Changes in histopathology and heteroplasmy rates over 8 years and effectiveness of taurine supplementation in a patient with mitochondrial nephropathy caused by MT-TL1 mutation: A case report

Abstract

The m.3243A > G mutation in the mitochondrially encoded tRNA leucine 1 (MT-TL1) gene is known to cause mitochondrial nephropathy. However, its long-term effects of the m.3243A > G mutation on renal histopathology or heteroplasmy rates remain unknown. Here we present the case of a female patient who underwent renal biopsy at 34 years of age to investigate the reason for a low estimated glomerular filtration rate (eGFR) of 47.9 mL/min/1.73 m². Light microscopy revealed nephrosclerosis with granular swollen epithelial cells (GSECs) in the renal tubules. Genetic testing revealed the m.3243A > G mutation in the MT-TL1 gene. Over a follow-up period of 8 years, the eGFR declined at a rate of 1.50 mL/min/1.73 m²/year. A second renal biopsy was performed at the age of 42 years; the patient's glomerular sclerosis rate had increased from 45.5% to 63.2%, and the frequency of GSECs in the collecting ducts had increased from 5.8% to 20.8%. Furthermore, the heteroplasmy rate in blood cells and urinary sediment cells increased from 9% to 20% and 20% to 53%, respectively. Taurine therapy was initiated just after the second kidney biopsy. To date, after approximately 3 years of taurine administration, the rate of eGFR decline has markedly decreased to 0.26 mL/min/1.73 m²/year. This experience suggests that an increased heteroplasmy rate may be associated with the progression of mitochondrial nephropathy caused by MT-TL1 mutation. Furthermore, our case is the first to suggest the effectiveness of taurine for mitochondrial nephropathy caused by the m.3243A > G mutation in the MT-TL1 gene.

1. Introduction

Several reports have shown that focal segmental glomerulosclerosis (FSGS) is a characteristic pathological change in mitochondrial nephropathies caused by mitochondrial DNA (mtDNA) mutations, such as those in the mitochondrial tRNA leucine 1 (MT-TL1) gene [[1], [2], [3]]. In such cases, the mitochondria in glomerular podocytes lose their normal function, and the resulting podocyte damage has been thought to be involved in the etiopathogenesis of FSGS lesions [4]. However, our prior analysis of 63 renal biopsy cases with the m.3243A > G mutation in MT-TL1 showed that 11.4% of these cases were diagnosed via histopathology as nephrosclerosis without FSGS lesions [3]. Although the lack of FSGS lesions in renal biopsy sections may be due to sampling errors, whether this error alone can explain the lack of FSGS lesions is debatable. The clinical manifestations and pathological changes of mitochondrial disease caused by mtDNA mutations are highly variable and are impacted by the heteroplasmy rate that differs among organs and cells [5].

The long-term effects of the m.3243A > G mutation on renal histopathology or heteroplasmy rates are unknown. In addition, although taurine is reportedly effective at treating mitochondrial diseases with MT-TL1 mutations that were already proven to cause a taurine modification defect in tRNALeu (UUR), as in m.3243A > G [6], its effect on mitochondrial nephropathy is unclear. In this context, we present a patient with an m.3243A > G mutation who underwent two renal biopsies performed 8 years apart, necessitated by a gradual decrease in estimated glomerular filtration rate (eGFR). We investigated the factors contributing to the progression of nephropathy in this patient by analyzing the changes in histopathology and heteroplasmy rates of mtDNA. Furthermore, this study is the first report verifying the effectiveness of taurine in such patients.

2. Case presentation

Herein, we describe the case of a 45-year-old Japanese woman with no significant medical history prior to the second pregnancy at 32 years of age. The patient was initially found to have proteinuria and a decreased renal function in week 27 of the second pregnancy and gave birth to the second child at 28 weeks of gestation due to an imminent risk of premature birth. One day after delivery, proteinuria was 1.2 g/day and eGFR was 48.3 mL/min/1.73 m², as calculated using the serum creatinine value and the Japanese equation (eGFR [mL/min/1.73 m²] = 194 × Age^−0.287 x Cre^−1.094 [x 0.739 if female]) [7]. Although the proteinuria disappeared 3 months after delivery, the eGFR was persistently <50 mL/min/1.73 m². At 34 years of age, 15 months after the second delivery, the patient was referred to our hospital by her primary doctor for decreased renal function. At the first visit to our hospital, the serum creatinine level was 1.12 mg/dL (eGFR: 46.0 mL/min/1.73 m²), without proteinuria. Three months after the first visit to us, the patient was admitted for a kidney biopsy to investigate the reason for persistent renal dysfunction.

