A Mouse Model of Renal Tubular Injury of Tyrosinemia Type 1

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J Am Soc Nephrol 11: 291–300, 2000 A Mouse Model of Renal Tubular Injury of Tyrosinemia Type 1: Development of de Toni Fanconi Syndrome and Apoptosis of Renal Tubular Cells in Fah/Hpd Double Mutant Mice

MAO-SEN SUN,* SHINZABURO HATTORI,† SHUJI KUBO,* HISATAKA AWATA,* ICHIRO MATSUDA,* and FUMIO ENDO* *Department of Pediatrics and †The College of Medical Sciences, Kumamoto University School of Medicine, Kumamoto, Japan.

Abstract. Hereditary tyrosinemia type 1 (HT1) (McKusick metabolite between HPD and FAH, was administered to the 276700), a severe autosomal recessive disorder of tyrosine FahϪ/ϪHpdϪ/Ϫ mice. Simultaneously, renal tubular function metabolism, is caused by mutations in the fumarylacetoacetate was impaired and Fanconi syndrome occurred. Apoptotic death hydrolase gene Fah (EC 3.7.1.2), which encodes the last en- of renal tubular cells, but not renal dysfunction, was prevented zyme in the tyrosine catabolic pathway. HT1 is characterized by pretreatment of the animals with YVAD, a specific inhibitor by severe progressive liver disease and renal tubular dysfunc- of caspases. In the homogentisate-treated FahϪ/ϪHpdϪ/Ϫ tion. Homozygous disruption of the gene encoding Fah in mice mice, massive amounts of succinylacetone were excreted into causes neonatal lethality (e.g., lethal Albino deletion c14CoS the urine, regardless of treatment with inhibitors. It is sug- mice), an event that limits use of this animal as a model for gested that apoptotic death of renal tubular cells, as induced by HT1. A new mouse model was developed with two genetic administration of homogentisate to FahϪ/ϪHpdϪ/Ϫ mice, was defects, Fah and 4-hydroxyphenylpyruvate dioxygenase (Hpd). caused by an intrinsic process, and that renal apoptosis and The FahϪ/ϪHpdϪ/Ϫ mice grew normally without evidence tubular dysfunctions in tubular cells occurred through different of liver and renal disease, and the phenotype is similar to that pathways. These observations shed light on the pathogenesis of in Fahϩ/ϩHpdϪ/Ϫ mice. The renal tubular cells of renal tubular injury in subjects with FAH deficiency. These FahϪ/ϪHpdϪ/Ϫ mice, particularly proximal tubular cells, un- FahϪ/ϪHpdϪ/Ϫ mice can serve as a model in experiments derwent rapid apoptosis when homogentisate, the intermediate related to renal tubular damage.

