Characterization of Iron Loading in the Hamp Knockout Rat

Savannah Nelson CALS Honors Program Honors Thesis Spring 2019 Food Science and Human Nutrition Department College of Agricultural and Life Sciences Faculty Advisor: Dr. James F. Collins

TABLE OF CONTENTS

Abstract…………………………………………………………………………………………...3

Introduction………………………………………………………………………………………4

Materials and Methods…………………………………………………………………………..7

Animal Experiments……………………………………………………………………..7

Non-Heme Iron Measurements…………………………………………………………8

Tissue Histology………………………………………………………………………….8

Statistical Analysis……………………………………………………………………….8

Results…………………………………………………………………………………………….9

Non-Heme Iron Measurements…………………………………………………………9

Tissue Histology………………………………………………………………………….9

Discussion…………………………………………………………………………………….....10

Literature Cited………………………………………………………………………………...13

Supporting Figures/Tables……………………………………………………………………..16

Figure 1: Non-Heme Tissue Iron Levels………………………………………………16

Figure 2: Tissue Histology……………………………………………………………...18

Figure 3: Late-Stage Tissue Histology………………………………………………...20

ABSTRACT

Iron is an essential mineral for several physiological functions. Since humans are unable to excrete iron, intestinal iron absorption must be tightly controlled by the hormone hepcidin.

Hepcidin agonists or mimetics offer attractive pharmaceutical targets for treating iron overload conditions. Previous research in our lab has shown that hepcidin (Hamp) ablation in rats causes iron overload, like that observed in human Type IIb hereditary hemochromatosis, by 9 weeks of age. However, the exact onset and progression of tissue iron accumulation is unknown. This study aimed to characterize iron loading between 3 and 9 weeks of age. Hamp-/- rats and wildtype controls (Hamp+/+) were sacrificed at 3, 4.5, 6, 7.5, and 9 weeks. Non-heme iron levels in the liver, heart, kidney, spleen, and pancreas were assessed by a colorimetric assay. Perls’

Prussian blue tissue staining revealed the location of iron deposition. Iron levels gradually

-/- increased in Hamp rats in the liver, starting at 4.5 weeks of age (females) and 6 weeks (males).

Iron status in wildtype controls remained relatively constant. With the determination of iron loading onset in the Hamp-/- rats, implications include the development and testing of therapeutic strategies in this pre-clinical rat model to reduce or prevent iron loading.

INTRODUCTION

Iron is a required micronutrient for humans and other mammals as it is used for multiple important processes such as cellular growth, energy metabolism, and the delivery of oxygen to tissues (Gulec, Anderson, & Collins, 2014). Iron is present in the diet associated with organic molecules such as heme (i.e. heme iron) (present in animal products) or in its free, ferric state as non-heme iron (found in plant and meat sources). Absorption of both types of dietary iron is highly regulated at the upper part of the small intestine due to the lack of an excretory mechanism. Non-heme iron absorption is important to study because it is more commonly found in food but is more difficult for the body to absorb (Milto, Suhodolo, Prokopieva, & Klimenteva,

2016). Many proteins regulate iron oxidation, reduction, and transport as it passes through the apical (top) and basolateral (bottom) membranes of enterocytes, the cells of the small intestine.

This ferric iron carries a +3 charge and must be reduced to ferrous iron with a +2 charge before it is transported into the enterocyte by a protein called divalent metal-ion transporter. Inside the cell during times of low demand, the storage protein ferritin can store iron (Gulec et al., 2014).

This iron can then be transported across the basolateral membrane and into the bloodstream via the transmembrane glycoprotein ferroportin, the only known mammalian iron exporter (Milto et al., 2016).

Ferroportin is in turn regulated by the peptide hormone hepcidin, derived from the liver.

Hepcidin can block iron flow into the bloodstream by binding to ferroportin, causing it to be internalized and degraded by lysosomes inside the cell (Nemeth et al., 2004). Once degraded, ferroportin can no longer export iron, so the net effect is a decrease in the release of absorbed iron into the blood. In the absence of hepcidin, ferroportin is fully functional, so iron can be released from intestinal enterocytes, and macrophages and, hepatocytes (which store excess iron)

and support various physiological functions, including, most importantly erythropoiesis in the bone marrow. Thus, it is seen that hepcidin is down-regulated in times of high demand for iron

(such as during erythropoiesis, when heme is synthesized and incorporated in red blood cells for oxygen transport), while it is upregulated when body iron stores are excessive (Milto et al.,

2016).

Mutations in hepcidin affecting its expression can lead to iron loading disorders in humans and other mammals. In humans, mutations in the HAMP (hepcidin antimicrobial peptide) gene can cause multi-organ iron overload in a condition known as Type IIb juvenile hereditary hemochromatosis (HH). This can lead to organ damage in the liver, heart, and other organs since the lack of hepcidin allows iron to escape enterocytes and migrate to these organs.

