EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Abstract
Mitochondrial disease is a heterogeneous group of disorders with variable clinical and laboratory manifestations. The most common mitochondrial DNA defect is the transition of adenine to guanine at position 3243 (m.3243A≥G) on the MT-TL1 gene, causing a systemic syndrome known as MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). The kidney is particularly susceptible to mitochondrial diseases due to its high oxygen consumption and abundance of mitochondria. Tubular cells and podocytes can be affected by these diseases, resulting in diverse clinical and laboratory manifestations. We reported a case of a 31-year-old female patient with bilateral sensorineural deafness diagnosed with the m.3243A≥G sequence variant in adulthood. At the time of diagnosis, she had end-stage renal disease secondary to focal segmental glomerulosclerosis. Her sister was diagnosed with MELAS syndrome, and mitochondrial disease was investigated. After 27 months on dialysis, our patient received a kidney transplant from a deceased donor and presented nonnephrotic range proteinuria within the first month after transplant. Despite developing de novo donor-specific antibodies after COVID-19, the function of the transplanted kidney remained stable. With adjustment to the maintenance immunosuppression therapy, there was a gradual decrease in the mean fluorescence intensity of de novo donor-specific antibodies. The graft function and proteinuria remained stable throughout a 5-year follow-up, which is similar to a follow-up reported in the literature. The kidney is especially vulnerable to mitochondrial diseases. In this report, posttransplant outcomes were satisfactory in a 5-year follow-up, similar to those reported by other authors.

Key words : Chronic kidney disease, Focal segmental glomerulosclerosis, Kidney transplantation, Mitochondrial diseases

Introduction
The mitochondrial respiratory chain is an enzymatic complex encoded by mitochondrial DNA (mtDNA) and nuclear DNA.¹ Mitochondrial diseases are a diverse group of disorders resulting from defects in mtDNA and nuclear DNA.¹ The first reports of mtDNA sequence variants occurred in the 1970s and were shown to involve the central nervous system, skeletal muscles, cardiac conduction system, hematopoietic system, pancreas, liver, endocrine system, and kidney.^2,3 The heterogeneity of these diseases results from heteroplasmy, ie, the coexistence of both mutated and healthy mtDNA in the cell, where the proportion of its copies defines the clinical phenotype and severity of the disease.^1,2 The transition of adenine to guanine at position 3243 of mtDNA (m.3243A≥G) in the MT-TL1 gene is considered the most common mtDNA defect and can clinically manifest as a systemic syndrome known as MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes).⁴

The current clinical criteria for MELAS diagnosis include at least 2 category A criteria and at least 2 category B criteria. Category A comprises neurological clinical symptoms and neuroimaging findings, such as headache with vomiting, seizures, hemiplegia, cortical blindness, and acute focal lesions involving the brain cortex.⁴ Category B is based on laboratory results showing increased plasma or cerebral spinal fluid lactate, mitochondrial abnormalities on muscle biopsy, and MELAS-related pathogenic sequence variation on genetic testing.⁴ In addition to the changes described by the acronym, this sequence variant can also be associated with other clinical manifestations, such as maternally inherited diabetes, deafness, hypertrophic cardiomyopathy, macular dystrophy, chronic progressive external ophthalmoplegia, gastrointestinal involvement, and increased risk of obstetric complications.⁵

The m.3243A≥G sequence variant can affect the kidneys, as seen in approximately 50% of children and 30% of adults who carry this sequence variant.⁵ The severity of the disease ranges from mild proteinuria to end-stage renal disease (ESRD), and the reasons for the different clinical courses remain unclear.⁵ These abnormalities in mtDNA trigger apoptotic signaling pathways in tubular epithelial cells, podocytes, or vascular smooth muscle cells, leading to structural and functional changes.⁶ Tubular cells present very high energy demand and are rich in mitochondria, and patients with mitochondrial disease usually present tubular dysfunction ranging from isolated wasting electrolytes to a complete Fanconi syndrome or tubulointerstitial nephritis.^1,7 Less frequently, renal tubular acidosis, Bartter-like syndrome, and hypercalciuria may develop. The podocytes are postmitotic cells rich in mitochondria and highly dependent on oxidative energy and can also be affected in cases of mitochondrial diseases.¹ Kidney biopsy of affected patients includes proximal tubular damage, abnormalities in the number, size, and configuration of mitochondria in proximal tubules, and focal segmental glomerulosclerosis (FSGS) with severe hyaline arteriolar deposits.^5,6

Here, we report a case of a patient diagnosed with the m.3243A≥G variant in adulthood, who experienced progressive kidney dysfunction that required hemodialysis and was undergoing a kidney transplant. We also reviewed the literature regarding renal involvement by the m.3243A≥G variant and the outcomes of kidney transplant in this population.

