Crystalline Nephropathy Due to 2,8-Dihydroxyadeninuria in a Transplanted Kidney: 2 Case Reports

Introduction

The renal condition 2,8-dihydroxyadeninuria is an autosomal recessive disorder caused by adenine phosphoribosyl transferase (APRT) deficiency, which most commonly manifests with nephrolithiasis. The enzyme APRT converts adenine and 5-phosphoribosyl-1-pyrophosphate (PRPP) into adenosine monophosphate (AMP) and pyrophosphate. In the absence of APRT, adenine is oxidized by xanthine oxidase into 2,8-dihydroxyadenine (DHA), a water-insoluble substance that can lead to formation of radiolucent DHA kidney stones [1,2]. DHA kidney stones are radiolucent; they are not visible on a standard X-ray because they are composed of DHA, a substance that does not readily absorb X-rays due to its low density. Computed tomography (CT) scan is considered the most reliable method for detecting these stones. APRT deficiency has also led to the development of chronic kidney disease and end-stage kidney disease in the absence of nephrolithiasis [3]. DHA nephropathy has also been shown to recur following kidney transplantation [4].

We present 2 patients with end-stage kidney disease who received kidney transplants and developed DHA crystalline nephropathy due to APRT deficiency early in their post-transplant course. The diagnosis of APRT deficiency had not been recognized prior to transplantation. Allograft biopsies demonstrated the presence of DHA crystal deposition with associated acute tubular injury. DHA crystalline nephropathy was effectively treated with a low-purine diet and xanthine oxidase inhibitors. Belatacept was added to the immunosuppressive regimen to allow reduction in tacrolimus dosages in an effort to limit long-term tubulointerstitial injury.

Case Reports

CASE 1:

A 72-year-old man of European ancestry with end-stage kidney disease presumed secondary to Type 2 diabetes mellitus began peritoneal dialysis in 2019. He developed peritonitis and converted to intermittent hemodialysis via a tunneled-dialysis catheter. There was no history of nephrolithiasis. Native kidney biopsy had not been performed. Eight months before his kidney transplant evaluation, he was found to have atrophic, poorly enhanced kidneys on CT scan in February 2020. He received a living donor kidney transplantation from his daughter in October 2020. Induction therapy included thymoglobulin (dose of 4.5 mg/kg) and steroids, followed by maintenance immunosuppression with tacrolimus, mycophenolate mofetil, and prednisone. The transplant was a single haplotype mismatch with no preformed donor-specific antibodies. He had slow graft function with a creatinine level of 4.0 mg/dL at discharge. Post-transplant, his urinalysis became unremarkable within a month. However, during the first month, he did have mild hematuria and mild pyuria, which are expected postoperative findings. Pre-transplant, his urinalysis showed mild pyuria, moderate blood parameters, and 1+ albumin.

At 6 weeks post-transplant, serum creatinine concentration remained elevated at 2.1–2.3 mg/dL with no apparent etiology for persistent allograft dysfunction. A kidney transplant biopsy was performed, revealing numerous polarizable pigmented brown intratubular crystals with associated acute tubular injury. The crystals were birefringent with fan-shaped needle appearance, brownish pigmentation with Hematoxylin and Eosin (H&E) and Periodic Acid-Schiff (PAS) stains, and intense silver-positive staining. There was no evidence of rejection. The differential diagnosis for this pattern of crystalline deposition includes triamterene-associated crystalline nephropathy and DHA crystalline nephropathy. The patient was not receiving any diuretic therapy. A diagnosis of DHA crystalline nephropathy was made, and he was started on allopurinol and a low-purine diet. To minimize tubular injury, belatacept was added to the maintenance immunosuppression regimen, and tacrolimus dosage was reduced with plans to wean it off completely in 6 months. He was continued on mycophenolate and prednisone.

To further confirm the diagnosis, a kidney APRT deficiency gene panel (Renasight™) was performed, which confirmed homozygous APRT deficiency with an autosomal recessive inheritance pattern. As a result, enzyme activity testing was deemed unnecessary.

The patient’s sister had chronic kidney disease of unclear etiology and was referred for genetic testing after the patient was diagnosed with this genetic disorder. Interestingly, she was found to have the same mutation and moving forward was properly managed. Other members of the family, including his daughter who donated the kidney, were advised to seek genetic counseling. The renal allograft biopsy is shown in Figure 1.