Hereditary tyrosinemia type 1 (HT1) (McKusick 276700), a Fah-deficient mice died, the kidney from newborn mice with severe autosomal recessive disorder of tyrosine metabolism FAH deficiency was investigated, and dilation and vesiculation (1,2), is caused by mutations in the fumarylacetoacetate hy- of the rough endoplasmic reticulum and Golgi apparatus in drolase gene Fah (EC 3.7.1.2) (3–5), encoding the last enzyme C14CoS/14CoS newborn mouse kidney were apparent (9). When in the tyrosine catabolic pathway (1,5). This disorder is char- the Fah-deficient mice were treated with 2-(2-nitro-4-triflu- acterized by severe progressive liver disease, including liver oromethylbenzoyl)-1,3-cyclohexanedione (NTBC), their life failure during infancy or in later life, chronic liver damage with was spared, and focal degeneration, regeneration of proximal a high incidence of hepatoma, and renal tubular dysfunction tubular epithelium, and aggregates of cytoplasmic microfila- characterized as de Toni Fanconi syndrome (1,2,6,7). Liver ments were observed (10). Mechanisms for cell injury or transplantation can save the life of HT1 patients, in some but cellular dysfunction of the renal tubular cells are not well not all patients, and liver transplantation led to improvement in understood. renal function (7,8). The precise mechanism of renal tubular The hereditary tyrosinemia type 3 model mice with genetic damage associated with FAH deficiency has required further deficiency of 4-hydroxyphenylpyruvic acid dioxygenase (Hpd; study. EC 1.13.11.27) on chromosome 5 (HpdϪ/Ϫ mice) have hyper- Microscopical tubular dilation, nephrocalcinosis, involution tyrosinemia without liver injury (11–13). Crossing heterozy- of epithelial cells, and some degree of glomerulosclerosis were gous c14CoS mice (Fahϩ/Ϫ) with Hpd-deficient mice, we de- noted in HT1 patients 18 mo to 17 yr of age (2,8). As neonatal veloped a new mutant combination that carried two genetic defects, FahϪ/Ϫ and HpdϪ/Ϫ (14). Although Fah-deficient mice, either c14CoS or target disrupted mice proved to be Received May 25, 1999. Accepted July 17, 1999. neonatally lethal, the FahϪ/ϪHpdϪ/Ϫ mice had a long survival Correspondence to Dr. Fumio Endo, Department of Pediatrics, Kumamoto ϩ without evidence of hepatic and renal injuries, yet their phe- University School of Medicine, Kumamoto 860-8556, Japan. Phone: 81 96 ϩ/ϩ Ϫ/Ϫ 373 5188; Fax: ϩ81 96 366 3471; E-mail: [email protected] notype is similar to Fah Hpd mice (14). We found that when homogentisate was administered, the 1046-6673/1102-0291 Ϫ/Ϫ Ϫ/Ϫ Journal of the American Society of Nephrology hepatocytes of the Fah Hpd mice undergo rapid apopto- Copyright © 2000 by the American Society of Nephrology sis by retrieval of the tyrosine catabolic pathway (Figure 1) and 292 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 291–300, 2000

volume of physiologic saline (n ϭ 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-␮m sections were rehydrated and stained with hematoxylin and eosin and periodic acid-Schiff. Using the one-and-a-half sections, the 3Ј-OH DNA ends generated by DNA fragmentation were detected using an in situ terminal deoxyribonucle- otidyltransferase-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 Figure 1. Tyrosine catabolic pathway in a mouse model of hereditary diamond knife followed by double staining with 5% uranyl acetate tyrosinemia. The principal metabolites in the pathway are tyrosine, and lead nitrate solution. The sections were examined under a H-300 4-hydroxyphenylpyruvate, homogentisate, maleylacetoacetate, and microscope (Hitachi, Tokyo, Japan). Three mice per intervention and fumarylacetoacetate. In hereditary tyrosinemia type III, 4-hydroxy- two to four blocks per kidney per mouse were used to observe the phenylpyruvate dioxygenase (HPD) is deficient, and in hereditary pathologic processes. tyrosinemia type I, fumarylacetoacetate hydrolase (FAH) is deficient. The Hpd-deficient mice carried a nonsense mutation in the Hpd gene on chromosome 5, and the Fah-deficient mice carried a large dele- Biochemical Analysis of Serum and Urine tion on chromosome 7 that disrupted the Fah gene. The mouse Blood samples were taken from the heart of the mice at 18 or 48 h model presented here carries deficiency of both Hpd and Fah after the administration of homogentisate, and the serum were sepa- (FahϪ/ϪHpdϪ/Ϫ mouse). rated and stored at Ϫ70°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 acute death follows (14). We now report that retrieval of the time. Serum levels of glucose, phosphate, blood urea nitrogen (BUN), Ϫ/Ϫ Ϫ/Ϫ tyrosine catabolic pathway in the Fah Hpd mice by and urinary creatinine, glucose, and phosphate were measured using administering homogentisate intraperitoneally resulted in renal an automated analyzer. Urinary glucose:creatinine ratio and phos- Fanconi syndrome, and renal proximal tubular cells underwent phate:creatinine ratio were calculated. Urinary concentration of suc- rapid apoptosis. These observations provide new insight into cinylacetone (SA) was determined by stable isotope dilution gas the disease process of HT1. chromatography-mass spectrometry assay, as described (16).