HH may also present itself comparable to other endocrine disorders such as diabetes and liver cirrhosis (Pietrangelo, 2010) or cause iron-deficiency anemia, which is harmful during periods of growth (Gehrke et al., 2003). Studies investigating hepcidin ablation in mice have found similar consequences of systemic iron overload; however, mice may not represent a complete model for human hereditary hemochromatosis since mice are unaffected by certain endocrine disorders

(Flores et al, 2017). Therefore, a rat model of this disease is warranted.

Novel hepcidin knockout Sprague-Dawley (outbred) rats have been developed at the

University of Florida to assess the regulation of iron metabolism and are only available in the laboratory of Dr. James Collins. These rats do not express hepcidin, so they are observed to develop iron overload in various organs due to the unregulated and excessive absorption of dietary iron. They represent a newly developed model of hereditary hemochromatosis (Flores et al., 2017).

It is known that these hepcidin knockout rats have normal body iron status at 3 weeks of age and become iron-loaded by 9 weeks of age (Flores et al., 2017). However, the point at which iron loading begins in each organ and its progression has yet to be investigated. This is significant because such information could be used in this pre-clinical, rat model of hereditary hemochromatosis as a prelude to human testing of alternatives to hepcidin for iron metabolism.

We hypothesize that hepcidin knockout rats will begin over-loading iron in the liver, heart, and pancreas soon after weaning as they begin to consume solid food, which contains more iron than the breast milk they were previously consuming.

MATERIALS AND METHODS Animal Experiments

All animal studies were approved by the University of Florida IACUC and performed upholding the guidelines of the American Association for Laboratory Animal Sciences.

A breeding colony of hepcidin (whole-body) knockout (Hamp-/-) Sprague-Dawley rats was already established at the University of Florida, from which heterozygous (Hamp+/-) animals were provided. Hamp+/- rats were interbred, from which the offspring were genotyped for selection of knockout and wildtype (Hamp+/+) rats. All pups were housed in the UF Food Science and Human Nutrition Animal Care Facility during the study. All animals were fed an iron- adequate, AIN-93G-based diet (Reeves, Nielson, & Fahey, 1993) and deionized tap water with ad libitum access.

Previous research indicating iron loading by 9 weeks of age led to the selection of a 6- week time period to assess the progression of iron accumulation. Starting at weaning (3 weeks of age) and occurring at ages of 4.5, 6, 7.5 and 9 weeks of age, 10 Hamp-/- rats (5 female, 5 male)

+/+ and 10 littermate controls (Hamp ) were sacrificed via CO2 narcosis and subsequent thoracotomy. Whole liver, kidney, pancreas, spleen, and heart samples were collected. All remaining tissues were held in -80°C storage until further analysis. Blood was collected from the posterior vena cava to determine hemoglobin (Hb) and hematocrit (Hct) levels. A HemoCue Hb analyzer (Hemocue AB) and a Readacrit Hct system (BD Sedi-CalTM) were used to measure these parameters, respectively, following manufacturers’ instructions.

Non-Heme Iron Measurement

To determine organ nonheme iron status at each time point, samples from the liver, kidney, heart, pancreas, and spleen were digested in acid solution (3M HCl and 10% trichloroacetic acid) overnight. Samples were analyzed by a colorimetric assay (chromogen- based) to quantify nonheme iron levels, adapted from a previously established method (Torrance

& Bothwell, 1968).

Tissue Histology

Small tissue samples from each organ were submitted for histology to the University of

Florida Molecular Pathology Core to assess the location of iron loading within the liver, pancreas, heart, kidney, and spleen. Tissue sections mounted to slides were stained using Perl’s

Prussian Blue staining method (Perls, 1867), then enhanced with DAB peroxidase substrate solution to visualize iron deposits in these tissues. Tissue slides were scanned at the Univeristy of

Florida Molecular Pathology Core. Images were viewed using Aperio ImageScope software

(Aperio ImageScope 12.4 for Windows 10, Leica Biosystems Imaging, Inc.).

Statistical Analysis

Nonheme iron data were analyzed using a two-way ANOVA test for genotype and time using GraphPad Prism Software. Data was transformed on a log10 scale to perform statistical analyses if there was a lack of homogeneity of variance in the data sets. Interaction was considered significant at p < 0.05. Results were portrayed in graphs to show trends in iron loading. Tukey’s multiple comparison test was used to identify significant variance between groups.

