or no increase in total body iron content. The term (J. Am. Soc. Nephrol. 1996; 7:128-134) hemochromatosis is used to denote chronic iron over- load leading to tissue damage. From the etiological standpoint, hemochromatosis is classified into pri- ABSTRACT mary and secondary types. Primary hemochromatosis We studied the urinary concentrating capacity in is caused by an autosomal recessive disorder of intes- experimental hemochromatosis. Sprague-Dawley tinal iron transport, whereas secondary hemochroma- rats were randomized into iron (Fe)-loaded (injected tosis is the result of other causes of excess iron sc with 1.2 g elemental iron/kg body weight as iron burden. dextran) and pair-fed control groups. The urinary Hemochromatosis is associated with a variety of concentrating ability was studied after 10 months of clinical disorders including hepatic cirrhosis (2,3), iron loading. At basal condition, urine osmolality cardiac involvement (2.4.5). various endocrinopathies (e.g., diabetes mellitus, hypothyroidism, hypopara- (Uosm) was significantly lower (P < 0.05) in the Fe- thyroidism, hypogonadism) (3,6-8), skin hyperpig- loaded rats compared with the control animals de- mentation, certain neoplasms (9,10), and impaired spite comparable urinary arginine-vasopressin (AVP) cellular immunity (11). Surprisingly, the available excretion in the two groups. Although 48-h water information on the effect of iron overload on renal deprivation resulted in comparable rises in plasma structure and function is quite limited. In a recent concentration and urinary excretion of AVP in the two study (12), acute iron overload was shown to have no groups, maximal Uosm in the Fe-loaded animals was demonstrable deleterious effect on kidney despite in- significantly lower than that seen in the control group creased lipid peroxidation. In another study, Master- (P < 0.01). Moreover, the observed urinary concen- angelo et al. (13) reported urinary concentrating defect trating defect could not be corrected by pharmaco- in a group of ten patients with thalassemia and pro- logical doses of exogenous AVP. There was no signif- longed iron-overload syndrome as a result of repeated blood transfusions. However, the mechanism of the icant difference in renal chloride, sodium, calcium, or reported defect was not clearly defined. Rats treated magnesium handling at either basal or sodium de- with iron dextran have been previously employed as pleted states. Histologic studies showed marked iron an experimental model of secondary hemochromato- deposition in the cortex and outer medulla accom- sis (14). The study presented here was designed to panied by mild tubular atrophy particularly in the examine the effect of iron dextran-induced hemochro- distal convoluted tubules. Thus, chronic experimental matosis on urinary concentrating capacity in rats. iron overload leads to nephrogenic diabetes insipi- dus marked by AVP-resistant urinary concentrating METHODS defect. Animals Key Words: Iron overload, vasopressin. osmolallty, hemosider- Male Sprague-Dawley rats (Charles River Inc., Wilmington. osis, renal sodium handling MA) weighing 340 to 380 g were randomIzed into iron- dextran (Fe) loaded and control groups. Animals placed in A lthough iron is an essential element for nearly all the Fe-loaded group received a single sc injection of an living organisms. its excessive accumulation can iron-dextran complex (Sigma Chemical Co., St. Louis, MO) at lead to severe cellular injury and organ damage. Until a dose of 1.2 g elemental iron/kg body weight and were recently, the true prevalence of iron overload in the allowed free access to food (Purina Rat Chow, Purina Mills, general population was not fully appreciated. It is now St. Louis, MO) and water. The dosage of iron employed here was based on the previously published studies (15,16) of rats known that approximately 10% of the American pop- with iron dextran-induced hemochromatosis. Animals as- signed to the control group received injections of the vehicle 1 Received September 27. 1994. Accepted September 1. 1995. alone and were pair-fed with their Fe-loaded counterparts. 2Correspondence to Dr. N.D. Vozirl, Division of Nephroiogy, Department of Pair-feeding was accomplished by limiting the amount of Medicine. uci Medical Center, 101 The City Drive. Orange. CA 92668-4088. food available to each control animal to that consumed 1046-6673/070 1.0 128503.00/0 Journal of the American Society of Nephroiogy during the preceding 24 h by its Fe-loaded counterpart. Copyright © 1996 by the American Society of Nephrology Animals were kept for 10 months in a university animal