Materials and Methods Statistical Analyses Animals For statistical analyses, we used unpaired t tests and one-way ϩ/ϩ Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ Male Fah Hpd mice and the male Fah Hpd mice, factorial ANOVA and multiple comparison tests. crossed by heterozygous c14CoS mice (Fahϩ/Ϫ) and homozygous HpdϪ/Ϫ mice, were used (14). Because the lethal dose of homogentisate is 400 mg/kg body wt for the FahϪ/ϪHpdϪ/Ϫ mouse (the Results median lethal dose is 300 mg/kg body wt), the Fahϩ/ϩHpdϪ/Ϫ mice Biochemical Data on Blood and Urine and FahϪ/ϪHpdϪ/Ϫ mice were given 100, 200, and 400 mg/kg of To evaluate renal tubular functions in the FahϪ/ϪHpdϪ/Ϫ neutralized homogentisate intraperitoneally and the kidneys were ex- mice after the administration of homogentisate, serum levels of cised 18 or 48 h after this injection. The control mice were injected glucose, phosphate, and BUN were measured after treatment at with the same volume of physiologic saline. All of the animals were appropriate intervals (Figure 2). Fahϩ/ϩHpdϪ/Ϫ mice served as freely fed during the experiments. the control animals. A significant reduction in serum glucose To evaluate the protective effects of an apoptosis inhibitor in vivo, Ϫ/Ϫ Ϫ/Ϫ Ϫ Ϫ Ϫ Ϫ levels was seen in the Fah Hpd mice treated with 200 the Fah / Hpd / mice were injected intraperitoneally with acetyl- Tyr-Val-Ala-Asp-CHO (YVAD) (200 mg/kg body wt in 500 ␮lof and 400 mg/kg homogentisate (Figure 2, A and B). In contrast, physiologic saline; n ϭ 4) (Takara, Japan), and 2 h later the mice were there was a significant increase in serum BUN after treatment given intraperitoneally 200 mg/kg (in 100 ␮l of physiologic saline) of with 200 and 400 mg/kg homogentisate (Figure 2, A and B) homogentisate, left for 48 h, and then the kidneys were excised. In and in serum phosphate after treatment with 400 mg/kg ho- Ϫ Ϫ Ϫ Ϫ contrast, the kidneys of FahϪ/ϪHpdϪ/Ϫ mice administered the same mogentisate to the Fah / Hpd / mice (Figure 2B). However, J Am Soc Nephrol 11: 291–300, 2000 Tyrosinemia Type 1 and Fah/Hpd Mice 293