A follow-up allograft biopsy performed 12 months later showed an absence of large intratubular crystals. The majority of remaining small intraepithelial crystals were observed within areas of fibrosis and scar formation. An additional image of the follow-up biopsy is shown in Figure 2. On the last follow-up at 24 months post-transplant, the patient’s creatinine was 1.42 mg/dL, and eGFR was 52 mL/min per 1.73 m2. The long-term immune suppression regimen included monthly belatacept, mycophenolate 180 mg twice daily, and prednisone 5 mg daily.

CASE 2:

A 68-year-old African-American man with end-stage kidney disease, presumed to be secondary to Type 2 diabetes mellitus and hypertension, also had a medical history, including nephrolithiasis with over 10 episodes of kidney stones, coronary artery disease, peripheral vascular disease, and renal cell carcinoma, status post-bilateral nephrectomy. Kidney stone composition analysis had not been performed. His mother also had a history of kidney stones and kidney failure at the age of 93 years.

The patient started hemodialysis in 2014. Pathology results from his previous nephrectomy showed clear-cell renal cell carcinoma with negative margins and no positive lymph nodes. Pathology findings from the non-neoplastic part of the kidney showed extensive chronic pyelonephritis and arteriolonephrosclerosis on H&E staining.

He underwent a transplant of a kidney from a 42-year-old African-American male donor (donation following cardiac death), with an elevated kidney donor profile index of 89% and 5-antigen mismatch. The recipient’s calculated panel reactive antibody was 52%. He had a pre-transplant donor-specific antibody (DSA) against B57 with mean fluorescence intensity (MFI) of 5508. The results of both cell-dependent cytotoxicity and flow-cytometry T- and B-cell crossmatch studies were negative. He received a total of 6 mg/kg of thymoglobulin and pulse steroid induction immunosuppression and maintenance immunosuppression with tacrolimus, mycophenolate mofetil, and prednisone.

His post-transplant course was complicated by postoperative hypotension and delayed graft function, requiring hemodialysis support. Kidney allograft ultrasound on postoperative day 2 did not show any peri-transplant fluid collections, abnormal vascular waveforms, or hydronephrosis. Repeat DSA showed an improvement with MFI decreased to 1100.

Due to persistent need for hemodialysis, the patient underwent an allograft biopsy on postoperative day 9. Light microscopy findings included widespread tubular epithelial injury manifested by variable degrees of tubular epithelial simplification, tubular ectasia, patchy cytoplasmic vacuolization, and reactive-appearing tubular epithelial nuclei. Numerous tubular lumens revealed birefringent, needle-shaped crystals organized in fan-shaped arrays. On H&E and PAS stains, they showed brownish pigmentation, and intense argyrophilia on Jones methenamine silver stain (Figure 3). These findings were consistent with crystalline nephropathy and suspicious for DHA crystalline nephropathy. There was no evidence of cellular or antibody-mediated rejection and no significant glomerular, tubulointerstitial, or vascular chronic injury. The patient was not receiving any diuretic therapy.

A presumptive diagnosis of recurrent DHA crystalline nephropathy was made, and the patient was started on febuxostat 40 mg daily, a low-purine diet, and daily dialysis for 3 days to enhance the clearance of DHA. He was discharged to receive maintenance hemodialysis with close follow-up. He received belatacept on postoperative day 16, and the tacrolimus trough goal was reduced to around 5 ng/mL. The patient was able to discontinue dialysis by postoperative day 21. Kidney gene panel (Renasight™) testing confirmed the diagnosis of APRT deficiency. He continued to have improvement in allograft function; febuxostat was increased to 80 mg daily, and a low-purine diet was continued. There was no strong indication for a repeat kidney biopsy since the patient demonstrated very stable and good graft function on the current management. His creatinine decreased to 1.3 mg/dL within the first year post-transplant. At the final follow-up visit, 10 months post-transplant, serum creatinine was 1.56 mg/dL and eGFR was 48 mL/min per 1.73 m². He continued to receive belatacept and low-dose tacrolimus with a trough level around 5 ng/mL. Additional immunosuppression included mycophenolate and prednisone.