128 Volume 7 - Number 1 1996 Zhou et a)

research facility. Arterial blood pressure was monitored by mm. to replace fluid losses during surgery. A 0.2- mL ahlquot using a rat- tail sphygmomanometer (Harvard Apparatus, of blood was then collected as blank, and 1.5 mL saline South Natick. MA). At the end of the 10-month observation containing inulin (20 gIL). sodium para-aminohippurate period, animals were placed in individual metabolic cages. (PAH, 15 g/L) and D-mannltol (30 g/L) was given as Iv bolus, Twenty-four-hour urine collection was obtained for the mea- followed by a sustained infusion at a rate of 3.9 mL/h. After surements of total volume and osmolailty. An ahiquot of urine a 90-min equilibration period, urine was collected under oil was adjusted to pH 4.5 with acetic acid and frozen at -70#{176}C for 30 min, and arterial blood samples were obtained at the for AVP determination. Blood was drawn from the orbital midpoint of the urine collection. Samples were used for the sinus for the measurements of hematocrit and osmolallty. measurements of inuhin and PAH clearances. The choice of a 10-month observation period was made on In addition, renal handling of chloride, sodium, calcium, the basis of the preliminary experiments, which showed no and magnesium was tested in five rats in each group. The significant difference between the two groups In urine vol- rats were placed in individual metabolic cages and 24-h ume or osmolailty at either basal or dehydrated condItions urine samples were collected. They were then subjected to after 4 months of iron-dextran admInistration (Table 1). salt depletion as follows. In brief, after two daily injections of Therefore, we extended the observations for an additional 6 furosemlde (10 mg/kg, ip), the rats were placed exclusively months. on a sodium-deficient diet (ICN Biomedicals. Inc., Cleveland, OH) for 7 days. The rats were given tap water ad Ilbitum. At Maximum Concentrating Ability the end of this period, 24-h urine samples were collected and urinary sodium, chloride, calcium, and magnesium were Ten rats in each group were placed in individual metabolic measured by standard laboratory techniques. Blood was also cages and allowed free access to food and water for 24 h. This drawn from the orbital sinus for the measurements of elec- was followed by a 48-h period during which water was trolytes. withheld. Immediately after the first 24-h water deprivation cycle, a micro-osmotic pump (Aiza Corporation, Palo Alto. CA) was implanted subcutaneously and was set to deliver a AVP Measurements synthetic AVP preparation at a continuous rate of 2.0 U/kg Plasma and urine AVP were measured by RIA using re- per 24 h. At the end of the experiments, the micro-osmotic agents obtained from Nichols Institute Diagnostics Inc. (San pumps were removed. The experIments were repeated 1 wk Juan Capistrano, CA). AVP was extracted from plasma sam- later without AVP infusion. Urine samples were collected ples with bentonite and assayed by the method of Skowsky et under oil during each experimental period, and urine volume al. (18) In a typical assay, 1 mL of plasma was extracted with was recorded. The samples were then stored at - 70#{176}Cforthe bentonite (3 mg). and the AVP bound to the bentonite was measurements of AVP and osmolailty. Blood was drawn eluted by using acidified acetone. The eluate was dried under Immediately after the 48-h water deprivation period for de- nitrogen. The AVP-contalning residue was reconstituted and termination of AVP and osmolahity. The choice of 48-h water assayed by RIA. In the RIA, an aliquot of the reconstituted deprivation was based on the observation of Bankir and material was mixed with rabbit antl-AVP and incubated for DeRouffignac (17), who showed that maxImal urinary con- 72 h at 4#{176}C.1125k] AVP was added and the incubation was centration may not be reached before 48 h in rats. continued for another 24 h. Bound/free separation was achieved by using a second antIbody (goat antirabbit -y Clearance Experiments globulin). Standard clearance experiments were performed as fol- For urine AVP measurement, the urine sample was ex- lows. Animals were anesthetized with an intraperitoneal tracted (1 mL) by usIng Sep-pak columns (Waters Inc., Injection of sodium pentobarbltal (50 mg/kg) and placed on a Milforb, MA). Sep-pak-bound AVP was eluted with acidified heating pad to maintain the body temperature at 37#{176}C.The acetonltrlle. The eluate was dried under nitrogen and recon- jugular vein, carotid artery, and the bladder were then stituted for RIA. Bound and free hormones were separated as cannulated with polyethylene catheters (PE-50). The animals described for the plasma AVP assay. Extraction efficiency were infused wIth 0.9% sailne solution. 10 mL/kg, for 10 was monitored routinely, and the final results were corrected for extraction efficiency (70%). The sensitivity of the assay is 1 pg/mL. In an attempt to avoid interassay variations, TABLE 1. Changes in urinary volume and osmolality samples were assayed in a single session. The intra-assay induced by Day 2 of dehydrafion in rats after 4 variation of the assay is 9%. months of iron-dextran administratlona Histologic Examinations urinary Volume Urinary Osmolality (mosmol/kg H20) Four animals in each group were exsanguinated for mor- Group(mL/24 h) phological studies. Immediately after exsanguination, the Day 0 Day 2 Day 0 Day 2 kidneys were removed and weighed. A small piece of the kidney was removed, weighed, placed on a glass plate. dried Fe.loadedb 14.4 ± 1.8 5.0 ± 0.9#{176}1620 ± 177 3419 ± 202#{176} in an oven at 70#{176}Cfor 24 h. and then weighed (dry weight).