Figure 2. Blood chemistry in the FahϪ/ϪHpdϪ/Ϫ mice. Representative data for serum levels of glucose, phosphate, and blood urea nitrogen (BUN) in the mice. Mice injected with neutralized homogentisate Figure 3. Effect of administration of homogentisate on the urinary solution (treated animals) or physiologic saline solution (untreated excretion of glucose and phosphate in the FahϪ/ϪHpdϪ/Ϫ mice. The animals) as described in Materials and Methods were examined. control animals were as follows: Ⅺ, Fahϩ/ϩHpdϪ/Ϫ mice treated with Blood samples were taken from the heart 48 h after the injection of 100 mg/kg homogentisate (n ϭ 3); ‚, Fahϩ/ϩHpdϪ/Ϫ mice treated 200 mg/kg body wt (A) or 18 h after the injection of 400 mg/kg body with 200 mg/kg homogentisate (n ϭ 6); E, Fahϩ/ϩHpdϪ/Ϫ mice wt (B). Numbers of mice treated were as follows: A, treated with 400 mg/kg homogentisate (n ϭ 6). The treated animals Ϫ Ϫ Ϫ Ϫ ϩ ϩ Ϫ Ϫ Fah / Hpd / mice, n ϭ 5; Fah / Hpd / mice, n ϭ 6; and B, were as follows: f, FahϪ/ϪHpdϪ/Ϫ mice treated with 100 mg/kg Ϫ Ϫ Ϫ Ϫ ϩ ϩ Ϫ Ϫ Fah / Hpd / mice, n ϭ 5; Fah / Hpd / mice, n ϭ 6. Untreated homogentisate (n ϭ 5); Œ, FahϪ/ϪHpdϪ/Ϫ treated with 200 Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Fah / Hpd / mice, n ϭ 4 (A and B). Ⅺ, untreated Fah / Hpd / mg/kg homogentisate (n ϭ 5); F, FahϪ/ϪHpdϪ/Ϫ treated with ϩ ϩ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ mice; u, treated Fah / Hpd / ; o, treated Fah / Hpd / mice. 400 mg/kg homogentisate (n ϭ 5). The arrow indicates the point of The asterisk indicates significant changes in the parameters of the injection of homogentisate. *P Ͻ 0.05; **P Ͻ 0.01; ***P Ͻ 0.001. Ϫ Ϫ Ϫ Ϫ ϩ ϩ Ϫ Ϫ treated Fah / Hpd / mice compared with treated Fah / Hpd / (A) Changes in glucose:creatinine ratio after the injection of 100, 200, mice *P Ͻ 0.05; **P Ͻ 0.01. and 400 mg/kg homogentisate are shown. Urinary excretion of glucose was significantly increased in the FahϪ/ϪHpdϪ/Ϫ mice compared with findings in the similarly treated Fahϩ/ϩHpdϪ/Ϫ mice. (B) Changes in phosphate:creatinine ratio after the injection of 100, 200, the levels of serum glucose, BUN, and phosphate in the Ϫ/Ϫ Ϫ/Ϫ and 400 mg/kg homogentisate are shown. The phosphate:creatinine Fah Hpd mice were not affected by 100 mg/kg ho- ratio was significantly high at 18 h after the injection of 400 mg/kg mogentisate (data not shown). Similarly, the level of serum homogentisate, at 24 h after the injection of 200 mg/kg homogenti- Ϫ/Ϫ Ϫ/Ϫ phosphate in the Fah Hpd mice was not affected in case sate, and at 36 h after the injection of 100 mg/kg homogentisate in of 200 mg/kg homogentisate (Figure 2A). In the control mice, FahϪ/ϪHpdϪ/Ϫ mice. the same treatment did not significantly change any of these parameters (Figure 2). In FahϪ/ϪHpdϪ/Ϫ mice and the control mice, urinary was markedly increased in the FahϪ/ϪHpdϪ/Ϫ mice after the levels of creatinine, glucose, and phosphate were measured administration of 100 to 400 mg/kg homogentisate. In con- before and after treatment with homogentisate. Figure 3, A trast, the urinary glucose:creatinine ratio was essentially and B, shows time courses of changes in urinary glucose and unchanged in the control (Figure 3A). Similarly, the urinary phosphate, respectively. The urinary glucose:creatinine ratio phosphate:creatinine ratio was significantly increased in the 294 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 291–300, 2000 treated FahϪ/ϪHpdϪ/Ϫ mice 18 to 48 h after the adminis- Representative electron microscopic views of proximal tu- tration (Figure 3B). These results suggest that in the bular cells from homogentisate-treated FahϪ/ϪHpdϪ/Ϫ mice FahϪ/ϪHpdϪ/Ϫmice, administration of homogentisate re- are shown in Figure 5, C through F. In the cytoplasm, droplets sulted in reduced reabsorption of glucose and phosphate by of fat (Figure 5D) and large lysosomes were present and many the renal tubules and that the degree of renal dysfunction in mitochondria were swollen (Figure 5, C and D). The breakage the treated FahϪ/ϪHpdϪ/Ϫ mice depended on the dose of and vacuolization were present at the brush border. In some homogentisate administered. cells, compaction and degradation of chromatin were present in association with convolution of the nuclear profile (Figure 5C). Histologic Investigations Thus, severe cellular damage of proximal tubular cells in the Renal sections from FahϪ/ϪHpdϪ/Ϫ mice treated with var- FahϪ/ϪHpdϪ/Ϫ mice occurred after treatment with 400 mg/kg ious doses of homogentisate showed pathologic features of homogentisate. Similar changes, albeit to a lesser extent, in- bleeding in vast areas, accumulation of mononuclear cells at cluding the characteristic nucleus of apoptosis, were seen the interstitium, proximal tubular dilation, and cytoplasmic in some epithelial cells of the proximal tubules from vacuolation. A representative view of the kidney sections ob- FahϪ/ϪHpdϪ/Ϫ mice after injection of 200 mg/kg homogenti- tained from the FahϪ/ϪHpdϪ/Ϫ mice given 400 mg/kg ho- sate (Figure 5E). However, in the FahϪ/ϪHpdϪ/Ϫ mice treated mogentisate is shown in Figure 4D. At the proximal tubule, the with 100 mg/kg homogentisate, there were no extensive ultra- nucleus of the epithelial cell was sometimes abnormal (Figure structural abnormalities of renal proximal tubular epithelial 4D). The appearance of glomeruli, interstitial cells, distal tu- cells (Figure 5F). These features were similar to findings in bular cells, and blood vessels was normal in all sections. control Fahϩ/ϩHpdϪ/Ϫ mice treated with various doses of The proximal tubular epithelial cells in the untreated homogentisate or untreated Fahϩ/ϩHpdϪ/Ϫ mice (Figure 5). FahϪ/ϪHpdϪ/Ϫ mice, and in the untreated or homogentisate- The most distal tubular and glomerular cells were normal in treated Fahϩ/ϩHpdϪ/Ϫ mice appeared unchanged (Figure 4, A treated FahϪ/ϪHpdϪ/Ϫ mice (data not shown). Some patho- through C). logic changes, including swollen mitochondria, vacuolization,