Discussion

In this report, we describe 2 cases of recurrent DHA nephropathy leading to allograft dysfunction during the early post-transplantation period. Characteristic crystalline deposition was noted in the allograft biopsies and the diagnosis of APRT deficiency was confirmed by genetic analysis. Both cases were managed with a xanthine oxidase inhibitor, low-purine diet, reduction in tacrolimus dose and use of belatacept for maintenance immunosuppression. Our cases are novel, incorporating a strategy of early diagnosis due to a high degree of suspicion, early confirmation by genetic analysis (using a next-generation sequencing gene panel), and use of belatacept to allow lower calcineurin inhibitor exposure in an effort to mitigate long-term tubulointerstitial injury.

The prevalence of DHA nephropathy varies based on ancestry. Type 1 APRT deficiency (complete enzyme deficiency) is most common in people of European ancestry, with an estimated incidence of homozygosity between 1 in 50 000 and 1 in 100 000. Type 2 APRT deficiency, characterized by partial enzyme activity, is more prevalent in Japanese populations. This autosomal recessive condition exhibits regional and genetic variation, with specific forms more frequently observed in certain populations. However, the true prevalence remains difficult to ascertain due to the underuse of genetic testing and a low index of suspicion.

DHA nephropathy is a rare condition which can affect native kidneys and which can recur in transplant allografts, leading to poor outcomes and high risk of allograft failure [4]. The diagnosis of DHA nephropathy is challenging and can potentially be missed if end-stage kidney disease is attributed to diabetes, hypertension, or other more common etiologies. Lack of native kidney biopsy and delays in diagnosis of chronic kidney disease until glomerular filtration rate has progressed to stage 4 or stage 5 chronic kidney disease often lead to a presumptive etiologic diagnosis without biopsy confirmation.

The diagnosis of DHA nephropathy can be made from kidney biopsy by light microscopy, utilizing standard staining techniques. DHA crystals are present in tubular lumens, tubular epithelial cell cytoplasm, and the interstitium. The crystals are reddish-brown on H&E and PAS stains, black on silver stain, blue on trichrome stain, and birefringent under polarized light. Calcium phosphate crystals, which are frequently present in early post-transplant allograft biopsies can be readily differentiated from DHA crystals. DHA crystals, however, show many morphologic similarities to calcium oxalate crystals by light microscopy. A helpful feature to identify DHA crystals is visible upon Jones methenamine silver staining, wherein DHA crystals stain black (silver positive), unlike calcium oxalate which is translucent (Figures 1, 2). DHA crystals cannot be distinguished from triamterene-associated crystals by light microscopy [5]. DHA can also be identified by stone analysis (which combines morphologic inspection by stereomicroscope with infrared spectroscopy). Crystals can also be investigated by Fourier transformed infrared microscopy when observed by microscopy in urine samples or renal biopsy in individuals with crystalline nephropathy. This is a highly sensitive and specific approach [6].

Suspected APRT deficiency can be confirmed by measuring APRT activity in erythrocyte lysates. The APRT activity test may also be useful in certain situations when crystalluria cannot be investigated due to anuria or non-availability of technique [6]. APRT gene analysis can also be utilized to confirm the diagnosis and to identify specific mutations [6]. Genetic testing to identify a pathogenic variant of the APRT gene in a homozygous state is the gold standard for diagnosis.

Urinalysis is an initial evaluation for APRT deficiency, as it helps detect DHA crystals in the urine, which are pathognomonic for the condition. Key findings in urinalysis include:

The differential diagnosis for DHA crystalline nephropathy includes several other crystalline nephropathies that may present with similar histopathological features. Key differentials include:

The most common complication of APRT deficiency in native kidneys is recurrent nephrolithiasis. However, progressive chronic kidney disease and acute kidney injury episodes, occurring in the absence of nephrolithiasis, have also been reported [3,7]. In contrast, recurrence of DHA-related disease in transplant kidneys presents as crystalline nephropathy early in the post-transplant course and can progress to allograft failure, if left untreated [3,8–10]. DHA crystalline nephropathy can be effectively treated and prevented by xanthine oxidase inhibitors [11,12]. The risk of allograft loss is very high if the diagnosis and subsequent xanthine oxidase inhibitor treatment are delayed. For example, Runolfsdottir et al compared renal outcomes in kidney transplant patients with previously diagnosed APRT deficiency. The median eGFR at 2 years post-transplant was 61.3 (24.0–90.0) mL/min/1.73 m2, when xanthine oxidase inhibitor therapy was initiated pretransplant, compared with 16.2 (10.0–39.0) mL/min/1.73 m2 (P=0.009) when it was not [7].

