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*
Department of Pediatrics, Kumamoto University School of Medicine,
Kumamoto, Japan.
The College of Medical Sciences, Kumamoto University School of Medicine,
Kumamoto, Japan.
Correspondence to Dr. Fumio Endo, Department of Pediatrics, Kumamoto University School of Medicine, Kumamoto 860-8556, Japan. Phone: +81 96 373 5188; Fax: +81 96 366 3471; E-mail: fendo{at}gpo.kumamoto-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Microscopical tubular dilation, nephrocalcinosis, involution of epithelial cells, and some degree of glomerulosclerosis were noted in HT1 patients 18 mo to 17 yr of age (2, 8). As neonatal Fah-deficient mice died, the kidney from newborn mice with FAH deficiency was investigated, and dilation and vesiculation of the rough endoplasmic reticulum and Golgi apparatus in C14CoS/14CoS newborn mouse kidney were apparent (9). When the Fah-deficient mice were treated with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), their life was spared, and focal degeneration, regeneration of proximal tubular epithelium, and aggregates of cytoplasmic microfilaments were observed (10). Mechanisms for cell injury or cellular dysfunction of the renal tubular cells are not well understood.
The hereditary tyrosinemia type 3 model mice with genetic deficiency of 4-hydroxyphenylpyruvic acid dioxygenase (Hpd; EC 1.13.11.27) on chromosome 5 (Hpd-/- mice) have hypertyrosinemia without liver injury (11, 12, 13). Crossing heterozygous c14CoS mice (Fah+/-) with Hpd-deficient mice, we developed a new mutant combination that carried two genetic defects, Fah-/- and Hpd-/- (14). Although Fah-deficient mice, either c14CoS or target disrupted mice proved to be neonatally lethal, the Fah-/- Hpd-/- mice had a long survival without evidence of hepatic and renal injuries, yet their phenotype is similar to Fah+/+ Hpd-/- mice (14).
We found that when homogentisate was administered, the hepatocytes of the Fah-/- Hpd-/- mice undergo rapid apoptosis by retrieval of the tyrosine catabolic pathway (Figure 1) and acute death follows (14). We now report that retrieval of the tyrosine catabolic pathway in the Fah-/- Hpd-/- mice by administering homogentisate intraperitoneally resulted in renal Fanconi syndrome, and renal proximal tubular cells underwent rapid apoptosis. These observations provide new insight into the disease process of HT1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To evaluate the protective effects of an apoptosis inhibitor in vivo, the Fah-/- Hpd-/- mice were injected intraperitoneally with acetyl-Tyr-Val-Ala-Asp-CHO (YVAD) (200 mg/kg body wt in 500 µl of physiologic saline; n = 4) (Takara, Japan), and 2 h later the mice were given intraperitoneally 200 mg/kg (in 100 µl of physiologic saline) of homogentisate, left for 48 h, and then the kidneys were excised. In contrast, the kidneys of Fah-/- Hpd-/- mice administered the same volume of physiologic saline (n = 2) as controls were excised 50 h after the injection.
Histology
For light microscopic examination, the kidneys fixed in 10% neutralized
formalin were dehydrated in 100% ethanol and embedded in paraffin wax at
58°C. The 1.5-µm sections were rehydrated and stained with hematoxylin
and eosin and periodic acid-Schiff. Using the one-and-a-half sections, the
3'-OH DNA ends generated by DNA fragmentation were detected using an
in situ terminal deoxyribonucleotidyltransferase-mediated
dUTP-digoxigenin nick end labeling assay (TUNEL)
(15) kit (ApopTag, Oncor,
Gaithersburg, MD), according to instructions from the manufacturer.