(n = 5) The ratio of dry over wet weight was multiplIed by the weight Controlb 12.6 ± 3.3 3.1 ± O,6c 1778 ± 355 4002 ± 427C of the whole kidney to calculate the dry weight of the whole stored for (n = 5) kidney. Another piece was removed and subse- quent determination of tissue iron content. The rest of the #{176} are means ± SE. Day 0. basal Condition; Day 2. after Day 2 of kidney was promptly sectioned and separately post-fixed in dehydration. b There was no significant difference between Fe-loaded and Control 10% buffered formalun. The fixed tissue was embedded in groups in either urine volume or osmolality at either Day 0 or Day 2 (P paraffin and sectioned. The sections were stained with Go- not significant). mon’s iron stain, Masson’s trlchrome stain, and hematoxylin C P < 0.05 versus Day 0. and eosin.

Journal of the American Society of Nephrology 129 Renal Concentrating Defect in Iron Overload

The degree of iron deposition was estimated by a semi- agents used were purchased from Sigma Chemical Co. (St. quantitative morphological analysis by using the following Louis, MO). scales: 0, none; 1+, mild; 2+, moderate, and; 3+, marked. The results of tissue damage were scored semiquantitatively Chemical Analyses on the basis of the percentage of atrophic tubules present in Plasma and urine mum concentrations were measured a total field of a single section. The degree of inflammatory colorimetrically for estimation of the GFR (20). LikewIse, a cell Inifitrate and interstitial fibrosis was rated as mild (1+), colonimetric method was used for measurements of serum moderate (2+), and severe (3+). The tubule segments were and urine PAIl. PAH clearance was used to evaluate renal roughly identified according to histologic features. Briefly, plasma flow (RPF) (21). Plasma and urine osmolalities were the proximal convoluted tubules exhibit a small uneven measured using a freezing point osmometer (Advanced In- lumen and contain a single layer of large cuboidal cells with struments, Inc.. Norwood. MA). intensely eosinophilic, granular cytoplasm. Also, the proxi- mal convoluted tubules are numerous in the cortex and Statistical Analysis contain well-developed brush borders. In contrast, distal convoluted tubules are fewer in number and exhibit a large For the evaluation of the data, t test was used. P values lumen with smaller cuboidal cells. The cytoplasm stains less equal to or less than 0.05 were considered to be statistically intensely, and the brush borders are not present. In addition, significant. Results are expressed as means ± SE. the thick descending and ascending segments of Henle’s loop in the medulla are similar to the proximal and distal convo- RESU LTS luted tubules, respectively. The thin segments of Henle’s loop Basal Condition are distinct because of their squamous epithelium lining. Likewise, collecting tubules can be recognized by the pres- Fe-loaded animals showed a marked reduction in ence of lightly stained cuboidal cells and visible cell mem- food intake and an insignificant weight gain during branes. the first month. They began to gain weight steadily thereafter. By design, food intake in the control rats Iron Determination was limited to the amount consumed by their Fe- Nonheme iron concentration in the kidney tissue was loaded counterparts. Consequently, body