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The study was approved by the University of Campinas Ethics Committee (CAAE No. 82891224.6.0000.5404). Written informed consent was obtained from the patients or their families to publish this case report.

Diagnosis and clinical course of the MELAS syndrome
A 31-year-old female patient with bilateral sensorineural deafness and a history of 2 miscarriages (1 in the second trimester and another in the first trimester) was referred to the nephrologist due to nephrotic-range proteinuria of 10.3 g/24 h, which was detected after her second pregnancy. Upon admission, she had been receiving treatment with an angiotensin-converting-enzyme inhibitor (ACEi) for 6 months. She reported no family history of kidney diseases. Laboratory investigation revealed a serum creatinine level of 76.0 μmol/L and serum albumin level of 4.5 g/L, and urinalysis showed proteinuria without hematuria or leukocyturia. The 24-hour urine protein test showed 3.1 g, with a urine output of 3100 mL. The results of serology tests for hepatitis B and C, syphilis, schistosomiasis, and human immunodeficiency virus were negative. Screening for antinuclear antibodies was negative, and serum complements C3 and C4 fractions were within reference ranges. Additionally, electrophoresis of serum and urinary proteins did not show any monoclonal peaks. The ophthalmological evaluation revealed no abnormalities.

A renal biopsy was performed to investigate the cause of proteinuria, and results showed FSGS in light microscopy, with the absence of immunoglobulins (IgG, IgA, and IgM), complement fractions (C3 and C1q), or light chains (kappa and lambda) in the immunofluorescence assay, as well as focal loss of pedicles by electron microscopy technique. After 2 years of follow-up, the ACEi was temporarily stopped due to pregnancy; however, the patient had a miscarriage at 20 weeks. During the 5-year follow-up, the patient had serum albumin within reference range and nonnephrotic proteinuria while taking ACEi. However, after this period, she developed nephrotic syndrome, with edema, serum albumin of 2.8 g/L, and a urinary protein-to-creatinine ratio (UPCR) of 7.4. Treatment with 1 mg/kg daily prednisone did not show any clinical or laboratory improvement. Her renal function progressively declined, leading to the need for renal replacement therapy 6 years after FSGS diagnosis. Just before starting dialysis, her 39-year-old sister, who has a bilateral sensorineural deafness, had a stroke. During the investigation, the sister was submitted to genetic testing, which found a sequence variant of the MT-TL1 gene, m.3243A≥G, with a heteroplasmy of 26%. This led to a molecular genetic test for our patient, which revealed the presence of the pathogenic sequence variant m.3243A≥G in the MT-TL1 gene, with no heteroplasmy described. Both the patient and her sister were diagnosed with MELAS syndrome after these results.

Kidney transplant
The patient remained on hemodialysis for 27 months. No antibodies against human leukocyte antigens (HLA) were detected during the pretransplant evaluation. She received a kidney from a 48-year-old male deceased donor with brain death due to head trauma, who had good kidney function at donation (serum creatinine of 57.5 μmol/L) and absence of histological abnormalities in the preimplantation kidney graft biopsy. The HLA compatibility showed 1 match in each A, B, and DR alleles. There were no surgical complications, and the cold ischemia time was 16.4 hours. The induction immunosuppression therapy included antithymocyte globulin and methylprednisolone, and the immunosuppression maintenance regimen contained cyclosporine, sodium mycophenolate, and steroids. The patient presented immediate graft function, and hospital discharge occurred on posttransplant day 6, with a serum creatinine of 221.9 μmol/L. On posttransplant day 15, the patient was asymptomatic with a serum creatinine of 119.3 μmol/L and UPCR 0.9.