For electron microscopic observations, about 1 mm3 of the kidney were rapidly fixed in 2% glutaraldehyde (Nacalai Tesque, Inc., Kyoto, Japan)-0.1 M cacodylate buffer (pH 7.2) on ice for 3 h. After washing in 0.1 M cacodylate buffer, the sections were post-fixed in 1% osmium (VIII) tetroxide (Nacalai Tesque, Inc.)-0.1 M cacodylate buffer on ice for 2 h, dehydrated in ethanol and propylene oxide, and embedded in epoxy resin. The ultrathin sections were cut on an AO-Reichert Ultracut E microtome (Vienna, Austria), using a glass or diamond knife followed by double staining with 5% uranyl acetate and lead nitrate solution. The sections were examined under a H-300 microscope (Hitachi, Tokyo, Japan). Three mice per intervention and two to four blocks per kidney per mouse were used to observe the pathologic processes.
Biochemical Analysis of Serum and Urine
Blood samples were taken from the heart of the mice at 18 or 48 h after the
administration of homogentisate, and the serum were separated and stored at
-70°C. Urine collections were made 6 h before administering homogentisate
and at 6, 12, 18, 24, 36, and 48 h after this treatment as spot urine samples.
Urine was obtained by massaging the lower abdomen of mice directed to the
meatus at the indicated time. Serum levels of glucose, phosphate, blood urea
nitrogen (BUN), and urinary creatinine, glucose, and phosphate were measured
using an automated analyzer. Urinary glucose:creatinine ratio and
phosphate:creatinine ratio were calculated. Urinary concentration of
succinylacetone (SA) was determined by stable isotope dilution gas
chromatography-mass spectrometry assay, as described
(16).
Statistical Analyses
For statistical analyses, we used unpaired t tests and one-way
factorial ANOVA and multiple comparison tests.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In Fah-/- Hpd-/- mice and the control mice, urinary levels of creatinine, glucose, and phosphate were measured before and after treatment with homogentisate. Figure 3, A and B, shows time courses of changes in urinary glucose and phosphate, respectively. The urinary glucose:creatinine ratio was markedly increased in the Fah-/- Hpd-/- mice after the administration of 100 to 400 mg/kg homogentisate. In contrast, the urinary glucose:creatinine ratio was essentially unchanged in the control (Figure 3A). Similarly, the urinary phosphate:creatinine ratio was significantly increased in the treated Fah-/- Hpd-/- mice 18 to 48 h after the administration (Figure 3B). These results suggest that in the Fah-/- Hpd-/- mice, administration of homogentisate resulted in reduced reabsorption of glucose and phosphate by the renal tubules and that the degree of renal dysfunction in the treated Fah-/- Hpd-/- mice depended on the dose of homogentisate administered.
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Histologic Investigations
Renal sections from Fah-/- Hpd-/- mice
treated with various doses of homogentisate showed pathologic features of
bleeding in vast areas, accumulation of mononuclear cells at the interstitium,
proximal tubular dilation, and cytoplasmic vacuolation. A representative view
of the kidney sections obtained from the Fah-/-
Hpd-/- mice given 400 mg/kg homogentisate is shown in
Figure 4D. At the proximal
tubule, the nucleus of the epithelial cell was sometimes abnormal
(Figure 4D). The appearance of
glomeruli, interstitial cells, distal tubular cells, and blood vessels was
normal in all sections. The proximal tubular epithelial cells in the untreated
Fah-/- Hpd-/- mice, and in the untreated or
homogentisate-treated Fah+/+ Hpd-/- mice
appeared unchanged (Figure 4, A through
C).