weight was assayed in the tissue homogenate by a modification of the virtually identical in the two groups throughout the colonimetric method described by Torrance and Bothwell observation period (Table 2). No differences were (19). In brief, a sample of kidney tissue weighing about 0.1 g found in systolic blood pressure (Fe-loaded, 131 ± 3.5 from each rat was cut Into four pieces and weighed accu- versus control, 133 ± 5.0 mm Hg) or hematocrit (45.9 rately. Each piece was transferred to a glass test tube and 1 ± 2.3 versus 42.4 ± 0.8%, respectively) between the mL of acid mixture (3 M hydrochloric acid and 0.61 M two groups. Mean water Intake (Fe-loaded, 24 ± 3 trichloroacetlc acid) was added. The tubes were kept in an versus control, 20 ± 3 mL) and urinary volume were oven for 20 h at 65#{176}C.After cooling to room temperature. 0.1 slightly and insignificantly higher in the Fe-loaded mL of the clear acid extract was transferred to another test tube and 5 mL of working chromogen reagents were added. group than in the control group (Table 2). Urine The working chromogen reagent was prepared by adding one osmolality (Uosm) was significantly (P < 0.001) lower volume of chromogen reagent (1.86 mM bathophenanthro- in the Fe-loaded rats as compared to the control group line sulfonate and 143 mM thioglycohic acid) to five volumes (Table 2). Plasma osmolality (Figure 1) and urinary of saturated sodium acetate and five volumes of iron-free AVP excretion were comparable in the two groups water. The contents of the tubes were then mixed and (Table 2). allowed to stand for at least 10 mm. The absorbance of the solutions were determined in a spectrophotometer at a wave- Effects of Water Deprivation length of 535 nm against a distilled water blank. The iron- standard solutions were likewise tested and a standard curve Water deprivation for 48 h led to a steady fall in body constructed. The tissue iron concentration was calculated weight and urine output in both groups (Table 2). according to the previously described method (19). All re- Although mean urine output in the Fe-loaded group

TABLE 2. Changes In body weight, urinary volume, osmolality, and AVP excretion induced by Day 2 dehydration In rats after 10 months of iron-dextran administration0

Urinary Volume Urinary Osmolality Urinary AVP Excretion Group Body W eight (g) (mL/24 h) (mosmol/kg H20) (ng/24 h) DayO Day2 DayO Day2 DayO Day2 DayO Day2

Fe-loaded 539 ± 21 490 ± 20k’ 22.0 ± 3.4 5.4 ± 0,6b 1098 ± 45C 2955 ± 148I.c 1.5 ± 0.1 5.8 ± (N= 10)

Control 535 ± 19 486 ± 18b 18.5 ± 1.3 4.7 ± 0,5” 1469 ± 64 4399 ± 276” 1.8 ± 0.4 7.2 ± (N= 10)

#{176}Data are means ± SE. Day 0. basal condition; Day 2. after Day 2 of dehydration. b P < 0.05 versus Day 0. C P < 0.001 versus control group.

130 Volume 7 ‘ Number 1 1996 Zhou et al

350 4

0 * I * Control

Fe-loaded 300

250 I NaClCa Mg 200 - Fe-loaded Control Figure 2. FractIonal urinary excretion of filtered load in five Figure 1. Plasma osmolality under basal condition (solid bar) Fe-loaded and five control rats. Brackets denote 1 SE. Frac- and after 48 h of dehydration (hatched bar) in ten Fe-loaded lional excretions of sodium, chloride, calcium, and magne- sium were comparable In the two groups. and ten control rats. Brackets denote 1 SE. * p < 0.01 versus basal level.