Physical examination at admission showed the following: height 160.0 cm, body weight 45.5 kg, body mass index 17.8, blood pressure 116/73 mmHg, and a regular heart rate of 67 bpm. Neurological examination results were normal. The patient had no headaches, edema, or skin lesions. The serum creatinine level was 1.08 mg/dL (eGFR: 47.9 mL/min/1.73 m²) (Table 1) without proteinuria (0.03 g/day) or hematuria (0–1/HPF). Except for hyperuricemia (uric acid: 6.5 mg/dL), all blood test results were normal (Table 1), and the serum lactate level was within the normal range (9.1 mg/dL). Blood tests did not suggest the existence of diabetes mellitus, the patient was not a smoker, and was not taking any prescribed medications. Audiometric tests revealed pure tone averages of 25.0 dB in the right and 20.0 dB in the left ears ([500 Hz + 1000 Hz x 2 + 2000 Hz]/4), indicating a normal hearing ability. An echocardiogram revealed a normal heart function. Brain magnetic resonance imaging (MRI) was not performed as neurological symptoms and stroke-like episodes were absent.

Table 1.

Laboratory tests on admission.

	1st biopsy	2nd biopsy
Age	34	42
Blood cell count
WBC (/μL)	3900	5400
RBC (/μL)	415 × 104	458 × 104
Platelet (/μl)	1,67,000	1,87,000
Blood chemistry
Albumin (g/dL)	4.9	4.7
Sodium (mEq/L)	139	140
Potassium (mEq/L)	4.1	4.2
Chloride (mEq/L)	101	108
BUN (mg/dL)	15.5	25.1
Cre (mg/dL)	1.08	1.48
eGFR (ml/min/1.73m2)	47.9	31.9
Uric acid (mg/dL)	6.5	7.8
Fasted blood glucose (mg/dL)	88	98
HbA1c (%)	5.7	5.8
Serum lactate (mg/dL)	9.1	12.5
Serum pyruvic acid (mg/dL)	0.46	0.86
Urinarysis
Glucose (test tape)	(−)	(−)
Protein (g/day)	0.03	0.02
RBC (/HPF)	0–1	0–1
NAG (IU/L)	5.2	4.6
b2-MG (mg/L)	37	75

WBC, white blood cells; RBC, red blood cells; BUN, blood urea nitrogen; Cre, creatinine; eGFR, estimated glomerular filtration rate; UA, uric acid; HbA1c, hemoglobin A1c by NGSP; NAG, N-acetylglutamate; b2-MG, microglobulin; HPF, high-power field. Bold texts indicate abnormal values.

The patient reported that her mother had died at 42 years of age due to diabetic nephropathy-induced uremia. Her brother was diagnosed with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) at 7 months of age and died at 37 years of age. The present patient was born after 36 weeks of gestation, with a birth weight of 2720 g. Both of her children are healthy with no symptoms; the first was born prematurely at 29 weeks of gestation, with a birth weight of 1566 g, and the second at 28 weeks of gestation with a birth weight of 1502 g. Neither showed proteinuria or renal dysfunction during health checkups.

The renal pathological findings of the present case are summarized in Table 2. Fig. 1A–C shows the light microscopy images of the first kidney biopsy. Renal pathology showed nephrosclerosis. Five glomeruli among observed eleven glomeruli had global sclerosis (45.5%), and two glomeruli were collapsed (18.2%). All other glomeruli showed almost normal appearance without any sclerotic lesions, tuft adhesions, or endocapillary proliferation. Age-inappropriate disarranged and irregularly sized vascular smooth muscle cells (AiDIVs) were also observed in the intralobular arteries (Fig. 1A) and afferent arterioles (Fig. 1B); arteriolar hyalinosis and intimal thickening were not observed. Furthermore, granular swollen epithelial cells (GSECs) were also apparent in the collecting ducts on Masson Trichrome staining (Fig. 1C). Electron microscopy examination showed an increase in the number of abnormal mitochondria in podocytes and collecting duct cells (Fig. 1D–F).