RESULTS

Non-heme Tissue Iron Levels

The interaction between the factors of genotype and time was found to be significant (at p

< 0.05) in both females and males in the liver and spleen (n=5 per timepoint) as well as in the kidneys for the males only (Figure 1, A-D & I). The concentration of nonheme iron in the liver showed a positive trend with respect to time in the knockout animals (Figure 1, A & B). For males (Figure 1, A), increases in liver iron accumulation in knockout animals were seen to be significantly different from their younger counterparts at 6 weeks of age. In females, (Figure 1,

B), this difference occurred at 6 weeks of age. There was no significant change in non-heme iron levels in males or females in pancreas and heart, nor in the kidneys of females (Figure 1, E-H &

J).

Tissue Histology

Visual evidence (using a DAB-enhanced Perls’ Prussian iron staining technique) of iron accumulation is seen in paraffin-embedded liver sections as early as 7.5 weeks in hepcidin knockout rats (Figure 2, D). Stained tissue sections of the spleen in a 7.5-week-old wildtype rat show the presence of iron (Figure 2, F). No evidence of iron is observed in stained sections of the pancreas, heart, and kidney of knockout and wildtype rats from 3 to 7.5 weeks of age (Figure 2,

I-T).

DISCUSSION

Iron is an essential mineral for the proper functioning of many organisms. Its metabolism is important to study because excesses of iron are known to cause damage to cells and their DNA

(due to oxidative stress), leading to programmed cell death (Gozzelino & Arosio, 2017).

Hepcidin functions to decrease the uptake of intestinal iron by internalizing the transport protein ferroportin in duodenal enterocytes. Diseases such as heredity hemochromatosis that are characterized by mutations in hepcidin lead to multi-visceral iron loading in humans and other mammals since iron uptake is no longer properly regulated.

Current management strategies for iron overload in humans include periodic phlebotomies to remove excess iron or the administration of iron chelators such as deferasirox

(Pietrangelo, 2010). Such methods may be limited in individuals who are resistant to blood draws or experience negative side effects during chelation therapy. Thus, attempts at manipulating the hepcidin pathway could serve as a better, pharmacological option to address the root metabolic problem and re-establish proper regulation of iron. Manipulation strategies previously studied to treat iron overload include the use of hepcidin agonists that mimic the function of hepcidin known as minihepcidins (Ruchala & Nemeth, 2014).

Since the 25-peptide hormone of hepcidin itself is too unstable and lacks a long enough half-life to be a suitable pharmacological agent, mimics have been developed as possible therapeutics. One analog, minihepcidin PR65, was developed based on the functional region of hepcidin that interacts with ferroportin. When tested in hepcidin knockout mice previously depleted of iron, Ramos et al. (2012) found that daily doses of PR65 prevented iron loading in the liver and heart, while increasing iron retention in the spleen and duodenum. When given to mice already iron-loaded, PR56 treatment was only observed to have a moderate effect, mainly

causing the redistribution of iron. Other investigators injected iron-depleted hepcidin knockout mice with a form of human hepcidin, palmitoyl-ri-hep9. They found a significantly lower liver iron accumulation in groups who received the minihepcidin over a diluent control (Preza et al.,

2011). However, neither of these analogs were tested in rats, and both studies used older knockout mice purposely depleted of iron via an iron-deficient diet rather than starting when the animals were first weaned from their mothers. Implementing minihepcidin therapy at a younger age, before iron buildup occurs, may be more useful to prevent tissue-iron accumulation rather than redistributing it.

The current study aimed to characterize early-stage iron loading in the hepcidin (whole- body) knockout rat in order to target a starting point for effective hepcidin therapy. Longer-term rat studies have already identified iron loading in multiple tissues (Flores et al., 2017), so this investigation aimed to determine its progression from 3 to 9 weeks of age, beginning when pups weaned. At this stage, iron accumulation was only seen to occur in the liver starting at about 4.5 to 6 weeks of age (Figure 1, A&B). The liver is the primary storage site for iron in humans

(Gozzelino & Arosio, 2017). This may suggest that iron loading into other tissues, such as the pancreas, spleen, heart, or kidney, does not occur until a later age when the liver is full iron loaded. When looking at iron stores in the spleen, wildtype animals had iron buildup through 9 weeks of age, while this not observed in the knockout animals (Figure 1, C & D). This may be attributed to the natural recycling of iron stores through splenic macrophages, accounting for

90% of iron recycling during red blood cell turnover (Gozzelino & Arosio, 2017).

Additionally, histological scans of various organs stained for iron prominently show iron in the knockout liver (Figure 2, C &D) and the 7.5-week wildtype spleen (Figure 2, F), as noted previously. Minimal iron is seen in the knockout heart (Figure 2, P). Iron accumulation in the

liver is seen to occur periportally, which is consistent with what is seen in older Hamp-/- rats. In the spleen, stains for iron reveal its location on the periphery. When compared to late-stage histology at 9 months of age (Figure 3), the same pattern of iron loading is observed (Flores et al,

2017). At these stages, iron can also be seen in the pancreas and heart.