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Representative electron microscopic views of proximal tubular cells from homogentisate-treated Fah-/- Hpd-/- mice are shown in Figure 5, C through F. In the cytoplasm, droplets of fat (Figure 5D) and large lysosomes were present and many mitochondria were swollen (Figure 5, C and D). The breakage and vacuolization were present at the brush border. In some cells, compaction and degradation of chromatin were present in association with convolution of the nuclear profile (Figure 5C). Thus, severe cellular damage of proximal tubular cells in the Fah-/- Hpd-/- mice occurred after treatment with 400 mg/kg homogentisate. Similar changes, albeit to a lesser extent, including the characteristic nucleus of apoptosis, were seen in some epithelial cells of the proximal tubules from Fah-/- Hpd-/- mice after injection of 200 mg/kg homogentisate (Figure 5E). However, in the Fah-/- Hpd-/- mice treated with 100 mg/kg homogentisate, there were no extensive ultrastructural abnormalities of renal proximal tubular epithelial cells (Figure 5F). These features were similar to findings in control Fah+/+ Hpd-/- mice treated with various doses of homogentisate or untreated Fah+/+ Hpd-/- mice (Figure 5). The most distal tubular and glomerular cells were normal in treated Fah-/- Hpd-/- mice (data not shown). Some pathologic changes, including swollen mitochondria, vacuolization, brush border loss, and large lysosomes, seem to be secondary damage in apoptotic cells. It may be that the apoptotic cells were inaccessible to phagocytes following extrusion into the lumen of the tubule or the extent of apoptotic cell death exceeded the phagocytic capacity.
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In Situ Detection of Apoptosis in Kidney Sections
Apoptosis was identified using the modified TUNEL assay. In the renal
sections obtained from Fah-/- Hpd-/- mice after
treatment with various doses of homogentisate, many nuclei showed evidence of
apoptosis (Figure 6). Most of
the apoptotic nuclei were located in the proximal tubules
(Figure 6, C, D, and F).
Counting of signal-positive cells in the kidney sections from the
homogentisate-treated Fah-/- Hpd-/- mice
revealed that administration of the 400 mg/kg homogentisate resulted in
apoptosis of approximately 80% of proximal tubular cells, as shown in
Table 1. When low doses of
homogentisate were administered, the ratio of positive cells decreased
(Table 1). The appearance of
apoptotic cells in the proximal renal tubules from the homogentisate-treated
Fah-/- Hpd-/- mice depended on the dose of
homogentisate administered. Few positive signals were present in distal
tubular nuclei, however, essentially no positive nucleus was found in
glomeruli, interstitial cells, or collecting duct cells
(Figure 6). Findings in the
untreated Fah-/- Hpd-/- mice were similar to
those seen in Fah+/+ Hpd-/- mice
(Figure 6, A, B, and E).
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Effects of a Caspase Inhibitor on Renal Tubular Cells
To evaluate in vivo effects of a caspase inhibitor on the kidney,
the Fah-/- Hpd-/- mice were preinjected
intraperitoneally with YVAD, then 2 h later, 200 mg/kg homogentisate was
administered. All mice survived for 48 h after the injection of homogentisate,
then were killed and the kidney was investigated. Renal sections from the
Fah-/- Hpd-/- mice pretreated with YVAD showed
few apoptotic nuclei, as determined using TUNEL assay
(Figure 6G,
Table 1). In addition,
histology of renal epithelial cells remained unchanged, as seen in the
hematoxylin and eosin and periodic acid-Schiff staining when YVAD was
administered before the administration of homogentisate (data not shown).
These results suggest that pretreatment with the caspase inhibitor can
effectively prevent cellular damage induced by homogentisate in the
Fah-/- Hpd-/- mice.