Table 2.

Comparison of histopathological findings and heteroplasmy rates of mtDNA.

	1st biopsy	2nd biopsy
Renal Pathology
Global sclerosis	5/11 (45.5%)	12/19 (63.2%)
Segmental sclerosis	0/11	0/19
Podocytes with abnormal mitochondria accumulation	+	+
GSECs in collecting ducts	16/274 (5.8%)	38/183 (20.8%)
Rate of m.3243 A > G heteroplasmy
Blood cells	9%	20%
Cells in urinary sediments	20%	53%
Kidney tissue	n.d.	75%

mtDNA, mitochondrial DNA; GSEC, granular swollen epithelial cell.

n.d., not done.

Fig. 1.

Microscopic histopathological examination of the first kidney biopsy specimen. (A) One glomerulus is globally sclerosed (arrow). Age-inappropriate disarranged and irregularly sized vascular smooth muscle cells (AiDIVs) can be observed in an interlobular artery (asterisk) on periodic acid-methenamine-silver and hematoxylin eosin (PAM-HE) staining. (B) Residual glomeruli show a normal appearance. The afferent arteriole of this glomerulus showed AiDIVs on PAM-HE staining. (C) Granular swollen epithelial cells (GSECs) can be observed among the renal tubules and in the collecting duct (arrows) on Masson Trichrome staining. (D) Mitochondria with abnormal cristae structures accumulate in a collecting duct cell (electron microscopy; magnification 1500 × ). (E) Mitochondria are increased in a podocyte (electron microscopy; magnification 1500 × ). (F) High magnification of the area enclosed in the square shown in (E). The mitochondrial cristae structures exhibit ripping and loss, and the mitochondria are enlarged (electron microscopy; magnification 8000 × ).

The presence of GSECs, podocytes with abnormal mitochondria, and a relevant family history prompted us to consider the possibility of mitochondrial nephropathy due to mtDNA mutation. We performed genetic analysis using mtDNA extracted from peripheral blood mononuclear cells and urine sediment cells [8]. The m.3243A > G mtDNA mutation was detected in both blood and urine sediment cells. Heteroplasmy rates of blood and urine sediment cells were 9% and 20%, respectively (Table 2).

After the first kidney biopsy and genetic analysis, the patient was followed up without prescribing any medications as there were no apparent clinical symptoms, and proteinuria was never detected (<0.15 g/g Cre). Urinary N-Acetylglucosamine and β2-microglobulin levels were within the normal range. However, after the first kidney biopsy, the patient became aware that her hearing ability was decreasing. In addition, the eGFR slowly but gradually decreased. Therefore, the patient was later admitted for a second kidney biopsy to investigate the cause of the gradually decreasing renal function.

On the 2nd admission, the patient's body weight was 45.5 kg, and blood pressure was 110/61 mmHg. Physical examination did not reveal any abnormalities; the serum creatinine level had increased to 1.48 mg/dL (eGFR: 31.9 mL/min/1.73 m²) without proteinuria (0.02 g/day). Although the uric acid level was high (6.5 mg/dL), all blood test results were normal and had not changed from the results at the first admission (Table 1). An audiometric test showed pure tone averages of 45.0 dB in the right and 40.0 dB in the left ears ([500 Hz + 1000 Hz x 2 + 2000 Hz]/4), indicating that the patient had experienced hearing loss. The echocardiogram was normal.

The renal pathological findings are summarized in Table 2. Light microscopy of the second biopsy specimen (containing 19 glomeruli) yielded 12 glomeruli with global sclerosis (63.2%) and one collapsing glomerulus; all other glomeruli appeared normal. There were more GSECs in the collecting ducts in this biopsy compared to the first biopsy (53% and 20%, respectively) (Fig. 2A, Table 2). AiDIVs were observed in the intralobular arteries and afferent arterioles, similar to those at the first biopsy (Fig. 2B and C). An electron microscopical analysis revealed accumulation of abnormal mitochondria in podocytes (Fig. 2D). In addition, vascular smooth muscle cells of arterioles were filled with abnormal mitochondria (Fig. 2E and F).

