This case occurred in a Japanese woman, who had been born at 39 weeks of gestation with a birthweight of 3210 g. She had no remarkable medical history until 29 years of age, at which point, an annual health checkup revealed proteinuria (1+) for the first time by a urine dipstick and a high serum creatinine (sCr) (1.01 mg/dL) level. Her estimated glomerular filtration rate (eGFR) using the Japanese Eq. (22) was calculated to be 54.0 ml/min/1.73 m2. At the same checkup, she was also found to have a slight hearing disturbance. In addition, she had a headache once a week since 30 years of age, and she had been diagnosed by a neurologist with migraine, which could not be controlled by triptan. Her health checkup at 32 years of age again showed proteinuria (1+) and an increased sCr (1.06 mg/dL). At 33 years of age, she was referred to the Yamanashi Prefectural Central Hospital by her primary doctor in order to examine the reason for her proteinuria and decreased eGFR. At the first visit, proteinuria (1.09 g/gCr), elevated sCr (1.28 mg/dL), and elevated uric acid (7.5 mg/dL) were detected. Therefore, febuxostat and sodium bicarbonate were prescribed for the treatment of hyperuricemia. Because proteinuria and decreased renal function continued to be detected, she was admitted for a kidney biopsy to investigate the reason for proteinuria and renal dysfunction 6 months after the first visit.
On admission, her blood pressure was 114/70 mmHg. Physical examination showed a height of 152 cm, body weight of 43.8 kg, and body mass index of 19.0. No crackles or murmurs were detected on chest auscultation. There were no abnormal neurological findings. Nether skin lesions and pitting edema were detected.
The laboratory data on admission are summarized in Table 1. The remarkable data were a decreased eGFR and proteinuria. The long × short axis of the kidneys measured 101 × 38 mm on the left and 104 × 50 mm on the right, indicating no renal atrophy in this patient. An electrocardiogram was normal, and echocardiography showed normal cardiac function. The standard pure tone hearing test showed a right-ear value of 38.8 dB and a left-ear value of 40.0 dB. She was thus diagnosed with sensorineural hearing loss by an otolaryngologist.
Table 1
Laboratory data on admission
Blood cell count
|
Blood chemistry
|
Immunology
|
Urinalysis
|
WBC
|
5400
|
/µL
|
TP
|
6.8
|
g/dL
|
CH50
|
54
|
U/mL
|
Gravity
|
1.01
|
|
RBC
|
373
|
× 104/µL
|
Alb
|
3.9
|
g/dL
|
C3
|
76.1
|
mg/dL
|
pH
|
7.0
|
|
Hb
|
11.5
|
g/dL
|
AST
|
21
|
IU/L
|
C4
|
19.3
|
mg/dL
|
RBC
|
1–4
|
/HPF
|
Ht
|
35.1
|
%
|
ALT
|
11
|
IU/L
|
IgG
|
1340.3
|
mg/dL
|
WBC
|
< 1
|
/HPF
|
MCV
|
94.1
|
fl
|
LDH
|
186
|
IU/L
|
IgA
|
225.7
|
mg/dL
|
Protein
|
2.04
|
g/gCr
|
MCHC
|
32.8
|
%
|
ALP
|
149
|
IU/L
|
IgM
|
180.1
|
mg/dL
|
NAG
|
8.6
|
U/L
|
Plt
|
25.7
|
× 10⁴/µL
|
Tbil
|
0.56
|
mg/dL
|
ANA
|
(-)
|
|
|
|
|
|
|
|
BUN
|
21.8
|
mg/dL
|
M protein
|
(-)
|
|
|
|
|
Coagulation
|
Cr
|
1.26
|
mg/dL
|
HBs Ag
|
(-)
|
|
|
|
|
APTT
|
34.0
|
sec
|
eGFR
|
40.8
|
mL/min/1.73m²
|
HCV Ab
|
(-)
|
|
|
|
|
PT
|
99.0
|
%
|
UA
|
5.4
|
mg/dL
|
|
|
|
|
|
|
INR
|
0.97
|
INR
|
Tcho
|
255
|
mg/dL
|
|
|
|
|
|
|
Fib
|
373.0
|
mg/dL
|
CRP
|
0.01
|
mg/dL
|
|
|
|
|
|
|
|
|
|
FBS
|
85
|
mg/dL
|
|
|
|
|
|
|
|
|
|
HbA1c
|
5.3
|
%
|
|
|
|
|
|
|
Aberrant values are underlined. |
WBC, white blood cells; RBC, red blood cells; Hb, hemoglobin; Hct, hematocrit; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration; Plt, platelet; APTT, activated partial thromboplastin time; PT, prothrombin time; INR, international normalized ratio; Fib, fibrinogen, TP, total protein; Alb, albumin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; LDH, lactate dehydrogenase; ALP, alkaline phosphatase; Tbil, total bilirubin; BUN, blood urea nitrogen; Cr, creatinine; eGFR, estimated glomerular filtration rate (by the Japanese equation); UA, uric acid; Tcho, total cholesterol; CRP, C-reactive protein; FBS, fasting blood sugar; HbA1c, hemoglobin A1c (NGSP); CH50, 50% hemolytic complement activity; C3, complement 3; IgG, immunoglobulin G; ANA, antinuclear antibody; M protein, monoclonal protein; HBs Ag, hepatitis B surface antigen; HCV Ab, hepatitis C virus antibody; NAG, N-acetyl glucosaminidase; HPF, high power field. |
Her parents had passed away owing to cancer (mother, breast cancer; father, colon cancer) without kidney disease or any hearing difficulty. Neither of her two older brothers had any abnormalities in their kidney function, urine, or hearing. She had a seven-year-old daughter and a five-year-old son. Her daughter’s birth weight had been 2840 g at 41 weeks, 2 days of gestation, and her son’s birth weight had been 2632 g at 39 weeks of gestation. Her daughter had been born by suction delivery, whereas her son had been born by normal delivery. Neither of her children had any urinary abnormalities or hearing difficulties at their medical checkups. However, the children also sometimes complained of headaches that had not yet been diagnosed by any doctor.