Intra- or extra-tubular crystalline precipitates can cause tubulointerstitial injury and lead to chronic kidney disease or allograft dysfunction [13,14]. Acute, sudden onset of crystal formation in the tubules can lead to crystal-induced tubular cell injury and interstitial inflammation, whereas a subacute gradual process of crystal formation can cause tubular obstruction and remodeling [13,14]. In the case of a kidney transplant for a patient with DHA nephropathy, acute intratubular crystal precipitation can explain delayed graft function or persistent allograft dysfunction. Our biopsy findings of significant tubular injury without evidence of obstructive changes support this hypothesis. Various mechanisms including direct and indirect cytotoxicity have been proposed in pathophysiology of tubular injury. Exposure to calcineurin inhibitors (CNIs) can contribute to further tubular injury and increase the risk of cell necrosis. Belatacept, a CNI-sparing agent, has been used in kidney transplantation to reduce interstitial fibrosis and tubular atrophy associated with CNI-related injury. While this drug has a broad clinical application for managing allografts with donor-derived injury and fibrosis, it is also emerging as a potential treatment for crystalline nephropathy. In our cases, belatacept was successfully used, contributing to favorable allograft outcomes.

The majority of cases of recurrent DHA nephropathy following kidney transplantation have occurred in patients in whom the diagnosis of APRT deficiency was not made prior to transplantation [15–20]. Zaidan et al reviewed 9 patients with recurrent DHA crystalline nephropathy, in all of whom the diagnosis had been missed prior to renal transplantation [12]. The diagnosis was confirmed at a median of 5 weeks after renal transplant. Patients had acute allograft dysfunction (n=1), acute-on-chronic allograft dysfunction (n=5), and delayed graft function (n = 2), while 1 patient had normal graft function at the time of diagnosis [12]. Therapy with allopurinol was initiated with an initial dose ranging from 100 to 400 mg/day and patients were followed up for 2 years. This study concluded that cases with early diagnosis and treatment have better outcomes in terms of graft function [12]. Similarly, Nasr et al reviewed 3 patients with APRT deficiency with no previous history of recurrent nephrolithiasis, 2 of whom were initially misdiagnosed [21]. Subsequently, all 3 progressed to end-stage renal disease, within 1 month of their renal biopsy in the first and second patients, and 9 months in the third. All 3 received kidney transplants with early recurrence of DHA nephropathy in the patient with previously unrecognized and untreated APRT deficiency. The extent of DHA crystals was markedly attenuated in the patients who received xanthine oxidase inhibitor therapy. This study highlights the importance of having 2,8-dihydroxyadeninuria as a differential diagnosis, even in the absence of a history of nephrolithiasis. Our patients received an early biopsy-proven diagnosis of DHA crystalline nephropathy in the allograft at 6 weeks and 2 weeks, respectively, leading to early initiation of appropriate therapy that resulted in long-term preservation of allograft function at 2 years of follow-up.

These cases also highlight the importance of accurate diagnosis of the etiology of kidney disease in patients with end-stage kidney disease. Given the high prevalence of diabetes and hypertension, which are the most common underlying etiologies of kidney disease in the United States, native kidney biopsy is not typically incorporated in chronic kidney disease management and a presumptive diagnosis based on clinical history is often made. For instance, patient 1 was presumed to have diabetic nephropathy, but on further review of his history, he was diagnosed with Type-2 diabetes mellitus 5 years prior to diagnosis of end-stage kidney disease and did not have other microvascular complications of diabetes including neuropathy or retinopathy. Patient 2 had an extensive history of kidney stones but lacked prior evaluation, such as stone composition analysis or 24-hour urine studies. Pre-transplant genetic testing can help identify high-risk patients with clinically suspected rare genetic disorders, such as APRT deficiency, thus preventing delayed diagnosis and ensuring better long-term transplant outcomes.

Conclusions

DHA nephropathy is a rare and under-recognized, yet preventable, cause of chronic kidney disease that can recur in transplanted kidneys and lead to allograft failure. Therefore, in transplant recipients with unexplained persistently elevated serum creatinine concentration, an allograft biopsy is indicated. The presence of crystals in the renal parenchyma should not be overlooked and DHA nephropathy should be considered in the differential diagnosis of crystalline nephropathy. A high index of suspicion and early diagnosis are important, as timely therapy can prevent irreversible damage.