Fig. 2.

Microscopic histopathological examination after the second kidney biopsy. (A) Most of the cells in the collecting duct (arrow) were classified as GSECs on Masson Trichrome staining. (B) One glomerulus is globally sclerosed (arrow). Another residual glomerulus appears normal while the vascular smooth muscle cells of the afferent arteriole of this glomerulus shows AiDIVs on PAM-HE staining. (C) AiDIVs can be observed in an arteriole (arrow) on PAM-HE staining. (D) Enlarged mitochondria can be observed in a podocyte (arrow; electron microscopy; magnification 10,000 × ). (E) A vascular smooth muscle cell (arrow) of an arteriole is filled with enlarged mitochondria (electron microscopy; magnification 2500 × ). (F) High magnification of the area enclosed in square (E). Cristae structures in accumulated mitochondria are disarranged. The crista structure of one mitochondrion exhibits a spiral shape (arrow; electron microscopy; magnification 30,000 × ).

Next, we analyzed heteroplasmy rates of mtDNA with m.3243A > G. The heteroplasmy rates in the peripheral blood mononuclear cells and urinary sediment cells were 20% and 53%, respectively, showing an increase over the 8 years (Table 2). The heteroplasmy rate of kidney tissue was 75%.

Due to the gradually decreasing eGFR and the confirmed renal tissue damage with progressively increasing global glomeruli sclerosis, we decided to administer taurine (12 g/day in three divided doses after each meal). The least squares regression line of the eGFR trend using all the values from the first visit date showed that the patient's eGFR was declining at a rate of 1.50 mL/min/1.73 m²/year prior to taurine treatment (Fig. 3). After a period of 1022 days (about 2.8 years) since taurine was first prescribed, the rate of eGFR decline markedly decreased to 0.26 mL/min/1.73 m²/year, as determined by linear regression using least square method (Fig. 3). The only drug prescribed to the patient during this 2.8-year period was taurine. The uric acid level normalized after taurine administration. However, there has been no improvement in hearing impairment.

Fig. 3.

Estimated glomerular filtration rate (eGFR) before (A) and after (B) taurine treatment. The slope is determined by a linear regression line using least square method. The horizontal axis shows the time in days, and the coefficient was multiplied by 365 to obtain the slope per year. The slope of eGFR prior to taurine administration was calculated using all eGFR measurements taken from the date of the first visit to the date of initiation of taurine administration, including the day of administration. The first renal biopsy was performed on day 99 and the second one was on day 3124. The slope of the post-taurine eGFR uses all measurements from the date of taurine administration to the present.

3. Discussion

FSGS cases with the m.3243A > G mutation often show an accumulation of abnormal mitochondria in podocytes upon electron microscopy, which suggests that mitochondrial dysfunction-induced podocytopathy may underline this disease [1,2,4]. However, our prior analysis showed that 11.4% of mitochondrial nephropathy cases with the m.3243A > G mutation were diagnosed as nephrosclerosis without FSGS lesions [3]. The pathogenesis of why nephrosclerotic lesions without FSGS occur in mitochondrial nephropathy remains unclear. Sampling error may mean that FSGS lesions were not included in the limited renal biopsy specimens. However, we believe that sampling error alone cannot explain our data, as renal biopsy was performed twice in our case. As observed upon electron microscopy, abnormal mitochondria accumulated in the vascular smooth muscle cells (Fig. 2E and F). This finding suggests that mitochondrial dysfunction may occur and cause cellular dysfunction in vascular smooth muscle cells. In fact, despite the absence of hypertension and in spite of the patient's age, the mesenteric vascular smooth muscle cells of the interlobular arteries were unequal in size and arrangement (Fig. 1, Fig. 3C). Thus, these age-inappropriate disarranged and irregularly sized vascular smooth muscle cells (AiDIVs) were assumed to cause abnormal glomerular hemodynamics, resulting in pathological changes similar to hypertension-induced nephrosclerosis.