Implications of these findings include the development of therapeutic techniques to curb iron overload before it causes systemic damage. It is important that mini-hepcidins be tested in this rat model at a younger age than the mouse studies conducted previously, so that previous iron stores before deficiency was induced do not affect the drug’s effectiveness. An optimized starting point of 4 weeks of age is suggested, since this study found 4.5-week-old female knockout animals to already be distinguished as more iron loaded than wildtype controls. During this early phase, efforts can be focused on preventing iron influx into the liver, since this organ seems to be the most affected.

Future studies should question additional advantages of a rat model for iron metabolism over the previously used mouse model. One such question is the ability of rats to absorb heme iron. Heme iron is found in meats complexed to myoglobin and hemoglobin and is a prevalent source of iron in the human diet since is more bioavailable when compared to inorganic iron.

However, evidence from Fillebeen et al. (2015) showed that mice fed a high-iron diet from heme did not experience increased iron concentration in the liver or blood. Furthermore, they did not observe increased production of hepcidin mRNA, suggesting that mice have a very low capacity to absorb heme iron. The hepcidin knockout rats used in the present study, along with wildtype controls, could be utilized to determine the capacity of a rat to absorb heme iron, as a closer model to human iron intake. Subsequently, tests for synthetic hepcidin in these models should be conducted to see if heme iron influences these pharmacological techniques.

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SUPPORTING FIGURES/TABLES Figure 1: Non-Heme Tissue Iron Levels A B

C D

E F

G H

I J

Figure 1. Non-heme (NH) iron levels increased in the livers of male & female KO rats The concentration of non-heme iron was measured by a colorimetric assay in the liver (A, B), spleen (C, D), pancreas (E, F), heart (G, H), and kidney (I, J). Data from male and female groups were analyzed separately. All animal numbers were n=5 /group. Box-and-Whisker plots designate the minimum value (lower whisker), lower quartile, median, upper quartile, and the maximum value (upper whisker). Variable analysis using 2-way ANOVA resulted in the following values: male & female livers (*genotype: p<0.0001; *time: p<0.0001; *genotype X time: p<0.0001); male spleen (*genotype: p<0.0001; time: p=0.0556; *genotype X time: p<0.0001); female spleen (*genotype: p<0.0001; *time: p<0.0001; *genotype X time: p<0.0001); male pancreas (genotype: p=0.08815; *time: p=0.0004; genotype X time: p=0.0998); female pancreas (genotype: p=0.1962; *time: p=0.0024; genotype X time: p=0.8692); male heart (genotype: p=0.1432; *time: p=0.0161; genotype X time: p=0.1434);); female heart (*genotype: p=0.0093; time: p=0.4474; genotype X time: p=0.6538); male kidney (*genotype: p=0.0242; *time: p=0.0051; *genotype X time: p=0.0029); and female kidney (*genotype: p=0.0201; *time: p<0.0001; genotype X time: p=0.1042). * = denotes significant value

Figure 2: Tissue Histology A B C D

3-week WT Liver 7.5-week WT Liver 3-week KO Liver 7.5-week KO Liver E F G H

3-week WT Spleen 7.5-week WT Spleen 3-week KO Spleen 7.5-week KO Spleen I J K L

3-week WT Pancreas 7.5-week WT Pancreas 3-week KO Pancreas 7.5-week KO Pancreas M N O P

3-week WT Heart 7.5-week WT Heart 3-week KO Heart 7.5-week KO Heart Q R S T

3-week WT Kidney 7.5-week WT Kidney 3-week KO Kidney 7.5-week KO Kidney 18

Figure 2. Tissue Histology shows iron accumulation. Representative images of DAB-enhanced Perls’ iron stain of paraffin-embedded tissue sections for the liver (A-D), spleen (E-H), pancreas (I-L), heart (M-P), and kidney (Q-T). Iron stores appear brown in color. Minimal iron accumulation is seen in the WT samples during this early stage. Iron buildup is seen in the 7.5-week-old knockout rat liver (B). The spleen shows iron loading on the periphery.

Figure 3: Late-Stage Tissue Histology A B

9-month WT Liver 9-month KO Liver C D

9-month WT Spleen 9-month KO Spleen E F

9-month WT Pancreas 9-month KO Pancreas G H

9-month WT Heart 9-month KO Heart

Figure 3. Comparisons made to late-stage iron accumulation show later iron build up in other tissues with age. Representative images of DAB-enhanced Perls’ iron stain paraffin-embedded tissue sections of the liver (A&B, not DAB-enhanced), spleen (C&D), pancreas (E&F), and heart (G&H) in 9- month-old rats. Iron stores appear brown in color. The pancreas and heart are seen to load iron unlike in the early characterization. The spleen still shows iron loading on the periphery.