pared with the basal condition. No significant differ- was higher than that seen in the control group both at ence was found in fractional excretion of either so- basal and dehydrated states, the difference did not dium (0.06 ± 0.01% in the control versus. 0.06 ± reach statistical significance. Uosm rose significantly 0.01% in the Fe-loaded rats) or chloride (0.60 ± 0.14% in both groups after 48 h of water deprivation. How- in the control versus 0.56 ± 0.13% in the Fe-loaded ever, the magnitude of the rise was significantly less in animals) after salt depletion. the Fe-loaded group than that in the control group (Table 2). The maximum Uosm after 48 h of dehydra- Tissue Iron Content tion without AVP infusion was not significantly differ- The iron content ent from those after high-dose AVP infusion (4180 ± in the renal tissue was markedly higher in the Fe-loaded group that in the control 262 versus 4399 ± 276 in the control and 2762 ± 142 than versus 2955 ± 148 mOsmol/Kg H20 in the Fe-loaded group (430.5 ± 39.3 versus 104.1 ± 16.8 g/g tissue, rats, respectively). Plasma osmolality increased sIgnif- respectively; N = 8 in each group, P < 0.00 1). icantly (P < 0.05) after dehydration in both groups. Although mean plasma osmolallty after 2 days of Kidney Size and Histology dehydration was higher in the Fe-loaded group than in Wet weights of the kidneys in the Fe-loaded group the control group, the difference did not reach statis- were comparable with those in the control animals tical significance (Figure 1). The changes in plasma (4.60 ± 0.70 versus 4.35 ± 0.25 g, N = 4 in each osmolality, together with those in body weight, indi- group). Likewise, no difference was found in the dry cate that the effect of dehydration on volume distribu- weight between the two groups (1.06 ± 0.16 in the tion was comparable in the two groups. At the end of Fe-loaded group versus 1.03 ± 0.08 g in the control the 48-h dehydration period, mean plasma AVP levels group, N = 4 in each group). were comparable in the two groups (201.7 ± 44.9 in The extent and pattern of iron deposits in the kid- the control versus 224 ± 66.4 pg/mL in the Fe-loaded neys are depicted In Table 3 and FIgure 3. The control animals, N = 10 in each group). Likewise, the mean rats showed only trace amounts of detectable iron by urinary AVP excretion rates obtained after 48 h of iron stain (Figure 3a). In contrast, the Fe-loaded rats water deprivation were similar in the two groups displayed marked iron deposits predominantly in the (Table 2). cortex and outer medulla. However, only small amounts of stainable iron were found In the glomeruli, Clearance Data and none were detected in the inner medulla (Figure The GFR in the Fe-loaded group was comparable to 3b). that in the control anImals (2.01 ± 0.32 versus 2.22 ± A mild tubular atrophy accompanied by mild-to- 0.40 mL/mln, N 6 in each group). Likewise, RPF was moderate interstitial fibrosis and lymphocytic inifitra- similar in the two groups (15.33 ± 2.93 versus 14.33 lion (1 + to 2+) were found in the cortex of Fe-loaded

± 2.57 mL/min, N = 6 in each group). rats. The atrophic changes were mainly confined to The results of renal sodium, chloride, calcium, and the thick ascending loops of Henie and distal convo- magnesium handling are presented in Figure 2. As luted tubules (Figure 3c and 3d). Focal proliferation of can be seen, fractional excretions of sodium, chloride, epithelial cells of Bowman’s capsule and glomerulo- calcium, and magnesium were comparable in the two sclerosis were found in one of the four Fe-loaded rats groups. Sodium depletion caused a tenfold decrease studied. No histologic abnormalities were found in the in the daily urinary excretion of sodium when corn- control animals (Figure 3a).

Journal of the American Society of Nephrology 131 Renal Concentrating Defect in Iron Overload

TABLE 3. Renal tissue iron deposition in Fe-loaded rats compared with the control group0

Medulla Bowman’s Glomeruli Cortex Outer Capsule inner T I I I I I

Fe-loaded ± 1+ 2-3+ 2+ 1+ 2-3+ ± 0

(N = 4) Control 0 0 ± 0 ± 0 0 0

(N = 4)

#{176}Scale is 0 to 3+. 1, tubule; I. interstitium.