In our case, two renal biopsies were performed with an intervening period of over 8 years. One weakness of this report was that the specimens from the first renal biopsy were not available for re-examination, and the mtDNA heteroplasmy rate could not be determined in the kidney. However, the rate of heteroplasmy increased markedly in the blood and urine sediment cells during this 8-year period, and the GSECs also increased between the first and second biopsy. This increase suggests that the rise in the heteroplasmy rate and the reduced mitochondrial function in the renal cells contributed to the progression of nephropathy. A previous report showed a relationship between the elevated m.3243A > G heteroplasmy rate in leukocytes and age-related changes [9], which may explain the age-inappropriate pathological changes in the renal lesions observed in our patient based on the increased heteroplasmy rate. Alternatively, other reports have shown that the heteroplasmy rates of the m.3243A > G pathogenic variant of MT-TL1 in leukocytes decrease over time [10,11], which differs from the results in our case, where the heteroplasmy rates in leukocytes and urine sediment cells increased over time. Although the reason for this cannot be clearly demonstrated, it has been speculated that the decline in quality control of mtDNA decreases with age and the heteroplasmy rate of mtDNA increases, and this impaired quality control of mtDNA may have been the cause in this case as well [[12], [13], [14]]. In the future, it will be necessary to investigate the change in heteroplasmy rate of mtDNA over time in mitochondrial nephropathy cases with this pathological variant in order to resolve this difference.

Prior to treatment, the eGFR in the present patient declined at a rate of 1.50 mL/min/1.73 m² per year. As the eGFR was 31.9 mL/min/1.73 m² at the time of the second renal biopsy, it was estimated that the patient would reach end-stage kidney disease (ESKD) (under 10 mL/min/1.73 m²) at approximately 60 years of age. The m.3243A > G mutation reduces the taurine modification of the leucine tRNA anticodon, resulting in the UUG codon not being appropriately recognized, leading to abnormal synthesis of mitochondrial proteins [15,16]. This reduction in tRNA function in cases with the pathogenic variants in MT-TL1 that were already proven to cause a taurine modification defect in tRNALeu (UUR), as in m.3243A > G, can be improved by taurine supplementation, which restores mitochondrial function [6]. So far, the patient has been taking taurine for approximately three years, during which time the rate of eGFR decline improved markedly to 0.26 mL/min per year (Fig. 3). Assuming that this rate of eGFR decline could be maintained, we calculated that the patient would reach ESKD (under 10 mL/min/1.73 m²) at approximately 115 years of age, because the current eGFR is about 28 mL/min/1.73 m². Although this is a single patient case report, it is the first study to show the long-term effects of taurine treatment on mitochondrial nephropathy caused by impaired taurine modification of mitochondrial tRNAs. It should be noted that this is an evaluation based on only one case treated with taurine, and the effect cannot be proven until additional cases are analyzed. Ultimately, randomized clinical trials with taurine supplementation are needed to address this research.

4. Conclusion

In conclusion, we believe that this case report provides three important insights into mitochondrial nephropathy. First, our analysis suggests that the etiopathogenesis of nephrosclerosis in mitochondrial nephropathy may be caused by abnormal mitochondrial function in vascular smooth muscle cells. Second, the heteroplasmy rates of mutated mtDNA increase over time, resulting in progression of nephropathy; these changes may be non-invasively captured by the analysis of urine sediment cells. Third, taurine administration may be effective at ameliorating MT-TL1 mutation-induced mitochondrial nephropathy, although further studies are needed to independently validate our findings.

Declarations

Author contribution statement

Toshiyuki Imasawa: Conceived and designed the experiments; Wrote the paper.

Hiroshi Kitamura, Yukiko Yatsuka, Yasushi Okazaki: Performed the experiments.

Takehiko Kawaguchi, Kei Murayama: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.

Funding statement

MD, PhD Toshiyuki Imasawa; Yasushi Okazaki; Kei Murayama were supported by The Japan Agency for Medical Research and Development, AMED [19ek0109273, 20ek0109468].

Data availability statement

Data will be made available on request.

Declaration of interest's statement

The authors declare no conflict of interest.

Ethics approval and consent to participate

This study was approved by the Ethics Committee of Chiba Children's Hospital [2014-11-05 (on 18th November 2014), 2017-024 (on 23rd August 2017), and 2020-015 (on 9th June 2020)]. The patient provided informed consent for publication of this case report.

Consent for publication

Written informed consent was obtained from the patient for the publication of this case report and all accompanying images.