Figure 4. Periodic acid-Schiff staining of kidney sections. The sections were obtained from Fahϩ/ϩHpdϪ/Ϫ mice (A and C) or FahϪ/ϪHpdϪ/Ϫ mice (B and D). A and B represent sections from the untreated animals. C and D show a representative view of the sections obtained from mice 18 h after the injection of 400 mg/kg homogentisate. Note the chromatic condensation near the nuclear membrane and the tubular dilation and cytoplasmic vacuolation (D). Magnification: ϫ297 in A; ϫ355 in B; ϫ354 in C; and ϫ312 in D. J Am Soc Nephrol 11: 291–300, 2000 Tyrosinemia Type 1 and Fah/Hpd Mice 295

Figure 5. Electron microscopic views of renal proximal tubular cells. Sections from an untreated Fahϩ/ϩHpdϪ/Ϫ mouse (A), an untreated FahϪ/ϪHpdϪ/Ϫ mouse (B), and treated FahϪ/ϪHpdϪ/Ϫ mice with 100 mg/kg (F), 200 mg/kg (E), and 400 mg/kg (C and D) of homogentisate are shown, respectively. Note the compaction, segregation, and homogenization of chromatin at the nuclear periphery (arrowhead; C, D, and E) and the broken nuclear envelope (C). At the cytoplasm, numerous mitochondria are swollen, cristae are curved and broken (C and D), and fatty droplets are numerous (D). Magnification: ϫ3520 in A; ϫ3200 in B; ϫ3500 in C; ϫ3000 in D; ϫ3600 in E; and ϫ4380 in F. 296 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 291–300, 2000 brush border loss, and large lysosomes, seem to be secondary sive apoptosis of tubular epithelial cells. SA is derived from damage in apoptotic cells. It may be that the apoptotic cells fumarylacetoacetate (FAA) and is excreted into the urine of were inaccessible to phagocytes following extrusion into the HT1 patients and in the homogentisate-treated FahϪ/ϪHpdϪ/Ϫ lumen of the tubule or the extent of apoptotic cell death mice (14). We confirmed that SA levels were markedly in- exceeded the phagocytic capacity. creased in FahϪ/ϪHpdϪ/Ϫ mice after the injection of homogentisate (204700 Ϯ 3360.67 nmol/mmol creatinine, n ϭ 4), In Situ Detection of Apoptosis in Kidney Sections whereas the untreated FahϪ/ϪHpdϪ/Ϫ mice showed low values Apoptosis was identified using the modified TUNEL assay. (150 Ϯ 14.75 nmol/mmol creatinine, n ϭ 2). SA levels In the renal sections obtained from FahϪ/ϪHpdϪ/Ϫ mice after in urine of YVAD-pretreated and homogentisate-treated treatment with various doses of homogentisate, many nuclei FahϪ/ϪHpdϪ/Ϫ mice remained at higher levels (14200 Ϯ showed evidence of apoptosis (Figure 6). Most of the apoptotic 2205.45 nmol/mmol creatinine, n ϭ 4). Therefore, the caspase nuclei were located in the proximal tubules (Figure 6, C, D, inhibitor YVAD did not alter the urinary excretion of SA in the and F). Counting of signal-positive cells in the kidney sections homogentisate-treated FahϪ/ϪHpdϪ/Ϫ mice. from the homogentisate-treated FahϪ/ϪHpdϪ/Ϫ mice revealed that administration of the 400 mg/kg homogentisate resulted in Discussion apoptosis of approximately 80% of proximal tubular cells, as In the present study, we investigated kidneys of a recently shown in Table 1. When low doses of homogentisate were developed mouse model of human HT1 with both FAH and administered, the ratio of positive cells decreased (Table 1). HPD deficiency. As expected from previous studies in the liver The appearance of apoptotic cells in the proximal renal tubules (14), kidneys from the FahϪ/ϪHpdϪ/Ϫ mice are phenotypically from the homogentisate-treated FahϪ/ϪHpdϪ/Ϫ mice depended normal in terms of morphology and function. This indicated on the dose of homogentisate administered. Few positive sig- that the introduction of a null mutation of Hpd prevented any nals were present in distal tubular nuclei, however, essentially pathology of the kidney associated with Fah deficiency. Our no positive nucleus was found in glomeruli, interstitial cells, previous study revealed that hepatocytes which lack Fah and or collecting duct cells (Figure 6). Findings in the untreated Hpd undergo rapid apoptosis when the tyrosine