In posttransplant month 2, she was diagnosed with posttransplant diabetes mellitus and started insulin treatment. The patient maintained a detected UPCR lower than 1.0 throughout the follow-up and started spironolactone therapy in posttransplant month 4. In posttransplant month 11, the patient tested positive for COVID-19 but did not show severe symptoms. At this time, de novo class II donor-specific antibodies (DSA) anti-DQ5 were detected, showing a mean fluorescence intensity (MFI) value of 10?742, with 26% class I and 85% class II panel reactive antibody. Due to the stable renal function and UPCR, the de novo DSA profile (isolated DQ), and recent infection, it was decided not to perform a renal biopsy or specific treatment for probable antibody-mediated rejection. Immunosuppression was adjusted by switching from cyclosporine to tacrolimus while maintaining mycophenolate and steroids. As the patient developed polycythemia, the antiproteinuric treatment was changed from spironolactone to enalapril to treat both conditions.

Five years after transplant, the patient experienced tremors in the extremities when the calcineurin inhibitor treatment was changed again, with the suspension of tacrolimus and reintroduction of cyclosporine. The graft function remained stable, with a serum creatinine level of 75.1 μmol/L and UPCR of 1.0. Additionally, the DSA anti-DQ5 intensity decreased to 1625 MFI, and there was also a decrease in class I and II panel reactive antibody (18% and 67%, respectively).

Discussion
We presented a case of a mitochondrial disease caused by the most common mtDNA sequence variant diagnosed in adulthood. The patient had been initially diagnosed with ESRD due to FSGS, and the mitochondrial disease was suspected only after her sister was diagnosed with MELAS syndrome. Mitochondrial disease is estimated to have a prevalence of approximately 1:85?000. Kidney problems are not common in these diseases, and many patients with these conditions may not be adequately diagnosed.¹ However, mitochondrial diseases should be suspected in patients with FSGS and a familial history of diabetes, neuromuscular disorders, or deafness.⁷ In this case, the pathogenic variant of mtDNA was identified only after the patient started renal replacement therapy.

Kidney impairment due to the m.3243A≥G variant is more frequent in female patients versus male patients, and most such cases are diagnosed during the second or third decade of life with nonnephrotic range proteinuria and renal dysfunction.⁸ Clinical manifestations of FSGS can be triggered or worsened with successive pregnancies.⁷ As reported in our case, after her third miscarriage, the patient experienced an abrupt increase in proteinuria, leading to steroid-resistant nephrotic syndrome and progressive renal dysfunction.

The kidney requires a high amount of oxygen to carry out functions such as reabsorption and excretion in the renal tubules and to maintain all podocyte functions.⁸ The cells involved in these processes are abundant in mitochondria, which makes them vulnerable to disease in the case of pathological variants of mtDNA or acquired mitochondrial dysfunction, as seen in diabetic nephropathy.⁸ The exact mechanism that leads to FSGS in the MELAS syndrome is unclear. The increased production of reactive oxygen species in patients with mtDNA sequence variant may affect the structure and function of glomerular cells and induce podocyte injury by alteration of apoptotic signaling pathways and cytoskeletons.⁹ Renal complications are uncommon in patients with MELAS syndrome.⁸ Nevertheless, patients with the mtDNA m.3243A≥G sequence variant can develop proteinuria and ESRD, often alongside other symptoms such as diabetes and sensorineural deafness,⁹ similar to the symptoms observed in this case. This clinical presentation may pose a challenge in diagnosis, and other conditions, such as Alport syndrome, should be considered in the differential diagnosis.

Histological evaluation of renal tissue can help to identify potential signs of mitochondrial disease. A detailed examination of the renal tubules is not often conducted, with more focus on the glomeruli in ultrastructural analysis of renal biopsies; however, a more detailed study of the renal tubules may reveal many mitochondrial disorders in patients with unexplained chronic tubulointerstitial nephropathy and declining renal function.

In the first report of a renal biopsy in a case involving mtDNA deletion, variations in the size of mitochondria were observed, alongside abnormal branching and disorientation of mitochondrial cristae, as well as electron-dense granular and fibrillary inclusions in the proximal tubule portion.³ In a subsequent study, podocyte lesions were observed in 4 female patients diagnosed with the mtDNA m.3243A≥G variant. Electron microscopy evaluation revealed binucleated or multinucleated podocytes with cell body attenuation, pseudocyst formation, and foot process effacement. There was a marked increase in the number of mitochondria with variation in size and shape, irregular outline, increased cristae, and lamellar structures within the podocyte cytoplasm.¹⁰