A percutaneous kidney biopsy under ultrasound guidance was performed. Light microscopy photographs of the renal biopsy specimens are shown in Fig. 1. Nine glomeruli were observed in the specimens. One glomerulus was globally sclerosed. Furthermore, segmental scleroses were observed at the perihilar area in two glomeruli, which was compatible with the perihilar variant in the Columbia classification of FSGS (23, 24). Interestingly, red-colored podocytes (ReCPos), whose cytoplasm was dyed red, were observed on AZAN staining. Interstitial fibrosis and tubular atrophy were observed in almost 30% of the area of the interstitium. Characteristically, granular swollen epithelial cells (GSECs), which have been reported as a specific finding of mitochondrial diseases (25, 26), were markedly observed in the distal tubular cells by AZAN staining (Fig. 1C). Such GSECs were emphasized by staining with anti-cytochrome c oxidase subunit 4 (COX IV) antibody (Mouse Anti-COX IV antibody [20E8C12]) (ab14744; Abcam PLC., Cambridge, UK) (Fig. 1D). COX IV staining is recognized as a loading control for mitochondria (26, 27). Furthermore, the sizes of the vascular smooth muscle cells in arterioles were irregular, and their arrangement was disorganized in a manner similar to that seen in older patients, despite the present patient being only 33 years old without any medical history that might cause arteriosclerosis (Fig. 1E). Such age-inappropriately disarranged and irregularly sized vascular smooth muscle cells (AiDIVs) were also noticeable in the interlobular arteries (Fig. 1F).
An immunofluorescent analysis revealed IgM and C1q deposits in the mesangial area (Fig. 1). In addition, an electron microscopic analysis (Fig. 2A-C) showed podocytes with increased mitochondria that had lost their normal cristae structure. We also detected cells with an increased content of such abnormal mitochondria among the parietal epithelial cells of the Bowman’s capsule (Fig. 2D). These renal pathology findings strongly suggested FSGS due to mitochondrial disease.
When the serum lactate and pyruvate concentrations were slightly elevated at 21.0 mg/dL (normal: 3.0–17.0) and 1.34 mg/dL (normal: 0.30–0.94), respectively. In addition, the sensorineural hearing difficulty and headache were also suggestive of mitochondrial disease. Therefore, we tried to perform a closer examination for mitochondrial disease.
After obtaining informed consent from the patient according to the protocol with permission from our ethics committee, genomic DNA extracted from her peripheral mononuclear blood cells and urine sediment cells was analyzed by targeted resequencing coupled with next-generation sequencing (NGS), followed by Sanger sequencing. Genomic DNA from the patient was subjected to the fragmentation and library preparation using the Lotus DNA Library Prep Kit (#10001074; Integrated Device Technology, Inc., San Jose, CA, USA) according to the manufacturer's instructions. Target enrichment was performed with xGen Human mtDNA Research Panel (#1075705; Integrated Device Technology) targeting the whole mtDNA and custom xGen® Predesigned Gene Capture Pools (Integrated Device Technology)/xGen Lockdown Probe pool (Integrated Device Technology) targeting the exons of 367 nuclear-encoded genes that cause mitochondrial diseases. The library was then sequenced on the Illumina MiSeq platform with MiSeq Reagent Kit v2 (MS-102-2002; Illumina, Inc., San Diego, CA, USA). As a result, a pathogenic mtDNA variant of m.13513 G > A was detected in both the patient’s blood cells and urine sediment cells (Fig. 3). The Minor Variant Finder (MVF) software program (Thermo Fisher Scientific, Waltham, MA, USA) revealed that the heteroplasmy rates of the blood cells and urine sediments were 10.3% (forward: 11.6%/reverse: 9.0%) and 62.2% (forward: 63.7%/reverse: 60.7%), respectively. Therefore, we diagnosed her renal dysfunction and proteinuria as being due to mitochondrial nephropathy caused by an MT-ND5 mutation.