catabolic path- FahϪ/ϪHpdϪ/Ϫ mice were similar to those seen in way was reopened at the step of HPD (14), and FAA is at least Fahϩ/ϩHpdϪ/Ϫ mice (Figure 6, A, B, and E). one of the chemicals that reacts with mitochondria and releases cytochrome c from the mitochondria (20). Recent advances in Effects of a Caspase Inhibitor on Renal Tubular Cells the understanding of mechanisms related to apoptosis indicated To evaluate in vivo effects of a caspase inhibitor on the that activation of caspases is one of the main processes leading kidney, the FahϪ/ϪHpdϪ/Ϫ mice were preinjected intraperito- to cell death (17–19). In the present study, apoptotic death of neally with YVAD, then 2 h later, 200 mg/kg homogentisate renal tubular epithelial cells was almost completely prevented was administered. All mice survived for 48 h after the injection by preadministration of the caspase inhibitor YVAD (Figure of homogentisate, then were killed and the kidney was inves- 6G, Table 1). This means that apoptosis of the epithelial cells tigated. Renal sections from the FahϪ/ϪHpdϪ/Ϫ mice pre- resulted from activation of caspases in the cells. treated with YVAD showed few apoptotic nuclei, as deter- Previous mouse models for HT1, either albino-lethal c14COS mined using TUNEL assay (Figure 6G, Table 1). In addition, or targeted disrupted FAH deficiency, were neonatally lethal histology of renal epithelial cells remained unchanged, as seen (21–27), or required treatment with NTBC (10), an effective in the hematoxylin and eosin and periodic acid-Schiff staining inhibitor for HPD activity. Although the neonatal c14COS mice when YVAD was administered before the administration showed no apparent kidney injury, abnormalities were charac- of homogentisate (data not shown). These results suggest terized by changes in membranous structures (9,10) and were that pretreatment with the caspase inhibitor can effectively limited to proximal epithelial cells of tubules (9,23), an area prevent cellular damage induced by homogentisate in the where FAH is physiologically expressed (23,25). In the NTBC- FahϪ/ϪHpdϪ/Ϫ mice. treated Fah-deficient mice, renal proximal tubular cells of the We next investigated the effects of a caspase inhibitor on kidney showed abnormalities in nuclei and lysosomes (10). In renal tubular functions in the FahϪ/ϪHpdϪ/Ϫ mice. We pread- addition, the caspase inhibitor YVAD effectively prevented the ministered YVAD, a potent, selective, cell-permeating inhibi- appearance of these changes in cellular structures of the Fah- tor of caspase-1 (17–19) to the FahϪ/ϪHpdϪ/Ϫ mice, then the deficient mice (Figure 6G). These results suggest that abnor- mice were treated with homogentisate. However, preadminis- malities in the structure of the cells noted in the earlier studies tration of YVAD did not alter the urinary levels of glucose and are signs of ensuing apoptotic death. Accordingly, the death phosphate (Figure 7). This evidence suggested that the im- signal was specific in triggering cell death processes. paired function of renal tubules was not overcome by pretreat- Previously, we analyzed hepatocellular apoptosis induced by ment of the homogentisate-treated FahϪ/ϪHpdϪ/Ϫ mice with homogentisate in FahϪ/ϪHpdϪ/Ϫ mice, and we suggested that the caspase inhibitor. It is notable that the serum BUN level cell death of hepatocytes that carried FAH deficiency was due remained high in the FahϪ/ϪHpdϪ/Ϫ mice pretreated by to intracellular events (14,20). For renal tubular cells, however, YVAD and treated by homogentisate (130.0 Ϯ 8.49 mg/dl, n ϭ there are at least two possibilities for the origin of cell injury: 4). Thus, elevation of serum BUN seen in homogentisate- The cell death is due entirely to an intracellular process, or is treated FahϪ/ϪHpdϪ/Ϫ mice is not directly related to the mas- initiated by chemicals derived from the extracellular metabo- J Am Soc Nephrol 11: 291–300, 2000 Tyrosinemia Type 1 and Fah/Hpd Mice 297