We next investigated the effects of a caspase inhibitor on renal tubular functions in the Fah-/- Hpd-/- mice. We preadministered YVAD, a potent, selective, cell-permeating inhibitor of caspase-1 (17, 18, 19) to the Fah-/- Hpd-/- mice, then the mice were treated with homogentisate. However, preadministration of YVAD did not alter the urinary levels of glucose and phosphate (Figure 7). This evidence suggested that the impaired function of renal tubules was not overcome by pretreatment of the homogentisate-treated Fah-/- Hpd-/- mice with the caspase inhibitor. It is notable that the serum BUN level remained high in the Fah-/- Hpd-/- mice pretreated by YVAD and treated by homogentisate (130.0 ± 8.49 mg/dl, n = 4). Thus, elevation of serum BUN seen in homogentisate-treated Fah-/- Hpd-/- mice is not directly related to the massive apoptosis of tubular epithelial cells. SA is derived from fumarylacetoacetate (FAA) and is excreted into the urine of HT1 patients and in the homogentisate-treated Fah-/- Hpd-/- mice (14). We confirmed that SA levels were markedly increased in Fah-/- Hpd-/- mice after the injection of homogentisate (204700 ± 3360.67 nmol/mmol creatinine, n = 4), whereas the untreated Fah-/- Hpd-/- mice showed low values (150 ± 14.75 nmol/mmol creatinine, n = 2). SA levels in urine of YVAD-pretreated and homogentisate-treated Fah-/- Hpd-/- mice remained at higher levels (14200 ± 2205.45 nmol/mmol creatinine, n = 4). Therefore, the caspase inhibitor YVAD did not alter the urinary excretion of SA in the homogentisate-treated Fah-/- Hpd-/- mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous mouse models for HT1, either albino-lethal c14COS or targeted disrupted FAH deficiency, were neonatally lethal (21, 22, 23, 24, 25, 26, 27), or required treatment with NTBC (10), an effective inhibitor for HPD activity. Although the neonatal c14COS mice showed no apparent kidney injury, abnormalities were characterized by changes in membranous structures (9, 10) and were limited to proximal epithelial cells of tubules (9, 23), an area where FAH is physiologically expressed (23, 25). In the NTBC-treated Fah-deficient mice, renal proximal tubular cells of the kidney showed abnormalities in nuclei and lysosomes (10). In addition, the caspase inhibitor YVAD effectively prevented the appearance of these changes in cellular structures of the Fah-deficient mice (Figure 6G). These results suggest that abnormalities in the structure of the cells noted in the earlier studies are signs of ensuing apoptotic death. Accordingly, the death signal was specific in triggering cell death processes.
Previously, we analyzed hepatocellular apoptosis induced by homogentisate in Fah-/- Hpd-/- mice, and we suggested that cell death of hepatocytes that carried FAH deficiency was due to intracellular events (14, 20). For renal tubular cells, however, there are at least two possibilities for the origin of cell injury: The cell death is due entirely to an intracellular process, or is initiated by chemicals derived from the extracellular metabolism, for example chemicals derived from glomerular filtrates. In this context, SA was present in the urine of HT1 patients (6, 7, 8, 28) and in the urine of Fah-deficient mice (10, 14, 20). However, SA did not cause cellular damage (29, 30). Other chemicals such as FAA or MAA were not detected in urine from FAH-deficient patients (6) or mice (25). In addition, apoptotic death is confined to proximal renal tubular cells where Fah is likely to be expressed. We speculate that intracellular metabolite(s) such as FAA may explain the cell death in proximal tubules.
Kidney pathology associated with HT1 includes glomerulosclerosis and interstitial fibrosis, and there frequently is a loss of the brush border and cytoplasmic vacuolation in ultrastructures of the tubular cells (2, 8). Such pathology in the renal tubular cells is likely to be related to occurrence of cell death signal in the cells. Because apoptotic cells are rapidly removed from the tubules, it may be difficult to detect such cells in kidney sections from patients. Long-term investigations of Fah-/- Hpd-/- mice given low doses of homogentisate may elucidate the precise process of renal damage seen in the HT1 patients.
Fanconi syndrome in HT1 patients
(2,
6) was reproduced in treated
Fah-/- Hpd-/- mice
(Figure 3), as reabsorption of
glucose and phosphate was impaired. Interestingly, treatment of the
Fah-/- Hpd-/- mice with the caspase inhibitor
YVAD did not prevent the development of Fanconi syndrome
(Figure 7), which means that
the cell death pathway and the metabolic process to Fanconi syndrome can be
separated in the nephropathy associated with FAH deficiency. If the pathway
for cell death and the biochemical process leading to Fanconi syndrome are
completely separable, one possible explanation is that Fanconi syndrome is
caused by chemicals derived from filtrates of glomerulus in this mouse model,
and in HT1 patients. Roth and colleagues extensively investigated the effects
of SA on renal tubules in rats
(29,
30,
31,
32). SA inhibited uptake of
methyl 
-D-glucose, but electron microscopic analysis revealed no
specific changes in morphology, even with high concentrations of SA
(29,
30). Thus, it is likely that
SA can act as a reversible inhibitor for cellular metabolism, but does not
cause cellular changes in morphology. It seems likely that Fanconi syndrome
seen in the homogentisate-treated Fah-/- Hpd-/-
mice is, at least partly, relevant to SA.