DISCUSSION ture, thereby excluding the role of altered medullary structure as a major factor. The available data on the effect of chronic iron Increased renal circulation, leading to medullary overload on renal function are limited to the reported washout, has been implicated in the genesis of uri- urinary concentrating defect in patients with thalas- nary concentration defect in the iron-overloaded pa- semIa treated with long-term blood transfusions (13). However, in addition to the abnormal renal tissue iron tients with thalassemia (13). This has been attributed deposition, several other factors could potentially con- to high cardiac output and low blood viscosity associ- tribute to the urinary concentrating defect in such ated with anemia. This mechanism cannot be impli- patients. These include the hemodynamic effects of cated in our Fe-loaded animals which lacked anemia severe persistent anemia, possible effects of blood- and had normal RPF. Likewise, the normality of renal borne viral infections (e.g., CMV), production of anti- handling of chloride, sodium, calcium and magne- bodies against antigenic determinants of donor plate- sium at basal and sodium-depleted states tends to lets and leukocytes, possible effects of urinary tract argue against a significant transport defect of the infection, and altered purine metabolism, to mention thick ascending Henle’s loop, which plays a major role only a few. In the study presented here, we adminis- in renal handling of these electrolytes (22-25). The tered parenteral iron dextran to otherwise normal rats available data do not allow a definitive conclusion as to produce severe iron overload without altering the to the possible role of altered medullary urea concen- erythrocyte mass or introducing other confounding tration in the genesis of the observed urinary concen- variables. trating defect in our Fe-loaded animals. It should be Our Fe-loaded animals showed a low urine osmola- noted that plasma urea concentration and urinary lity at baseline and an impaired urinary concentrating urea excretion rate in the Fe-loaded group was com- capacity after a 48-h water deprivation test. The ob- parable with those in the control group (data not served urinary concentrating defect was the result of shown), implying normal urea production rate in nephrogenic diabetes insipidus rather than vasopres- these animals. Nonetheless, we cannot exclude the sin deficiency. This conclusion is based on several partial role of suboptiinal medullary urea concentra- observations. First, the 24-h urinary AVP excretion, tion in our Fe-loaded animals exhibiting AVP resis- which is a reliable indicator of its production rate, was tance. This is because of the dual action of vasopres- normal in the Fe-loaded rats. Second, the Fe-loaded sin on both water and urea transport in the collecting group showed a marked urinary concentrating defect ducts, where the latter contributes to maintenance of during the water deprivation test despite a compara- high medullary urea concentration. On the basis of ble rise in plasma AVP concentration as that seen in these observations, the AVP-resistant urinary concen- the control group. Finally, the observed urinary con- trating defect in the Fe-loaded group appears to be the centrating defect could not be corrected by pharma- result of a functional defect of the vasopressin-depen- cological doses of exogenous AVP, implying an im- dent water transport in the medullary collecting ducts paired renal response to AVP. despite the lack of discernible light microscopic ab- The observed vasopressin-resistant urinary concen- normalities in this segment. Hypercalcemia, hypoka- trating defect may be the result of either impaired lemia, and a variety of drugs and poisons can cause water permeability of the collecting ducts and/or in- nephrogenic diabetes insipidus. However, serum cal- ability to generate and maintain medullary hyperto- cium and potassium were normal in our animals (data nicity. The latter can, in turn, occur as a result of not shown) and, with the exception of excess iron, the either distortion of normal medullary architecture, animals were not exposed to any other agents. medullary washout phenomenon. impaired sodium! This study revealed severe (2 + to 3+) iron deposi- chloride transport in the ascending Henle’s loop, or tion in the cortex and outer medulla along with tubu- insufficient medullary urea concentration. The histo- lar atrophy and interstitial fibrosis and lymphocyte logical examination of the kidneys from our Fe-loaded infiltration in the renal cortex of Fe-loaded animals. animals revealed virtually normal medullary architec- These observations are basically consistent with the

132 Volume 7 ‘ Number 1 1996 Zhou et al

Figure 3. Photographs of renal histology obtained from control (A, (top left)) and Fe-loaded (B (top right), C (lower left), D (lower right)) rats. Increased iron deposits were observed In some tubular epithella of the outer medulla (m), meduliary ray and cortex

(C) (iron stain, Panel B x25). Fe-loaded animals exhibited interstitial fibrosis (arrowhead) and lymphocyte infiltration (-*, Panel