Figure 6. Homogentisate-induced apoptosis of kidney cells in the FahϪ/ϪHpdϪ/Ϫ mice and effects of a caspase inhibitor. The renal sections were obtained from variously treated mice and stained for DNA fragmentation, as described in Materials and Methods. The controls are untreated Fahϩ/ϩHpdϪ/Ϫ mice (A), untreated FahϪ/ϪHpdϪ/Ϫ mice (B), and Fahϩ/ϩHpdϪ/Ϫ mice treated with 400 mg/kg homogentisate (E). In the experimental animals, FahϪ/ϪHpdϪ/Ϫ mice were treated with 100 mg/kg homogentisate (C), 200 mg/kg homogentisate (D), or 400 mg/kg homogentisate (F). A section from acetyl-Tyr-Val-Ala-Asp-CHO (YVAD)-pretreated FahϪ/ϪHpdϪ/Ϫ and homogentisate-treated (200 mg/kg body wt) mice is shown in G. The arrowheads indicate apoptotic cells. Magnification: ϫ318 in A through G. 298 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 291–300, 2000

Table 1. Percentages of apoptotic cells in proximal renal tubules of FahϪ/ϪHpdϪ/Ϫ mice after the administration of homogentisate

Genotype

Parameter ϩ/ϩ Ϫ/ Fah Hpd Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫb Ϫ Fah Hpd Fah Hpd

Dose of homogentisate administered (mg/mouse) 2.5 5 10 2.5 5 10 5 Preadministration of YVAD No No No No No No Yes No. of mice 3 6 6 5 5 5 4 Percentages of positive nuclei (mean) Ͻ1 Ͻ1 Ͻ1 6.9 49 83.8 Ͻ1 (1 to 14.1) (32.6 to 67.9) (61.9 to 94.7)