Other studies have reported 4 cases with the mtDNA m.3243A≥G variant, with renal histological findings of typical FSGS with severe arteriolar thickening, possibly related to abnormal mitochondria in arteriolar myocytes.^9-11 However, electron microscopy showed an increased number of abnormal mitochondria in the podocytes of some patients, with no description of the equivalent finding in the arteriolar myocytes.⁹ Some authors have also described significant vascular changes, such as unusual hyaline lesions, indicating myocyte necrosis in afferent arterioles and small arteries.^7,12 These arteriolar lesions produce glomerular hypertension and hyperperfusion, causing secondary epithelial cell damage and, ultimately, FSGS.⁷

Apart from glomerular, tubular, and vascular involvement, a previous report of a male patient with kidney neoplasia (oncocytoma and chromophobe carcinoma) and the mtDNA m.3243A≥G sequence variant has suggested a potential link between cancer and MELAS syndrome. Accumulation of mitochondria in renal oncocytomas could be associated with mtDNA sequence variants, and heteroplasmic mtDNA sequence variants are found in chromophobe renal cell carcinomas.¹² Therefore, kidney neoplasia surveillance should be performed in patients affected by MELAS syndrome.

After evaluation of the ultrastructural images of our patient’s renal biopsy, we did not find evidence of the mitochondrial changes that have been described in other studies. The quality of the images was insufficient for this purpose, because the tissue samples were obtained from deparaffinized tissue. When the diagnosis of MELAS syndrome was established, our patient already had advanced kidney disease, with a serum creatinine of 229.7 μmol/L and an estimated glomerular filtration rate of 18 mL/min. Hence, a new biopsy to investigate mitochondrial abnormalities was not performed, because of (1) the procedural risks in a chronic kidney, (2) the chance of extensive chronic lesions that would hinder an adequate tissue analysis, and (3) the reduced benefit on the therapeutical management.

It is essential to emphasize the role of genetic knowledge to diagnose and monitor FSGS caused by sequence variants. In our case, genetic testing was not initially performed, so the patient was primarily misdiagnosed with idiopathic FSGS and treated with corticosteroids. Genetic FSGS can be linked to susceptibility variants, such as the APOL1 gene, or high-penetrance variants with Mendelian inheritance for nuclear genes or maternal inheritance for genes encoded by mtDNA.¹³ Extrarenal manifestations can provide a clinical clue that a patient could have a sequence variant in a particular gene and facilitate an accurate diagnosis. However, the routine genetic testing for patients with FSGS remains uncertain, because such testing is known to be helpful to identify a cause for FSGS in only a minority of cases, such as infants, young children, and patients with syndromic disease or a family history.¹⁴ The most appropriate and cost-effective method for genetic testing is the use of panels that focus on early-onset FSGS or adult-onset FSGS. Nonetheless, a scan of the whole exome may replace selected panels in the future. Treatment for genetic FSGS is generally conservative and based on renin-angiotensin-aldosterone system antagonism. There is no evidence to support corticosteroids in genetic FSGS, and calcineurin inhibitors may only benefit a minority of patients.¹⁴

In a previous study by de Laat and colleagues, 5 cases of kidney transplant recipients with the m.3243A≥G variant were reported.⁵ Over a 5-year posttransplant follow-up, the recipients had stable graft function and no proteinuria. Seidowsky and colleagues and Guéry and colleagues also reported cases of successful renal transplant in patients with the same mitochondrial sequence variant.^9,11 In our present case, the patient also remained with stable graft function during the follow-up. However, we observed the onset of nonnephrotic range proteinuria within the first month after transplant, which remained stable. A routine anti-HLA antibody screening was performed after the patient presented with COVID-19, which revealed the detection of class II DSA. A graft biopsy was not conducted, so it was not possible to confirm whether an acute antibody-mediated rejection had occurred versus a persistent low-grade proteinuria, indicating a chronic antibody-mediated rejection. Despite the observation of de novo DSA, therapy involved an increase in the maintenance of immunosuppression therapy without specific treatment with intravenous immunoglobulin or plasma exchange. This decision was based on previous studies performed in our center that have shown a poor response of class HLA-DQ DSA MFI to this treatment.¹⁵ During follow-up, we observed a gradual reduction in the DSA MFI and maintenance of graft function.

Conclusions
The kidney is especially vulnerable to mitochondrial diseases because it consumes a large amount of oxygen and is rich in mitochondria. The clinical and laboratory characteristics of the pathogenic m.3243A≥G sequence variant are diverse and remain a challenge in the diagnosis. In this report, post-transplant outcomes were satisfactory in a 5-year follow-up, similar to the outcomes reported by other authors.

Volume: 23 Issue: 2 February 2025

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