C xlOO, H&E). In addition, mild glomerulosclerosls (arrowhead) and tubular atrophy with iron deposition (-*, Panel D xlOO, Iron stain) were also found in Fe-loaded rats. No abnormalities were observed in the control rats except rare Iron deposits in the interstitium (_*, Panel A x250, iron stain). findings of two autopsy studies. First, Landing et a!. The precise mechanism by which chronic iron over- (26) found hemosiderin deposits in visceral and pan- load leads to these abnormalities is unclear. However, etal glomerular epithelial cells in patients with hemo- it may be the result of iron-dependent lipid peroxida- siderosis. They also showed a greater hemosiderin tion injury as previously shown to occur in liver of deposition in the straight segment of the proximal man and experimental animals with iron overload tubules and distal convoluted tubules than in the (28,29). In fact, increased lipid peroxidation has been collecting ducts. In a second study. Pardo-Mindan et shown in the kidneys of rats 12 months after admin- a!. (27) reported proximal tubular atrophy and inter- istration of high-dose (800 to 1600 mg/kg) but not stitial fibrosis in a group of patients with prosthetic low-dose (30 mg/kg) iron dextran (15). It is, therefore, heart valves accompanied by severe iron overload. tempting to speculate that this mechanism may be in However, those with mild or moderate iron overload part responsible for histological and functional injury did not show altered kidney architecture. to the kidneys in chronic iron overload.

Journal of the American Society of Nephrology 133 Renal Concentrating Defect in Iron Overload

It should be noted that iron toxicity appears to be a therapy. CRC Crit Rev Clin Lab Sci 1983; 19:205-266. dose- and time-dependent process (2.12.30). Because 11. Dc Sousa M: Immune cell functions in iron overload. Clin Exp Immunol 1989:75:1-6. animals included in the present study were exposed to 12. Galleano M, Farre SM, Turrens JF, Puntarulo 5: ResIs- a heavy iron load for a prolonged period, the results tance of rat kidney mitochondrial membranes to oxida- may not be necessarily applicable to less severe states tion induced by acute iron overload. Toxicology 1994:88: of iron overload. Moreover, the rise in iron burden in 14 1-149. 13. Masterangelo F, Lopez T, Rizzelli S. Manisco G, Corlia clinical hemochromatosis is a gradual process and as C. Alfonso L: Function of the kidney in adult patients such differs from that produced by a one-time, large- with Cooley’s disease. Nephron 1975;14:229-236. dose iron administration employed here and by other 14. Younes M, Ebezhard I, Lemoine E: Effect of iron over- load on spontaneous and xenobiotic-lnduced lipid per- investigators. Nonetheless, this model has been oxidation in vivo. J Appl Toxicol 1989:9:103-108. widely used in studies of liver disease in chronic iron 15. Golberg L, Martin LE, BatchelorA: Biochemical changes overload and found to be highly relevant. The study in the tissue of animals injected with Iron: 3. Lipid presented here lends further support for the relevance peroxidation. Biochem J 1962;83:291-298. 16. Dlllard CJ, Downey JE, Tappel AL: Effect of antioxidants of this model by revealing renal functional and histo- on lipid peroxidation In iron loaded rats. Lipids 1984:19: logical changes mimicking those described in man 127-133. with clinical iron overload (26.27). 17. Bankir L, DeRouffignac C: Urinary concentrating ability: Insights from comparative anatomy. Am J Physiol 1985; In conclusion, maximum urinary concentrating 21 9:R643-R666. ability was markedly impaired in rats with chronic 18. Skowsky WR, Rosenbloom AA, Fisher DA: Radloimmu- experimental iron overload. This was the result of an noassay measurement of arginine vasopressin in serum. acquired AVP-resistant nephrogenic diabetes insipl- Development and application. J Clin Endocrinol Metab 1974:38:278-287. dus as evidenced by normal urinary AVP excretion, 19. Torrance JD, Bothwell TH: Tissue iron stores. In: Cook appropriate AVP response to water deprivation, and J, Ed. Iron. New York: Churchifi Livingston; 1980:90- lack of response to exogenous AVP. Histological stud- 115. 20. Davidson WD, Sackner MA: Simplification of the an- ies showed significant iron deposition in the cortex throne method for the determination of insulin in clear- and outer medulla and mild tubular atrophy particu- ance studies. 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