a The percentages of signal-positive cells after TUNEL staining were calculated based on counting in 20 random fields of one slice from each kidney. Ranges are given in parentheses. b The caspase inhibitor YVAD was injected 2 h before the administration of homogentisate. lism, for example chemicals derived from glomerular filtrates. The possibility that abnormalities in intracellular metab- In this context, SA was present in the urine of HT1 patients olism caused by FAH deficiency lead to Fanconi syndrome (6–8,28) and in the urine of Fah-deficient mice (10,14,20). has to be considered. In hepatocytes of albino lethal mice or However, SA did not cause cellular damage (29,30). Other HT1 patients (33), expression of hepatocyte-specific and chemicals such as FAA or MAA were not detected in urine developmentally regulated transcription factors, including from FAH-deficient patients (6) or mice (25). In addition, G6Pase, HNF1, HNF4, and C/EBP (21,24,26), are reduced. apoptotic death is confined to proximal renal tubular cells These changes may contribute to the impaired develop- where Fah is likely to be expressed. We speculate that intra- ment of metabolic functions of the liver. Observations of cellular metabolite(s) such as FAA may explain the cell death the expression of phosphoenol pyruvate carboxykinase in proximal tubules. (21,23,26) and menadione oxidoreductase (26) in renal tu- Kidney pathology associated with HT1 includes glomerulo- bules from albino lethal mice suggested that similar changes sclerosis and interstitial fibrosis, and there frequently is a loss in the expression of transcription factors occur in renal of the brush border and cytoplasmic vacuolation in ultrastruc- tubular cells when FAH is deficient. In this context, it is tures of the tubular cells (2,8). Such pathology in the renal tubular interesting that targeted disruption of HNF-1 in mice re- cells is likely to be related to occurrence of cell death signal in the sulted in Fanconi syndrome and was characterized by cells. Because apoptotic cells are rapidly removed from the tu- marked glucosuria. Accordingly, it is possible that Fanconi bules, it may be difficult to detect such cells in kidney sections syndrome in the FahϪ/ϪHpdϪ/Ϫ mice is partly due to ab- from patients. Long-term investigations of FahϪ/ϪHpdϪ/Ϫ mice normal expression of transcription factors. given low doses of homogentisate may elucidate the precise There are observations that apoptosis of kidney cells under- process of renal damage seen in the HT1 patients. lies the pathogenesis of kidney diseases, conditions that in- Fanconi syndrome in HT1 patients (2,6) was reproduced in clude polycystic kidney disease (34), glomerulonephritis treated FahϪ/ϪHpdϪ/Ϫ mice (Figure 3), as reabsorption of (35,36), exposure to toxic agents (37–39), and some cases of glucose and phosphate was impaired. Interestingly, treatment kidney transplantation (40). However, clear relationships be- of the FahϪ/ϪHpdϪ/Ϫ mice with the caspase inhibitor YVAD tween Fanconi syndrome and apoptosis have not been demon- did not prevent the development of Fanconi syndrome (Figure strated (41). We found that renal tubular damage in HT1 7), which means that the cell death pathway and the metabolic involves cell death due to apoptosis. As discussed above, process to Fanconi syndrome can be separated in the nephrop- apoptosis associated with Fah deficiency is caused by interac- athy associated with FAH deficiency. If the pathway for cell tion between FAA and mitochondria. Changes in morphology death and the biochemical process leading to Fanconi syn- of proximal cells after injection of homogentisate are charac- drome are completely separable, one possible explanation is terized by abnormalities in mitochondria, disruption of the that Fanconi syndrome is caused by chemicals derived from nuclear membrane, and fragmentation of nuclei. These changes filtrates of glomerulus in this mouse model, and in HT1 pa- are obviously relevant to apoptosis and are completely pre- tients. Roth and colleagues extensively investigated the effects vented by administration of a caspase inhibitor before the of SA on renal tubules in rats (29–32). SA inhibited uptake of administration of homogentisate. We suggest that the apoptosis methyl ␣-D-glucose, but electron microscopic analysis revealed of renal tubular epithelial cells observed in our study is likely no specific changes in morphology, even with high concentra- due to the accumulation of FAA in epithelial cells. tions of SA (29,30). Thus, it is likely that SA can act as a reversible inhibitor for cellular metabolism, but does not cause Acknowledgments cellular changes in morphology. It seems likely that Fanconi This work was supported by a Grant-in-Aid for Scientific Research Ϫ/Ϫ Ϫ/Ϫ syndrome seen in the homogentisate-treated Fah Hpd from the Ministry of Education, Science, Sports, and Culture of Japan, mice is, at least partly, relevant to SA. the Ministry of Health and Welfare (Japan), and a Grant-in-Aid from J Am Soc Nephrol 11: 291–300, 2000 Tyrosinemia Type 1 and Fah/Hpd Mice 299

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