The possibility that abnormalities in intracellular metabolism caused by FAH deficiency lead to Fanconi syndrome has to be considered. In hepatocytes of albino lethal mice or HT1 patients (33), expression of hepatocyte-specific and developmentally regulated transcription factors, including G6Pase, HNF1, HNF4, and C/EBP (21, 24, 26), are reduced. These changes may contribute to the impaired development of metabolic functions of the liver. Observations of the expression of phosphoenol pyruvate carboxykinase (21, 23, 26) and menadione oxidoreductase (26) in renal tubules from albino lethal mice suggested that similar changes in the expression of transcription factors occur in renal tubular cells when FAH is deficient. In this context, it is interesting that targeted disruption of HNF-1 in mice resulted in Fanconi syndrome and was characterized by marked glucosuria. Accordingly, it is possible that Fanconi syndrome in the Fah-/- Hpd-/- mice is partly due to abnormal expression of transcription factors.
There are observations that apoptosis of kidney cells underlies the pathogenesis of kidney diseases, conditions that include polycystic kidney disease (34), glomerulonephritis (35, 36), exposure to toxic agents (37, 38, 39), and some cases of kidney transplantation (40). However, clear relationships between Fanconi syndrome and apoptosis have not been demonstrated (41). We found that renal tubular damage in HT1 involves cell death due to apoptosis. As discussed above, apoptosis associated with Fah deficiency is caused by interaction between FAA and mitochondria. Changes in morphology of proximal cells after injection of homogentisate are characterized by abnormalities in mitochondria, disruption of the nuclear membrane, and fragmentation of nuclei. These changes are obviously relevant to apoptosis and are completely prevented by administration of a caspase inhibitor before the administration of homogentisate. We suggest that the apoptosis of renal tubular epithelial cells observed in our study is likely due to the accumulation of FAA in epithelial cells.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-D-glucoside uptake by the rat renal tubule. Biochem Biophys Acta
820: 140
-146, 1985[Medline]
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, untreated Fah-/- Hpd-/- mice; [UNK],
treated Fah+/+ Hpd-/-; [UNK], treated
Fah-/- Hpd-/- mice. The asterisk indicates
significant changes in the parameters of the treated Fah-/-
Hpd-/- mice compared with treated Fah+/+
Hpd-/- mice *P < 0.05;
**P < 0.01.
, Fah+/+ Hpd-/- mice treated with 200
mg/kg homogentisate (n = 6); 
, Fah+/+
Hpd-/- mice treated with 400 mg/kg homogentisate
(n = 6). The treated animals were as follows: 
,
Fah-/- Hpd-/- mice treated with 100 mg/kg
homogentisate (n = 5); 
, Fah-/-
Hpd-/- treated with 200 mg/kg homogentisate (n =
5); [UNK], Fah-/- Hpd-/- treated with 400 mg/kg
homogentisate (n = 5). The arrow indicates the point of injection of
homogentisate. *P < 0.05; **P < 0.01;
***P < 0.001. (A) Changes in glucose: creatinine ratio
after the injection of 100, 200, and 400 mg/kg homogentisate are shown.
Urinary excretion of glucose was significantly increased in the
Fah-/- Hpd-/- mice compared with findings in
the similarly treated Fah+/+ Hpd-/- mice. (B)
Changes in phosphate:creatinine ratio after the injection of 100, 200, and 400
mg/kg homogentisate are shown. The phosphate:creatinine ratio was
significantly high at 18 h after the injection of 400 mg/kg homogentisate, at
24 h after the injection of 200 mg/kg homogentisate, and at 36 h after the
injection of 100 mg/kg homogentisate in Fah-/-
Hpd-/- mice.
